Protective effect of sesamol against ⁶⁰Co γ-ray-induced hematopoietic and gastrointestinal injury in C57BL/6 male mice.

Abstract Protection of γ-ray-induced injury in hematopoietic and gastrointestinal (GI) systems is the rationale behind developing radioprotectors. The objective of this study, therefore, was to investigate the radioprotective efficacy and mechanisms underlying sesamol in amelioration of γ-ray-induced hematopoietic and GI injury in mice. C57BL/6 male mice were pre-treated with a single dose (100 or 50 mg/kg, 30 min prior) of sesamol through the intraperitoneal route and exposed to LD50/30 (7.5 Gy) and sublethal (5 Gy) dose of γ-radiation. Thirty-day survival against 7.5 Gy was monitored. Sesamol (100 mg/kg) pre-treatment reduced radiation-induced mortality and resulted survival of about 100% against 7.5 Gy of γ-irradiation. Whole-body irradiation drastically depleted hematopoietic progenitor stem cells in bone marrow, B cells, T cell subpopulations, and splenocyte proliferation in the spleen on day 4, which were significantly protected in sesamol pre-treated mice. This was associated with a decrease of radiation-induced micronuclei (MN) and apoptosis in bone marrow and spleen, respectively. Sesamol pre-treatment inhibited lipid peroxidation, translocation of gut bacteria to spleen, liver, and kidney, and enhanced regeneration of crypt cells in the GI system. In addition, sesamol pre-treatment reduced the radiation-induced pattern of expression of p53 and Bax apoptotic proteins in the bone marrow, spleen, and GI. This reduction in apoptotic proteins was associated with the increased anti-apoptotic-Bcl-x and PCNA proteins. Further, assessment of antioxidant capacity using ABTS and DPPH assays revealed that sesamol treatment alleviated total antioxidant capacity in spleen and GI tissue. In conclusion, the results of the present study suggested that sesamol as a single prophylactic dose protects hematopoietic and GI systems against γ-radiation-induced injury in mice.


Introduction
Exposures to ionizing radiation cause oxidative injury to almost all organs depending upon the radiosensitivity of the organs, radiation dose, and dose rate [1,2]. These damages result in multi-organ dysfunction, which can lead to acute radiation syndrome (ARS) and long-term health eff ects, for example, cancer or pulmonary fi brosis [3,4]. ARS includes hematopoietic (2)(3)(4)(5)(6), gastrointestinal (6)(7)(8), and cerebrovascular ( Ͼ 8 Gy) sub-syndromes [5]. The hematopoietic and gastrointestinal (GI) subsyndromes are manifested by enormous loss of hematopoietic progenitor stem cells (HPSCs) in bone marrow and impairment of crypt cell regeneration in the GI tract, respectively. Therefore, strategies for developing prophylactic agents as radioprotectors necessarily require an investigation of hematopoietic and GI injury [5].
The possibility of occurrence of ARS during planned radiation exposure exists in critical operations, military warfare, radiation environment, nuclear reactors, and radiotherapy. At present, no radioprotector is available to known for its role in antiaging, chemoprevention, neuroprotection, and hepatoprotection [12 -14]. Sesamol is structurally composed of both phenolic and benzodioxole groups, which are responsible for the antioxidative and radioprotective properties, respectively [15]. Sesamol scavenges free radicals, ROS, and nitrogen species by donating hydrogen atoms and enabling electron transfer to the radical center [15]. In addition, sesamol has been demonstrated to have properties that decrease radiationinduced micronuclei (MN), dicentric frequencies, thiobarbituric acid reactive substances (TBARS) and DNA strand breaks, and increase GSH, SOD, CAT and GPx in a concentration/dose-dependent manner in cultured lymphocytes [16,17]. Further, sesamol pre-treatment increased the survival of lethally irradiated mice by inhibiting radiation-induced DNA strand breaks in lymphocytes and lipid peroxidation, and enhancing the level of antioxidant enzymes (GSH, GST, catalase) [18,19]. In view of the promising results, our research group has investigated in detail the antiradical properties of sesamol, along with other known antioxidants. These studies have revealed strong radical-scavenging properties of sesamol in comparison to other reference antioxidant molecules including melatonin, a potential antioxidant-based radioprotector [20]. Further, studies using plasmid DNA (pBR322) and calf thymus DNA, and the in vitro V79 cell line, showed 20 times higher protection and greater DMF (dose-modifying factor) than melatonin, possibly be due to its strong free radical-scavenging property [21].
Based on our earlier studies on sesamol ' s antioxidant properties and in vitro results [20,21], as well as literature reports [16,18,19] on radioprotection, we have selected sesamol for further in vivo investigations. The objective of this study, therefore, was to investigate the radioprotective potential of sesamol in whole-body γ -irradiated (5 Gy and 7.5 Gy) C57BL/6 male mice, a recommended animal model for developing radioprotectors [22]. A single dose of sesamol (50 -100 mg/kg, 1/10th -1/5th of LD 50 ) was selected based on the estimated LD 50 dose for the same strain (unpublished data). Sesamol pre-treatment (100 mg/kg) provided 100% survival against LD 50/30 (7.5 Gy) dose of γ -radiation. Thus, subsequent studies have been designed at 100 mg/kg of sesamol against LD 50/30 (7.5 Gy) and a sublethal (5 Gy) dose of γ -irradiation, to understand the mechanism underlying observed radioprotection in the most radiosensitive organs -the bone marrow, spleen, and GI system [23,24]. We have performed assays of colony forming units (CFU), micronuclei (MN), and cell cycle in the bone marrow; studied immunophenotyping (CD4, CD8), B cells, and apoptosis in spleen; and performed studies on histology, along with lipid peroxidation, and carried out western blotting of apoptotic and anti-apoptotic proteins in the GI system. In addition, gut bacterial translocation to the spleen, liver, and kidney was measured. The results suggest that sesamol pre-treatment protects from radiation-induced injuries in hematopoietic and GI systems in C57BL6 mice. These fi ndings can be exploited to develop sesamol-based radioprotectors.

Animals
Male C57BL/6 mice (8 -10 weeks old) were issued and randomly divided into diff erent groups one week prior to the study, to allow acclimatization. This mouse model is one of the recommended models for developing countermeasures to radiation [22]. Animals not treated (with drug or radiation) served as sham control. Animals treated with sesamol (100 mg/kg body weight) served as sesamol control (sesamol treatment). Mice treated with radiation alone (5 Gy and 7.5 Gy, at 1 Gy/min, whole-body γ -radiation exposure) served as radiation control. The fourth group consisted of sesamol pre-treated mice that received sesamol (100 mg/kg body weight) intra-peritoneally before 30 min of 5 Gy and 7.5 Gy (1 Gy/min) with wholebody γ -radiation exposure. Animals were distributed, with maximum of six per polypropylene cage containing certifi ed paddy husk as bedding, and provided certifi ed food and acidifi ed water ad libitum throughout the experiment. Animals were housed in a pre-maintained room with a 12-h light/dark cycle at a temperature of 23 Ϯ 2 ° C and relative humidity of 55 Ϯ 5%. All protocols used in this experiment were approved by the Committee on the Ethics of Animal Experiments of the Institute of Nuclear Medicine and Allied Sciences. The Institutional Ethical Committee number under which this study was performed is INM/IEAC/2012/06. All eff orts were made to minimize suff ering during sacrifi ce of the animal through cervical dislocation.

