Sodium selenite, radioprotection, X-ray irradiation, esophageal cells, apoptosis, irradiation-induced damage Running title: PUSPITASARI et al: PROTECTIVE EFFECTS OF SODIUM SELENITE AGAINST IRRADIATION- INDUCED CELL DAMAGE

The administration of radioprotective compounds is one approach to prevent radiation damage in normal tissues. Thus, radioprotective compounds are important in clinical radiotherapy. Selenium is one radioprotective compounds that has been widely used in clinical studies of radiotherapy. However, evidence regarding the effectiveness of selenium use in radiotherapy and the mechanisms through which selenium reduces the side effects of radiotherapy remains insufficient. To provide further evidence of the effectiveness of selenium in radiotherapy, this study investigated the protective effects of sodium selenite supplementation prior to X-ray irradiation treatment in noncancerous human esophageal cells (CHEK-1). Sodium selenite supplementation increased glutathione peroxidase 1 (GPx-1) activity in a doseand timedependent manner. The sodium selenite dose that resulted in the most GPx-1 activity was 50 nM for 72 h. The half maximal inhibitory concentration (IC50) of sodium selenite in the cells was 3.6 μM. Sodium selenite supplementation increased the survival rate of the cells in a dose-dependent manner and increased cell viability at 72 h post irradiation (p<0.05). Combined treatment of 50 nM sodium selenite and 2 Gy Xray irradiation decreased sub-G1 cells compared with 2 Gy X-ray irradiation alone from 5.9 to 4.2% (p<0.05) and increased G1 cells from 58.8 to 62.1%. Western blot analysis revealed that treatment with 2 Gy X-ray irradiation increased the expression level of cleaved poly ADP ribose polymerase (PARP) (p<0.05). In addition, combined treatment of 50 nM sodium selenite and 2 Gy X-ray irradiation reduced the expression of cleaved PARP protein compared with 2 Gy X-ray irradiation alone. Our results suggest that 50 nM sodium selenite supplementation for 72 h before irradiation can protect CHEK-1 cells from irradiation-induced damage by inhibiting irradiation-induced apoptosis. Sodium selenite is a prospective radioprotective compound for normal cells in clinical radiotherapy. Introduction Radiotherapy is one of the most common and effective treatments for cancer (1). Greater than 40% of cancer patients require radiotherapy during the management of their disease (2). Although clinical radiotherapy treatment planning and delivery technologies have improved, the toxicity of radiotherapy to normal tissues and organs remains a problem (2, 3). Thus, radioprotective compounds are very important in clinical radiotherapy (3) and the administration of radioprotective compounds has been suggested as one approach for preventing radiation damage in normal tissues (4, 5). Selenium is a trace element that has fundamental importance in human biology (6). Selenium detoxifies reactive oxygen species (ROS) produced by irradiation treatment (4, 7). Selenium acts in human antioxidant systems in the form of selenocyctein (SeCys), which is incorporated into various selenoproteins (8, 9). At least 25 selenoproteins have been identified in humans, including glutathione peroxidase (GPx), thioredoxinreductases (TrxR), iodothyroninedeiodinase, and the selenoproteins P, W and R (10). In nature, selenium exists in many chemical forms. The most well studied forms are selenomethionine (SeMet), sodium selenite, selenium methylselenocysteine, 1,4-phenylenebis (methylene) selenocyanate (p-XSC), and methylseleninic acid (MSA) (9). Sodium selenite is the chemical form of selenium that was used for radiotherapy supplementation clinical studies between 1987 and 2012 (7). Despite being broadly used as a complementary medicine during radiotherapy (11, 12), evidence regarding the effectiveness of selenium use in radiotherapy and the mechanism by which selenium reduces the side effects of radiotherapy remains insufficient (7). Schleicher et al (13) and Hehr et al (14) performed in vitro studies of selenium and radiotherapy and found that sodium selenite has potential as a protective agent for normal tissues during radiotherapy (15). However, the mechanisms of this protection have not been revealed. Diamond et al (16) reported that low level supplementation of culture media with selenium in the form of sodium selenite significantly protected CHO-AA8 cells, a hamster ovary-derived cell line, from radiation-induced mutagenesis. Eckers et al (17) also reported that overexpression of selenoprotein P (SEPP1) suppressed late radiation-induced ROS