Preparation and administration of sesamol
Sesamol was freshly prepared by mixing in soybean oil. A single prophylactic dose of sesamol (50 or 100 mg/kg body weight) or vehicle (soya bean oil) in a volume of 0.2 ml was administered intraperitoneally using a 26-gauge needle, 30 minutes before of whole-body γ -irradiation.

Gamma-irradiation
Animals were placed in a plastic cage and whole-body γ -radiation exposure was carried out using a 6 ° Co teletherapy unit (Bhabatron II, Bangalore, India) at a dose rate of 1 Gy/min, to a total dose of 5 Gy and 7.5 Gy. After irradiation, mice were returned to the animal house and monitored. The dose rate of 6 ° Co γ -rays source (Bhabatron II) was calibrated using physical dosimetry by the radiation safety offi cer of our Institute.

Immunomagnetic selection of HPSCs and colony forming unit assay in bone marrow
Animals were sacrifi ced under aseptic conditions, and both the femurs were dissected out. After removing extra tissue from the femur, bone marrow cells were extruded by cutting the epiphyseal ends and fl ushing using a 26-gauge needle with 5 mL of recommended media (PBS supplemented with 2% FBS). Single-cell suspensions of bone marrow were prepared by gentle pipetting using a sterile Pasteur-pipette (BD Biosciences, San Diego, CA, USA) and passing through a 100 μ m nylon mesh strainer (BD Biosciences, San Diego, CA, USA). The marrow cells were centrifuged twice at 500 ϫ g at 4 ° C for 5 min, and cell numbers were maintained by a hemocytometer (Neubauer, Marienfeld, Germany) using an inverted microscope (4200, Meiji, Japan).
HPSCs (Sca-1 ϩ cells) were isolated from whole bone marrow suspensions using the purple EasySep magnet and Mouse Sca-1 Positive Selection Kit (Stem Cell Technology, Canada), following the manufacturer ' s protocol. Sca-1 ϩ cells (3.6 ϫ 10 3 cells) in a volume of 300 μ L of recommended media were mixed (by gentle vortex) with 3 mL of MethoCult TM media (Stem Cell Technology, Canada). Using a Luer lock-fi tting syringe and a 16-gauge blunt-end needle (Stem Cell Technology, Canada), 1.1 mL of media was dispensed into a pre-marked 35 mm culture dish (Stem Cell Technology, Canada). A 100 mm Petri dish containing two 35 mm culture dishes with lid and a third uncovered 35 mm culture dish fi lled with sterile water were placed, and incubated for 7 -14 days at 37 ° C, 5% CO 2 , with Ն 95% humidity. On day 7, the whole 35 mm Petri dish was scanned in a 60 mm gridded scoring Petri dish (Stem Cell Technology, Canada) under low power (40 ϫ and 100 ϫ ), and individual colony types (CFU-E, CFU-GM and CFU-GEMM) were identifi ed using an inverted microscope. For each sample, two 35 mm culture dishes were scored, and three mice were used to generate each data point. The total CFU indicated the sum of CFU-E, CFU-GM, and CFU-GEMM.

Bone marrow micronucleus assay
Animals were sacrifi ced by cervical dislocation on the 1st, 4th, 7th, 15th and 30th days post irradiation. Both the femurs of the animal were dissected and the bone marrow immediately fl ushed out with pre-chilled PBS. For the MN assay, the bone marrow cell suspension was processed according to Schmid ' s method, with minor modifi cations [25]. Briefl y, after centrifugation at 1000 rpm for 10 min, the supernatant was discarded and the cell pellet was resuspended in 2 to 3 drops of FBS. The cell suspension was smeared on clean dry slides, as well as air-dried, and then fi xed with methanol. The slides were stained with 4% Giemsa (1/6) diluted in Sorensen ' s phosphate buff er (sodium phosphate monobasic and sodium phosphate dibasic, pH 6.8) for 10 -15 min, followed by washing with the same buff er. All slides were coded by an independent person and scored under 100 ϫ objectives with a light microscope (Primo Star, Carl Zeiss, Germany). From each mouse, a minimum of 1000 nucleated cells were scored for MN analysis. The frequency of MN per cell was calculated with respect to total nucleated cells scored. The representative images of MN and apoptotic cells are shown in Figure 3. All slides were decoded after completion of scoring and analysis.

Bone marrow cell cycle analysis
Bone marrow cells of animals sacrifi ced on the 1st, 4th, 7th, 15th and 30th days post irradiation were fi xed with methanol. After overnight fi xing, 1 ϫ 10 6 cells were washed with PBS, and then treated with 200 μ g/ml of RNase at 37 ° C for 30 min. Cells were washed with PBS and stained with PI (50 μ g/ml). After 20 min, cell cycle analysess were carried out using a BD FACSCalibur 3CB (BD Biosciences, USA) to measure the diff erent phases of the cell cycle.

Flow cytometric analysis of immunophenotyping and assay for apoptosis in splenocytes
Animals were sacrifi ced by cervical dislocation on the 4th and 21st days post irradiation. The spleen was removed, cleaned, and morphologically observed. The spleen was then minced using sterile slides in a Petri dish containing pre-chilled PBS. Single-cell suspensions were obtained by fi ltration with a 100 μ m nylon mesh strainer (BD Biosciences, San Diego, CA, USA), and RBCs were lysed using BD FACS TM lysing solution (BD Biosciences, San Diego, CA, USA). The single-cell suspension was used for the analysis of CD19 as a B cell marker and CD4 and CD8 as T cell subpopulation markers, and apoptosis was assayed by fl ow cytometry (LSR II, BD, USA).
The Annexin V-FITC apoptosis detection kit was used, following the manufacturer ' s instructions (BD Biosciences, San Diego, CA, USA). In brief, singe-cell suspensions (1 ϫ 10 6 cells) were mixed with 1 mL of binding buff er, and 100 μ l of this suspension was again mixed with 5 μ l each of Annexin V-FITC and propidium iodide (PI) for 15 min at room temperature. In each sample, 1 ϫ 10 4 cells were analyzed by fl ow cytometry.

Splenic lymphocyte proliferation assay
To assess the eff ect of sesamol pre-treatment on splenocyte proliferation in irradiated mice, mitogen (Con A)stimulated proliferating splenocytes were evaluated by trypan blue dye exclusion and carboxyfl uorescein succinimidyl ester (CFSE) staining [26]. Briefl y, animals were sacrifi ced on day 4 post irradiation by cervical dislocation. Under aseptic conditions, the spleen was dissected out and minced using a pair of sterile frosted slides in autoclaved pre-chilled PBS supplemented with 10% RPMI-1640 and 2% FBS. Single-cell splenocyte suspensions were obtained by passing through a 100 μ m nylon mesh strainer (BD Biosciences, San Diego, CA, USA), and RBCs were lysed using BD FACS TM lysing solution (BD Biosciences, San Diego, CA, USA) for 10 min at room temperature. After centrifugation, splenocytes were washed with pre-chilled PBS and resuspended in complete RPMI-1640 media supplemented with 25 mM HEPES (pH 7.4), 50 mM 2-mercaptoethanol, 100 U/ml Pen Strep (penicillin and streptomycin), and 10% FBS. For direct assessment of splenocyte proliferation, the splenocytes (5 ϫ 10 6 cell/ml) were cultured in the presence of Con A (5 μ g/ml) and incubated in a humidifi ed atmospheric condition with 5% CO 2 at 37 ° C for 72 h. The splenocyte proliferation was examined by direct counting of viable cells after trypan blue dye exclusion. Alternatively, for assessment of proliferating splenocytes by CFSE staining, the Con A-stimulated splenocytes (5 ϫ 10 6 cell/ml) as described above, were incubated with 1 μ M CFSE. The splenocytes were washed twice with PBS supplemented with 10% RPMI-1640 and the pellet resuspended in PBS. The single-cell suspensions were acquired in a fl ow cytometer (LSR II, BD, USA), and mean fl uorescence intensity (MFI) was assessed using FACS Diva software.

Histological examination in the GI tract
To evaluate the radiation-induced oxidative damage and recovery in the GI tract, mice were sacrifi ced on the 1st, 4th, 7th, 15th and 30th days post irradiation and the jejunum dissected out, cleaned in pre-chilled PBS, and fi xed in 10% formalin (v/v) at room temperature. Five micronthick sections were cut and placed on pre-cleaned slides. The sections were stained with hematoxylin and eosin (H & E), mounted and analyzed using an upright motorized compound microscope with a DIC attached and a digital imaging system (Axio, Imager M2, Zeiss, Germany). Qualitative changes in the jejunum of the GI tract were assessed by visualizing the submucosal crypt and jejunal villi. To assess the quantitative changes in the jejunum of the GI tract, ten circumferences were scored per slide for mean crypt and villi numbers, with three mice per group, and a total of 3 ϫ 10 circumferences per group were used to generate each data point. Ten villi were measured in each section and ten circumferences per slide for a total of 10 ϫ 10 villi per mice, and a total of three mice were used to generate each data point for mean villi length. To avoid bias in analysis, all slides were coded prior to staining by a person not involved in the analysis, and decoded after completion of analysis.

Analysis of gut bacterial translocation
To analyze the eff ect of sesamol pre-treatment on the translocation of gut bacteria, bacterial loads were measured as CFUs by culturing spleen, kidney, and liver homogenates on sheep blood agar plates, as described elsewhere [27]. Briefl y, mice were killed by cervical dislocation and their abdomens were soaked with 70% alcohol. The spleen, kidney, and liver were collected aseptically on the 9th, 17th, and 21st days post irradiation, with a sterile scissor for each organ. These organs were immediately ground in broth (5 mL each) and incubated aerobically for 24 h at 37 ° C. After incubation, the organ homogenates were spread with glass rods onto sheep blood agar plates and were incubated for 16 h at 37 ° C to detect the presence of bacterial CFUs.

Assay of thiobarbituric acid reactive substances in GI tract
To assess the level of thiobarbituric acid reactive substances (TBARS) production in GI tract (jejunum), mice were given whole-body γ -irradiation (5 Gy and 7.5 Gy), and 3 h post irradiation, the jejuna were dissected out by cervical dislocation. Tissue was homogenized in prechilled PBS (10% w/v) using a tissue homogenizer (OMNI TH, USA). TBARS were measured following the standard protocol specifi ed by Ohkawa and coworkers [28] in homogenate. TBARS were represented as nmol per gram of wet tissue weight.

Extraction of protein and western blot analysis in GI Tract, spleen, and bone marrow cells
The mouse jejuna, spleen, and both femurs were dissected out 3 h post irradiation (5 Gy and/or 7.5 Gy), and extra fat tissue was cleaned in pre-chilled PBS on ice. The jejuna and spleen were homogenized in cold RIPA buff er (50 mM Tris-HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Tween-100, 5 mM EDTA, 1 mM EGTA, and 1 mM PMSF) containing a protease inhibitor cocktail using a tissue homogenizer. Bone marrow cells were fl ushed out in pre-chilled PBS, as per the protocol described in the previous section, and lysed in cold RIPA buff er containing protease inhibitor cocktail. The tissue and cell homogenates were centrifuged at 10,000 RPM for 15 min at 4 ° C. In the same supernatant, protein was measured by the Bradford method [29] and concentration estimated using the BSA standard curve. An equal amount of proteins was separated using 12% SDS-PAGE followed by transfer onto a nylon membrane (Sigma-Aldrich Co.) for 55 min. The membranes were blocked using TBST buff er (0.2 M Tris-base, 1.5 M NaCl, 0.1% Tween-20) supplemented with a solution containing 5% skimmed milk, for 1 h, on a shaker at room temperature. Membranes were then incubated with primary antibodies ( β -actin, p53, Bax, Bcl-x and PCNA) overnight on the shaker at 4 ° C. After washing twice with TBST buff er, the membranes were incubated with secondary antibody conjugated with peroxidase for 1 h at room temperature, on the shaker. The membranes were again washed twice with TBST buff er and bands were visualized using ECL chemiluminescence reagent. The intensity of each protein band was measured using Gel Doc XR (Bio-Rad, USA).

Analysis of total antioxidant capacity in the GI tract and spleen
The total antioxidant capacity in the GI tract and spleen was measured in mice treated with sesamol and compared with that of control mice. The GI tract and spleen were removed and cleaned in pre-chilled PBS on ice. Tissue homogenates were prepared in pre-chilled PBS (10% w/v) with a tissue homogenizer, and centrifuged at 12000 g at 4 ° C for 15 min. The supernatant was stored at Ϫ 80 ° C for further analysis.
ABTS radical scavenging activity of the GI tract and spleen was determined spectrophotometrically (Cary100-Bio, Varian, Australia) using the method described elsewhere [30]. The decrease in absorbance of the ABTS radical at 732 nm was measured using 30 min of reaction kinetics. The ABTS • ϩ scavenging capacity of the GI tract and spleen were expressed as TEAC (Trolox Equivalent Antioxidant Capacity)/mg of wet tissue. The amount of Trolox ( μ mol) corresponds to 1 mg of wet tissue.
The DPPH radical scavenging capacity in the GI tract and spleen was measured following the modifi ed procedure [20]. Briefl y, prior to analysis, a 200 μ M DPPH stock solution was prepared in methanol. DPPH working solution (60 μ M) in a volume of 2 mL was mixed with the biological sample (100 μ L) and the decrease in absorbance continuously monitored for 30 min using a spectrophotometer at 515 nm. The DPPH radical scavenging activity is expressed as percentage inhibition per mg of wet tissue, as mentioned below.
Where A control is the initial concentration of the DPPH and A sample is absorbance of the remaining concentration of DPPH in the presence of antioxidant.

Statistical analysis
The mean values and standard errors or percentage of the present data were analyzed and reported. The percentages of survival among groups were analyzed using the Kaplan -Meier statistical graph. For the survival study, p -values at the end of the 10th, 20th, and 30th days post irradiation were analyzed using Log-rank (Mantel -Cox) and Gehan -Breslow -Wilcoxon statistics. Pairwise comparisons were made between groups (inter and intra) using the Student ' s t-test and ANOVA (analysis of variance). Statistically signifi cant diff erences between groups were considered if the value of p ˂ 0.05.