accumulation and protected normal human fibroblasts from radiation-induced toxicity. Tak and his coworker (18) found that when U937 cells, a human leukemic monocyte lymphoma cell line, were exposed to 2 Gy of γ-irradiation, a distinct difference was noted between cells that were or were not pretreated with ebselen with respect to apoptotic features and mitochondrial function. However, more evidence is required to determine the mechanisms which selenium supplementation helps prevent the side effects of radiotherapy before it can be recommended as a cancer adjuvant radiotherapy. Given that sodium selenite is the only chemical form of selenium that has been used in clinical studies in this context, this study investigated the protective effects of sodium selenite supplementation on noncancerous human esophageal cells before X-ray irradiation. Materials and Methods Cell culture Cells of the immortalized noncancerous human esophageal cell line CHEK-1 (19), were maintained in RPMI-1640 (Wako, Japan) with 10% fetal bovine serum (Hyclone, Utah, USA) and 1% penicillinstreptomycin (Gibco, New York, USA) at 37°C in a 5% CO2 humidified chamber. The culture medium was replaced every 3 days, and the cells were passaged on a weekly basis using a 1:5 splitting ratio. Selenium supplementation Sodium selenite (Sigma, St Louis, USA) was the chemical form of selenium that was used for supplementation. The dose of sodium selenite supplementation ranged from 0-200 nM, and the time of incubation ranged from 24 to 72 h after 18 h of initial seeding. Supplementation with a dose of 50 nM for 72 h before irradiation treatment was used for the cell viability assay, cell cycle analysis and Western blot analysis. Irradiation Irradiation was performed using an X-Ray irradiation machine (Titan-225S, Shimadzu, Japan) at a rate of 1.3 Gy/min. The dose of irradiation was 2 Gy based on the common fractionation dose for radiotherapy. Protein extraction for GPx-1 activity assay and Western blot analysis CHEK-1 cells were supplemented with sodium selenite for 72 h, washed twice with phosphate-buffered saline (PBS) and harvested. Then, proteins were extracted using RIPA buffer (Sigma, St. Louis, USA) with 10% protein inhibitor (Sigma, St. Louis, USA). Protein concentrations were determined using a Bio-Rad DC protein assay kit (Bio-Rad, Tokyo, Japan) following the method of Lowry (20). The extracted sample was stored at -80°C until the day of analysis. GPx-1 activity assay The enzymatic activity of GPx-1 in CHEK-1 cell homogenates was determined using the method of Paglia and Valentine with some modification (21). GPx-1 activity was indirectly monitored using spectrophotometric methods to observe the reduction of oxidized glutathione using nicotinamide adenine dinucleotide phosphate (NADPH) as the reducing agent. GPx-1 activity was quantified by measuring the change in NADPH absorbance at 340 nm and was expressed as the change in NADPH absorbance (∆ mM NADPH) over time (min) and with different levels of protein (mg) in the presence of the substrate tertbutyl hydroperoxide. Absorbance was observed using a microplate reader (SpectraMax Plus 384, Molecular Devices, CA, USA). Cytotoxicity assay Cytotoxicity of sodium selenite in CHEK-1 cells was examined with various concentrations of sodium selenite (0-8 μM) using a colorimetric assay. Briefly, cells (2×10 in 50 μl/well) were seeded in 96-well plates. After 18 h of initial cell seeding, sodium selenite solutions were added, and the cells were incubated for 72 h. The cell proliferation rate and half maximal inhibitory (IC50) were then determined using a cell counting kit (Dojindo Lab., Tokyo, Japan) according to the manufacturer’s instructions. Absorbance was measured using a micro plate reader (SpectraMax Plus 384, Molecular Devices, CA, USA). Clonogenic assay CHEK-1 cells were supplemented with various concentrations of sodium selenite (0-200 nM) for 72 h after 18 h of initial seeding and were then irradiated with 2 Gy X-ray irradiation. The cells (500 cells/4 ml medium) were seeded in 25cm tissue culture flasks (Falcon, NJ, USA) immediately following irradiation. After 14 days of culture at 37C, the cells were washed with PBS, fixed and stained with 0.5% crystal violet in H2O:methanol (1:1) for 30 min at room temperature. Then, the cells were washed with tap water and air‐dried. The total number of colonies with >50 cells was counted using a Binocular Light microscope (Olympus Corp). After counting the colonies, plating efficiency (PE) and survival fraction (SF) can be calculated using the following equations (22, 23):