Results
Radioprotective effi cacy of sesamol against 60 Co γ -irradiation in C57BL/6 male mice Sesamol or vehicle (soya bean oil) was administered intraperitoneally, 30 min prior to 7.5 Gy of whole-body γ -radiation exposure. Sesamol (100 mg/kg) pre-treatment showed survival of about 100% at LD 50/30 dose of 7.5 Gy, whereas a lower dose of sesamol (50 mg/kg) resulted in 70% survival ( Figure 1). The vehicle did not exhibit protection and followed an almost similar pattern of death of mice in the irradiated group (Figure 1). The survival study was analyzed on the 10th, 20th, and 30th days post irradiation, with respective percent survival and p -value ( Figure 1). The survival data in Figure 1 showed signifi cant protection by sesamol (100 mg/kg) pre-treatment ( p Ͻ 0.05), in comparison to mice treated with 7.5 Gy alone, on 20th and 30th day post irradiation. In the present survival study, the fi rst animal died on day 9 in the group exposed to radiation alone, which was expected because of hematopoietic and GI injury. Whole-body radiation dose of 7.5 Gy is known to cause such injuries [1 -4]. Therefore, signifi cant survival by sesamol pre-treatment could be due to overcoming the hematopoietic and GI injury. Thus, we have selected 100 mg/kg of sesamol for further evaluation in radiosensitive organs.

Eff ect of sesamol on radiation-induced HPSC depletion in bone marrow
HPSCs are highly sensitive to radiation-induced cellular injury [5]. Therefore, a CFU assay of femoral HPSCs (CFU-E, CFU-GM and CFU-GEMM) was performed to assess the eff ect of sesamol on radiation-induced hematopoietic injury in mice irradiated with 5 Gy and 7.5 Gy. Whole-body radiation exposure to 5 Gy depicted signifi cant ( p Ͻ 0.001) femoral HPSC depletion and reached a nadir on day 4 in 7.5 Gy-irradiated mice ( Figure 2). Interestingly, sesamol pre-treatment signifi cantly expanded recovery of femoral HPSCs in 5 Gy and 7.5 Gy ( p Ͻ 0.5) whole-body irradiated mice ( Figure 2). The CFU assay confi rmed that sesamol pre-treatment expands recovery of femoral HPSCs, thereby contributing to overcoming hematopoietic injury in irradiated mice.

Eff ect of sesamol on radiation-induced micronuclei in bone marrow
The MN frequency was determined in nucleated bone marrow cells of all experimental animals. The results have shown signifi cant increase in the frequency of MN in 7.5 Gyirradiated mice, in comparison to control mice ( p Ͻ 0.001), 24 h post irradiation. Pre-treatment with sesamol reduced this elevated frequency in irradiated mice. Most importantly, sesamol (100 mg/kg) did not increase MN frequency in bone marrow cells (Figure 3). Irradiation with 7.5 Gy increased apoptotic frequency in bone marrow cells on the 1st day post irradiation, and the frequency was signifi cantly higher ( p Ͻ 0.001) in comparison to control. However, sesamol pretreatment signifi cantly ( p Ͻ 0.05) reduced radiation-induced apoptotic cell frequency in irradiated mice (Figure 3). On the 7th and 15th days post irradiation, 7.5 Gy-irradiated mice showed very few cells in microscopic slides; only 20 -30 cells without MN were observed. On the 30th day post irradiation, all 7.5 Gy-irradiated mice were dead. Pre-treatment with sesamol reduced the MN frequency near to control on 15th and 30th days post irradiation in irradiated mice. The diff erence was non-signifi cant ( p Ͼ 0.05) in sesamol pre-treated mice compared with control mice. The plot of MN/cell versus post irradiation days for diff erent groups showed the protective eff ect of sesamol ( Figure 3). Therefore, results suggest that sesamol pre-treatment provided signifi cant protection to bone marrow cells against radiation-induced cellular injury.

Eff ects of sesamol on radiation-induced cellular perturbation in bone marrow
The analysis of cell cycle proliferation in bone marrow showed signifi cant ( p Ͻ 0.05) increase in the percentage of cell death in irradiated mice on the 1st day post irradiation. This further increased at later post irradiation time points and ultimately led to the death of mice, as observed till the 30th day. Sesamol pre-treatment reduced cell death on the 1st day, but it was signifi cant ( p Ͻ 0.001) on the 7th day in irradiated mice ( Figure 4). The increase in cell death ( Figure 4A) was associated mainly with the decrease in G1 phase population ( Figure 4B), which was significantly reversed in sesamol pre-treated mice (Figure 4). This reduction of cellular death at later time points was associated with the survival of 100% of the animals till the 30th day of observation (Figure 1).

Morphological examination of spleen
Morphological examination of splenic injury against γ -irradiation was performed and showed hemorrhagic spleens in γ -irradiated (7.5 Gy) mice on day 21, and this was completely absent in sesamol pre-treated mice ( Figures 5A -D). Therefore, sesamol pre-treatment visibly showed protection against radiation-induced injury in spleen.

Eff ect of sesamol on radiation-induced depletion in B cell and T cell subpopulations
The indirect staining of mouse splenocytes showed signifi cant recovery ( p Ͻ 0.01) of CD19 (B cell), CD4, and CD8 populations in irradiated mice pre-treated with sesamol on day 4 ( Figure 6). As expected, the percentage of CD19 cells was clearly low in mice treated with radiation alone, in comparison to control ( p Ͻ 0.001), while an increase was noted in the sesamol pre-treated mice in comparison to mice treated with radiation alone ( p Ͻ 0.001) Figure 1. Radioprotective dose -response of sesamol against 60 Co γ -irradiation with Kaplan -Meier survival statistical graph. Male C57BL/6 mice were injected with 50 or 100 mg/kg of sesamol intra-peritoneally, 30 min prior to 7.5 Gy ( n ϭ 10) whole-body γ -radiation, and periodically monitored in a 30-day survival study. Statistically signifi cant diff erences between groups at the 10th, 20th and 30th days post irradiation were analyzed using log-rank (Mantel -Cox) and Gehan -Breslow -Wilcoxon statistics. @ indicates groups compared with the group treated with 7.5 Gy alone.
( Figure 6A). In addition, the percentage of CD4 was signifi cantly ( p Ͻ 0.001) depleted by γ -irradiation (7.5 Gy), and hence, the ratio of CD4/CD8 decreased on day 4 post irradiation. Sesamol pre-treatment signifi cantly ( p Ͻ 0.01) increased CD4/CD8 ratio on day 4 in comparison to that in mice treated with radiation alone ( Figure 6B). The percentage of CD4 and CD8 further decreased on day 21 in comparison to day 4, after exposures to 7.5 Gy of radiation. In contrast, signifi cantly ( p Ͻ 0.01) higher numbers of CD4 and CD8 were observed on day 21 in comparison to day 4 in sesamol pre-treated mice, therefore, showing recovery in the CD4/CD8 ratio ( Figure 6B

Eff ect of sesamol on radiation-induced apoptosis in spleen
Gamma-radiation induced signifi cant apoptosis ( p Ͻ 0.001) on day 4 as analyzed fl ow cytometrically by Annexin V-FITC and PI staining in mice treated with radiation alone. Sesamol pre-treatment signifi cantly reduced ( p Ͻ 0.001) apoptosis on day 4 in 7.5 Gy-irradiated mice. In addition, there was an increase ( p Ͻ 0.01) in the apoptosis on day 21 in comparison to day 4, after exposure to 7.5 Gy of radiation, which was reversed in sesamol pretreated mice. The basal levels of apoptosis in control mice and mice treated with sesamol alone were found to be similar ( Figure 5E). The results suggest that reduction of apoptosis by sesamol pre-treatment might be associated with the increase of B cell and T cell subpopulations in irradiated mice ( Figure 6). Figure 2. Eff ects of sesamol on femoral HPSCs in mice exposed to γ -radiation (5 Gy and 7.5 Gy, 1 Gy/min) after the 4th day post-irradiation. Femoral bone marrow cells were collected under aseptic conditions by cutting the epiphyseal ends and using 26-gauge needle in 5 mL of recommended media. Femoral HPSCs (Sca-1 ϩ cells) were isolated using purple EasySep magnet and mouse Sca-1 positive selection kit. Sca-1 ϩ cells were mixed with MethoCult TM media and transferred into 35 mm culture dishes using a Luer lock-fi tting syringe and a 16-gauge blunt-end needle. All culture dishes were incubated for 7 -14 days at 37 ° C, 5% CO 2 , with Ն 95% humidity in a CO 2 incubator. On day 7, a 35 mm culture dish was placed in a 60 mm gridded scoring Petri dish and scanned under low power (40x and 100 ϫ ) using an inverted microscope. Individual colonies like CFU-E, CFU-GM, and CFU-GEMM were identifi ed in each of the 35 mm culture dishes. Two culture dishes were scored for a single mouse and three mice were used to generate each data point. Total CFU indicated the sum of CFU-E, CFU-GM, and CFU-GEMM. * p Ͻ 0.001 vs Control, $ p Ͻ 0.05 vs 5 Gy, # p Ͻ 0.05 vs 7.5 Gy.

Eff ect of sesamol on radiation-induced proliferation in splenocytes
In order to determine the splenocyte proliferation in 7.5 Gy-irradiated mice, we evaluated PCNA (a nuclear protein and a co-factor for DNA polymerase δ ) in 7.5 Gy-irradiated mice, 3 h post irradiation by western blot. A significant decrease in the level of PCNA protein expression was found in 7.5 Gy-irradiated mice in comparison to control ( Figure 7A). Sesamol pre-treated mice expressed higher levels of PCNA protein on 7.5 Gy irradiation ( Figure 7A). This result indicated that sesamol pre-treatment enhances the expression pattern of PCNA protein, which plays an important role in the processes of proliferation and repair of splenocytes in irradiated mice.
Alternatively, the eff ect of sesamol on the lymphoproliferative capacity of splenocytes in 7.5 Gy-irradiated mice was assessed on day 4 post irradiation by the trypan Figure 3. Sesamol reduced radiation-induced micronuclei and apoptotic cell in bone marrow of mice exposed to γ -irradiation (7.5 Gy, 1 Gy/min). Bone marrow cells were collected on diff erent days after irradiation. After smear preparation on slides, cells were stained with Giemsa and observed under 100 ϫ (oil) objective in a light microscope for micronuclei and apoptotic cells. Panels A, B, and C show one, two, and three micronuclei in a cell, respectively. Panels D, E, and F show apoptotic cells. Panel G shows micronuclei frequency in diff erent groups for post-irradiation days. Panel H represents the percentage of apoptotic cells 24 h post irradiation. * p Ͻ 0.001 vs 7.5 Gy (Day 1), $ p Ͻ 0.001 vs 7.5 Gy (Day 1), @ p Ͻ 0.05 vs 7.5 Gy. Figure 4. Eff ect of sesamol on radiation-induced cell cycle perturbation in bone marrow cells of mice exposed to γ -irradiation (7.5 Gy, 1 Gy/min). Bone marrow cells were collected on the 1st, 4th, 7th, 15th and 30th days post irradiation. After fi xation, cells were stained with propidium iodide and analyzed using fl ow cytometry, for diff erent phases of the cell cycle. Panel A: % Cell Death; Panel B: % Cells in G 1 Phase; Panel C: % Cells in S Phase; Panel D: % Cells in G 2 /M Phase. * p Ͻ 0.05 vs Con and Ses, $ p Ͻ 0.001 vs Con and Ses, @ p Ͻ 0.001 vs $ .

S. Khan et al.
blue dye and CFSE staining methods. By direct enumeration under an inverted microscope (4200, Meiji, Japan), we observed a signifi cant proliferation of Con A-stimulated splenocytes from control and sesamol pretreated mice, in comparison to 7.5 Gy-irradiated mice ( Figure 7B). In addition, MFI of irradiated mice were signifi cantly ( p Ͻ 0.01) increased in comparison to control mice. However, sesamol pre-treatment signifi cantly ( p Ͻ 0.01) decreased MFI and reached levels near control, in comparison to irradiated mice ( Figure 7C). This reduction in MFI was associated with the proliferating splenocytes that underwent division. Thus, results suggest that sesamol pre-treatment enhances the proliferation of splenocytes, which might have led to recovery in the B cell and T cell subpopulations ( Figure 6).

Eff ect of sesamol on radiation-induced damage in the GI tract
In addition to the hematopoietic system, the GI system is also highly sensitive to ionizing radiation. Radiation-induced GI injury is primarily manifested by the death of epithelial crypt cells. The epithelial crypts of the GI region are the highest proliferating cells and most susceptible to ionizing radiation [31]. Thus, counting of the epithelial crypts is considered the gold standard method to measure GI injury. It is believed that GI injury is initiated at supralethal doses of radiation exposure, but the several recent investigations have revealed GI injury at a much lower radiation dose, also indicating dependency on the radiation dose. Therefore, in this study at the LD 50/30 , a dose of 7.5 Gy was selected to assess the prophylactic role of sesamol in ameliorating GI injury. Qualitative analysis showed ruptured villi tips and lower numbers of villi and crypts on day 4 in 7.5 Gy-irradiated mice (Figure 8C, E, G and I). Our observations depicted normal histopathological architecture with slight loss of crypts and villi on day 4 in sesamol pre-treated mice ( Figure 8D, F, H and J). Sesamol treatment alone did not result in any alterations in the histological architecture of control mice ( Figure 8A -B).
Quantitative analysis in the GI tract includes enumeration of crypts and villi, and measuring the length of the villi. A signifi cant ( p Ͻ 0.001) decrease in viable crypts, villi numbers, and villi lengths was observed on day 4 in 7.5 Gy-irradiated mice ( Figure 8K -L). Sesamol pre-treatment increased ( p Ͻ 0.001) viable crypts, and the number and length of the villi, between day 4 and day 7 in the mice exposed to 7.5 Gy of whole-body radiation ( Figure  8K -M). Interestingly, no changes in these parameters were found in control mice treated with sesamol alone till the 30th day of observations ( Figure 8K -M). Thus, sesamol pre-treatment protected GI injury by promoting the regeneration of crypts, leading to increase in villi number and length ( Figure 8).

Eff ect of sesamol on translocation of radiation-induced gut bacteria
The eff ect of sesamol pre-treatment on intestinal mucosal integrity was analyzed by evaluating the translocation of gut bacteria to spleen, liver, and kidney through bacterial CFUs. After exposure to 7.5 Gy of radiation, only a few bacterial CFUs were found on day 9, and no bacterial CFUs were found on day 17 or 21 in sesamol pre-treated mice (Figure 9). In contrast, a large number of bacterial CFUs were found on days 9, 17, and 21 in 7.5 Gyirradiated mice (Figure 9). However, bacterial CFUs were found at the maximum on day 17 and then decreased in irradiated mice (Figure 9). The number of bacterial CFUs found were higher in the spleen. Thus, the hierarchy of bacterial CFUs in descending order can be demonstrated as spleen Ͼ liver Ͼ kidney (Figure 9). This result indicates that sesamol pre-treatment results in relatively normal intestinal mucosal integrity in irradiated mice on post-irradiation days.

Eff ect of sesamol on radiation-induced TBARS in the GI tract
The TBARS level in the GI tract was measured 3 h post irradiation, and showed signifi cant increase in 5 Gy ( p Ͻ 0.01) and 7.5 Gy ( p Ͻ 0.001) γ -irradiated mice. However, sesamol pre-treatment reduced the TBARS level ( p Ͻ 0.01) in irradiated mice (5 Gy and 7.5 Gy). Treatment with sesamol alone did not cause any changes in the TBARS level of the GI tract ( Figure 10). This result suggests that sesamol pre-treatment decreases the TBARS induced by ionizing radiation in normal mice GI tract. Figure 5. Eff ect of sesamol on splenocytes hemorrhage, and apoptosis in mice exposed to γ -radiation (7.5 Gy, 1 Gy/min), at the 4th and 21st days post irradiation. The spleen was observed and single-cell suspensions prepared in pre-chilled PBS. Splenocyte single-cell suspensions were stained with Annexin-V-FITC and propidium iodide and analyzed using fl ow cytometry. Panels A and B are Control and Sesamol, respectively. Panels C and D are 7.5 Gy and Ses ϩ 7.5 Gy on the 21st post-irradiation day, respectively. Panel E is the comparison of % apoptosis between groups. The arrow indicates the point of hemorrhage. @ p Ͻ 0.001 vs control group. $ p Ͻ 0.01 and p Ͻ 0.001 vs @ and ¥ , respectively. @ p Ͻ 0.05 vs ¥ .

Eff ect of sesamol on radiation-induced p53 and related apoptotic proteins in the GI tract, spleen, and bone marrow cells
It is well documented that the p53 protein plays a vital role in the regulation of radiation-induced apoptotic signaling pathways [32]. To investigate the possible role of sesamol in the regulation of radiation-induced p53-dependent upstream regulators of apoptotic pathways, we examined the diff erences in the expression of pro-apoptosis (p53 and Bax) and anti-apoptosis (Bcl-x) proteins by western blot, 3 h post irradiation in the GI tract ( Figure 11A), spleen ( Figure 11B), and bone marrow cells ( Figure 11C). Treatment with sesamol did not cause any changes in the expression of these proteins ( Figure 11A -C). In addition, mice irradiated with 5 Gy and 7.5 Gy showed enhanced expression of p53 ( p Ͻ 0.0001) and Bax ( p Ͻ 0.001), in comparison to control, in a radiation dose-dependent manner. Sesamol pre-treatment lowered p53 ( p Ͻ 0.001) and Bax ( p Ͻ 0.001) protein expression patterns in 5 Gy and 7.5 Gy-irradiated mice ( Figure 11A). The level of the antiapoptotic Bcl-x protein in mice was signifi cantly decreased ( p Ͻ 0.01) following exposure to 5 and 7.5 Gy radiation. Sesamol pre-treatment signifi cantly ( p Ͻ 0.01) increased the Bcl-x protein expression pattern in 5 Gy and 7.5 Gytreated mice. This increased expression of Bcl-x protein was associated with a relatively decreased Bax/Bcl-x ratio in sesamol pre-treated mice, and the ratio was decreased by approximately 2-and 6-fold in 5 Gy-and 7.5 Gy-irradiated mice, respectively ( Figure 11A).
The molecular mechanism of a new drug plays a vital role in the drug approval process and is a necessity as per the United State FDA ' s " Animal Effi cacy Rule " . Therefore, it is crucial to understand the mechanism of a new drug in more than one organ. To prove this hypothesis, we have also measured the expression pattern of p53, Bax, and Bcl-x in spleen ( Figure 11B) and bone marrow cells ( Figure 11C) of whole-body 7.5 Gy-irradiated mice. Our fi ndings demonstrated that sesamol pre-treatment also decreased the expression pattern of apoptotic proteins-p53 and Bax in the spleen ( Figure 11B) and bone marrow cells ( Figure 11C). The decrease of apoptotic proteins was associated with the increase of anti-apoptotic protein-Bcl-x, and accordingly, a decrease of Bax/Bcl-x ratio in spleen and bone marrow cells ( Figure 11B and C). Thus, here it is hypothesized that part of the radioprotective eff ects of sesamol may be due to the inhibition of the expression pattern of p53-mediated apoptotic proteins in the GI tract, spleen, and bone marrow cells of whole-body irradiated mice.

Eff ect of sesamol on total antioxidant capacity in the GI tract and spleen
Total antioxidant capacity (TAC) was measured in the GI tract and spleen after 30 minutes after sesamol treatment Figure 6. Eff ect of sesamol on splenic B cell and T cell subpopulations (CD4/CD8) in mice exposed to γ -radiation (7.5 Gy, 1 Gy/min). Spleen from each mouse was dissected out and cleaned, and single-cell suspensions were prepared in pre-chilled PBS. Single cells were stained with PE-CD19, FITC-CD4, and PE-CD8, as well as acquired in fl ow cytometry. Panel A: Comparison of B cell population between groups; Panel B: Comparison of T cell subpopulation between groups. * p Ͻ 0.001, $ p Ͻ 0.01, @ p Ͻ 0.05. through the intraperitoneal route. The TAC was measured using ABTS ( Figure 12A and B) and DPPH ( Figure 12C and D) radical assays. Sesamol treatment signifi cantly ( p Ͻ 0.01) enhanced gastrointestinal TAC, in comparison to that of control mice ( Figure 12A and C). The calculated values of TEAC/mg of wet tissue in the GI tract were found to be 6.5 and 10.1 for control and sesamol-treated mice, respectively ( Figure 12A). The values of percentage inhibition of DPPH radical in the GI tract were 55.5 and 72.2, for control and sesamol-treated mice, respectively ( Figure  12C). In addition, sesamol treatment also signifi cantly increased ( p Ͻ 0.01) the TAC of spleen in comparison to control ( Figure 12B and D). The calculated values of TEAC/ mg of wet tissue in spleen were 7.0 and 8.2, for control and sesamol-treated mice, respectively ( Figure 12B). However, the values for percentage inhibition of DPPH radical in spleen were 22.6 and 38.5, for control and sesamol-treated mice, respectively ( Figure 12D). Thus, the results indicated that the increase of TAC could overcome the imbalance between pro-oxidants and anti-oxidants in irradiated mice.

Discussion
Poor effi cacy and toxicity are the two major impediments in developing radioprotectors. A large number of compounds, both natural and synthetic, have demonstrated potential through in vitro and in vivo model systems [5,7,8]. Cytotoxicity in diff erent organs and systemic levels limit the further progress of these potential candidates. Most of the research on radioprotectors has been focused on the effi cacy and mechanism related to the lethal dose of radiation in small animals. However, exposure to a lethal dose of radiation is less likely in planned radiation exposure scenarios, for example, fi rst-responders of radiation emergency, military operations in radiation zones, and also radiotherapy patients for protection of normal tissue. Therefore, radioprotective effi cacy of potential candidates needs to be evaluated at lower radiation doses. In this study, therefore, we have performed radioprotective effi cacy and pre-clinical evaluations at a safe dose of sesamol (100 mg/kg) (1/5th of LD 50 dose) in C57BL/6, based on the estimated LD 50 drug dose in the same strain (unpublished data).
In an earlier study, Parihar et al. reported survival of about 70% at a sesamol dose ranging from 50 -100 mg/kg in lethally (9 Gy) whole-body-irradiated Swiss albino mice [18]. In the present study, sesamol pre-treatment using 100 and 50 mg/kg provided 100% and 70% survival in 7.5 Gy (LD 50/30 ) whole-body-irradiated C57BL/6 male mice, respectively (Figure 1). This mice strain is a recommended animal model for developing radioprotectors [22]. On careful observations of the previous study in Swiss Figure 7. Eff ect of sesamol on splenocyte proliferation in mice exposed to 60 Co ( γ -irradiation (7.5 Gy, 1 Gy/min). The expression pattern of splenic PCNA protein was analyzed 3 h post irradiation by western blot. Further, on day 4 post irradiation, the spleen was dissected out under aseptic condition. The splenocytes were cultured in the presence of Con A under humidifi ed atmospheric condition. After 72 h, the lymphoproliferation capacity of the splenocytes was enumerated by trypan blue dye exclusion and CFSE staining. Panel A: Comparison of splenic PCNA expression between groups; Panel B: Comparison of proliferating splenocytes between groups; Panel C: Comparison of MFI between groups. * p Ͻ 0.001, $ p Ͻ 0.01. albino mice, sesamol dosage of 50 mg/kg showed higher survival as compared to the next higher dosage of 100 mg/ kg. This dose -response variation in survival following exposure to lethal radiation could be due to toxicity caused by sesamol itself in this strain, which was not evaluated. Thus, the observation of 100% survival at 100 mg/kg (1/5th of the LD 50 drug dose) was novel, and the overall survivability could be due to strain variability. Parihar et al. evaluated GI injury at a supra-lethal radiation dose of 15 Gy using only qualitative analysis at single time points (72 h), and revealed signifi cant protection by sesamol pre-treatment (50 mg/kg), which was not suffi cient to assess the recovery end point. In this study, therefore, we have performed both qualitative and quantitative analyses to assess the radiation-induced GI injury in 7.5 Gy wholebody-irradiated mice and found signifi cant recovery as a function of post irradiation days (Figure 8). Further, Parihar et al. had reported benefi ts of sesamol in lowering splenic injury by evaluating the splenic CFU and splenic index [18]. Thus, we have made an attempt to measure B cell ( Figure 6A) and T cell subpopulations (CD4 and CD8) ( Figure 6B) and apoptosis ( Figure 5E) in the spleen, which plays an important role in immunomodulation. In addition, we have also measured proliferation of splenocytes in presence of Con A by trypan blue dye exclusion ( Figure 7B) and CFSE staining ( Figure 7C). These results (Figures 1, 5 -8) are complementary to the previous study [18], and further strengthen the radioprotective potential Figure 8. Eff ect of sesamol on jejunum crypt number, villus number, and length, in mice exposed to 60 Co (-irradiation (7.5 Gy, 1 Gy/min). Jejuna were collected on the 1st, 4th, 7th, 15th and 30th days post irradiation. After fi xation, processing, and dehydration, cross sections of jejunum (5 μ m) were stained with H & E, and crypt number, villus number and villus height were analyzed. Representative photographs (1st -30th Days) for crypt number, villus number and villus height are shown (original magnifi cation 100 ϫ ). Panels A and B are control and sesamol, respectively. Panels C, E, G, and I are 7.5 Gy on the 1st, 4th, 7th and 15th days, respectively. Panels D, F, H, and J are Ses ϩ 7.5 Gy on the 1st, 4th, 7th and 30th days, respectively. Panel K: Comparison of Mean Crypt Numbers between groups; Panel L: Comparison of Mean Villi Length between groups; Panel M: Comparison of Mean Villi Numbers between groups. No survivors in radiation group for day 30. * p Ͻ 0.001 vs Control group. $ p Ͻ 0.001 vs 7.5 Gy on corresponding days. of sesamol. Kanimozhi et al. reported signifi cant protection against radiation-induced DNA strand breaks in blood lymphocytes by sesamol pre-treatment (100 mg/kg) in 7.5 Gy-irradiated mice [19]. Radiation-induced injuries in hematopoietic systems are manifested by the loss of HPSCs in bone marrow and T cell subpopulations in spleen, whereas GI injury is manifested by the impairment of crypt cell regeneration. These organs (bone marrow, spleen, and GI) are more sensitive to radiation-induced injury in male C57BL/6 mice, in comparison to those of the C3H/He strain [23,24]. As a the fi rst step, we have undertaken qualitative and quantitative investigations, revealing that sesamol pre-treatment provided signifi cant protection to these organs against radiation-induced injury in 5 Gy and 7.5 Gy whole-body γ -irradiated mice ( Figures  2 -12).
Whole-body ionizing radiation exposure can cause hematopoietic and GI sub-syndromes depending on the radiation type, dose, dose rate, and exposure conditions [33]. The hematopoietic sub-syndrome has been reported as a manifestation of the depletion of spared HPSCs, committed to erythrocytes, granulocyte macrophages, granulocyte erythrocyte macrophage megakaryote in bone marrow of whole-body irradiated murine model [5]. In addition, the hematopoietic sub-syndrome is also known to complicate into sepsis, and this is a primary cause of mortality in the early phase in animal models. Several cytokines, including growth factors, have been reported to protect mice from radiation-induced mortality when administered as a prophylactic dose. These cytokines and growth factors off er protection by primarily orchestrating and stimulating the host ' s innate immune response against ionizing radiation [34,35]. Therefore, cytokines and growth factors are expected to expand the restoration of HPSCs in bone marrow after radiation exposure, and have been demonstrated in pre-clinical and clinical investigations [34,36]. The growth factors GCSF (granulocyte colony-stimulating factor) and GM-CSF (granulocytemacrophage colony-stimulating factor) are approved for treatment of acute myelosuppression [37,38]. In the present study, femoral HPSCs were measured through the CFU assay and demonstrated signifi cant radiation dose-dependent depletion in whole-body irradiated mice. Sesamol pre-treatment has demonstrated a potential for restoration of femoral HPSCs committed to erythrocytes, granulocytes, macrophages and megakaryotes in irradiated mice (5 Gy and 7.5 Gy) ( Figure 2). In addition, MN and cell death in bone marrow cells were also observed. Radiation-induced MN and apoptotic cells increased 24 h following radiation, and led to cell death at later time points. This was clearly observed as an increased percentage of cell death and MN frequency (Figures 3 -4). Cell cycle analysis depicted about 90% cell death on the 4th day ( Figure 4A), and appeared to have contributed to animal death observed during observation period of 30 days post irradiation. Sesamol pre-treatment reduced radiationinduced MN frequency and apoptosis to levels near control on the 15th day post irradiation (Figure 3). Further, sesamol pre-treatment also signifi cantly reduced bone marrow cell death, as seen in the cell cycle analysis Figure 9. Eff ect of sesamol on gut bacterial translocation in mice exposed to γ -radiation (7.5 Gy, 1 Gy/min). Spleen, liver, and kidney were aseptically collected on days 9, 17 and 21 post irradiation and then immediately treated with broth media for further processing. After 24 h, homogenates were spread on a sheep blood agar plate using a glass rod and incubated in a bacterial incubator for 16 h. Total bacterial CFUs indicate the sum total of spleen, liver, and kidney bacterial CFUs. * p Ͻ 0.0001 vs Con, Ses ϩ 7.5 Gy and Mel ϩ 7.5 Gy.
( Figure 4). The microscopic observations of MN frequency showed a very low number of cells on day 4 in 7.5 Gy irradiated mice. This was expected because maximum (about 90%) cell death was analyzed by cell cycle analysis and very few HPSCs were found through a CFU assay on the 4th day post irradiation (Figures 2 -4). Therefore, sesamol pre-treatment facilitated survival of irradiated mice by enhancing the restoration of femoral HPSCs, leading to immunomodulation.
The hematopoietic tissue (spleen) is composed of lymphocytes and their subpopulations having diff erential radiosensitivity. After exposure to ionizing radiation, the distribution of T cell subpopulations may change, and can cause immunomodulation [39]. Loss of T cell subpopulations (Th and Tc) results in thrombocytopenia or hemorrhage, and can cause malfunction of the adaptive immune response [3]. Among the cells that comprise the adaptive immune response, CD4 and CD8 are vital, with functional relevance in cell-mediated immunity, while CD19 is relevant in humoral immunity. Studies have reported apoptosis mainly in the white pulp region of the spleen due to the increased expression pattern of p53 and Bax proteins in 2 -3 Gy whole-body irradiated mice [40,41]. In the present study, higher levels of apoptotic cells (Annexin-V-FITC) were detected in the spleen after exposures to 7.5 Gy of whole-body radiation ( Figure 5E). At the same time, irradiation also reduced the maximum numbers of B cell ( Figure 6A) and T cell subpopulations (Th and Tc) in the spleen ( Figure 6B). This increase in apoptosis ( Figure 5E) and decrease in lymphocyte populations appears to have contributed to the observed hemorrhage in the spleen of 7.5 Gy-irradiated mice ( Figure 5C). However, sesamol pre-treatment inhibited apoptosis ( Figure 5E) and enhanced recovery of B cell and T cell subpopulations (Figure 6), resulting in the protection of spleen from hemorrhage (Figure 5D). Our fi ndings also suggest that sesamol treatment through the intraperitoneal route signifi cantly enhances TAC in hematopoietic tissue ( Figure 12). Enhancement of tissue TAC by sesamol is expected to decrease free radical burden and can reduce apoptosis and increase the B cell and T cell populations. Increase in T cell subpopulations is likely to facilitate the recovery of the T cell subpopulation ratio (CD4/CD8) by lowering the expression pattern of p53, Bax, and Bax/Bcl-2 ratio [41].
PCNA is a nuclear protein and a co-factor for DNA polymerase δ , which is involved in the RAD6-dependent DNA repair pathway in response to oxidative DNA damage. In particular, PCNA level rises to the maximum during the S-phase of the cell cycle, which is an important indicator of cell proliferation. Our results show a higher level of PCNA protein expression in irradiated mice pretreated with sesamol ( Figure 7A). In addition, sesamol pre-treated mice also show higher lymphoproliferative capacity of splenocytes in the presence of mitogen (Con A), as has been directly and indirectly enumerated by trypan blue dye exclusion ( Figure 7B) and CFSE staining ( Figure 7C), respectively. Overall, these results demonstrate that an increased expression of PCNA protein and enhanced proliferation of splenocytes may be an important mechanism for the protection of spleen in irradiated mice pre-treated with sesamol.
The GI system is prone to radiation-induced injury and is most sensitive, next to the hematopoietic system [42]. In the GI tract, epithelial crypt cells are the highest proliferating cells [30,43], and this makes them the most susceptible to ionizing radiation [31]. Therefore, death of epithelial crypts is manifested as the pathogenesis of the GI sub-syndrome [43]. The GI sub-syndrome is believed to appear at 10 Gy and above in mice, but recent studies have also reported GI injury at lower radiation doses [43]. Apoptosis has been reported in the intestinal crypts (stem cell region) within 3 -6 h following exposure to radiation of 1 Gy [31], and increases subsequently with radiation dose [43]. Ionizing radiation-induced free radicals mediate oxidative damages, for example, lipid peroxidation, DNA strand breaks, and subsequent alteration in the antiapoptotic and pro-apoptotic proteins, manifested as malabsorption in the GI tract (acute bowel reaction and radiation proctitis) [30,31,44 -46]. Ultimately these eff ects lead to fl uid and electrolyte imbalance, bacteremia, endotoxemia, and subsequent death of epithelial crypts [47]. Therefore, protection of the epithelial crypt cells is of utmost importance for the prevention of radiation-induced pathogenesis of the GI sub-syndrome. A number of compounds have demonstrated potential for protection against radiation injury in GI in animal models. These are nutraceuticals (vitamin E and vitamin A) and nutritional compounds, methyl xanthenes, interleukin -11, prostanoids (prostaglandins, prostacyclins), and other biological and chemical agents [48]. However, no specifi c agent is available for protection of the GI system. Injury in the GI is observed as early as 30 min after radiation exposure. Further, repair and regeneration of epi- Figure 10. Eff ect of sesamol on TBARS of GI tract (jejunum) in mice exposed to γ -irradiation (5 Gy and 7.5 Gy, 1 Gy/min) 3 h post irradiation. The GI tract was dissected out, cleaned, and prepared as a homogenate in pre-chilled PBS (10% w/v). TBARS product in jejunum was measured using a spectrophotometer and represented as nmol/gm of wet tissue weight. * p Ͻ 0.01, * * p Ͻ 0.001 vs Control group. $ p Ͻ 0.01 vs 5 Gy and 7.5 Gy groups. Ø p Ͻ 0.05 for 5 Gy vs 7.5 Gy groups. thelial crypt cells in the radiation-exposed GI tract may be completed within 3 -days [49]. Therefore, in this study, early measurement of radiation-induced GI injury was initiated after 3 h for lipid peroxidation and altered expression patterns of anti-apoptotic and pro-apoptotic proteins in mice irradiated with sub-lethal (5 Gy) and LD 50/30 (7.5 Gy) doses. Histopathological changes and gut bacterial translocation were assessed from day 1 and day 9 in 7.5 Gy-irradiated mice, respectively. Our data has shown that sesamol pre-treatment can signifi cantly lower lipid peroxidation in the GI tract of mice irradiated with 5 Gy ( p Ͻ 0.01) and 7.5 Gy ( p Ͻ 0.001) ( Figure 10). In addition, more number of surviving crypts and villi as well as increased villi lengths were observed in sesamol pretreated mice (Figure 8). Further, sesamol treatment increased GI tissue ' s TAC (Figure 12), which might have led to an increased scavenging of ROS, and hence decreased ROS-mediated injury in irradiated mice.
Elucidation of the molecular mechanism of a radioprotective drug in more than one organ is crucial and plays a vital role in the process of approval of a new drug. To our knowledge, no study has reported the molecular mechanism of sesamol in the amelioration of γ -ray-induced GI tract, spleen and bone marrow injury in mice. Therefore, Figure 11. Eff ect of sesamol on radiation-induced modifi cations of the GI tract (jejunum), spleen, and bone marrow cells, and apoptotic and anti-apoptotic protein expression pattern in mice exposed to γ -irradiation 3 h post irradiation. The diff erences in the expression pattern of p53, Bax, and Bcl-x were analyzed by western blot, as described in Materials and Methods. Panel A: GI Tract; Panel B: Spleen; Panel C: Bone Marrow Cells. * * p Ͻ 0.0001, * p Ͻ 0.05, $ p Ͻ 0.001, @ p Ͻ 0.01.