Experimentally determined relative biological effectiveness of cyclotron-based epithermal neutrons designed for clinical BNCT: in vitro study.

A neutron beam for boron neutron capture therapy (BNCT) of deep-seated tumours is designed to maintain a high flux of epithermal neutrons, while keeping the thermal and fast neutron component as low as possible. These neutrons (thermal and fast) have a high relative biological effectiveness in comparison with high energy photon beams used for conventional X-ray radiotherapy. In the past, neutrons for the purpose of BNCT were generated using nuclear reactors. However, there are various challenges that arise when installing a reactor in a hospital environment. From 2006, the Kyoto University Research Reactor Institute, in collaboration with Sumitomo Heavy Industries, began the development of an accelerator-based neutron source for clinical BNCT in a bid to overcome the shortcomings of a nuclear reactor-based neutron source. Following installation and beam performance testing, in vitro studies were performed to assess the biological effect of the neutron beam. Four different cell lines were prepared and irradiated using the accelerator-based neutron source. Following neutron and gamma ray irradiation, the survival curve for each cell line was calculated. The biological end point to determine the relative biological effectiveness (RBE) was set to 10% cell survival, and the D10 for each cell line was determined. The RBE of the accelerator-based neutron beam was evaluated to be 2.62.

Pharmacokinetic Study of 14C-Radiolabeled p-Boronophenylalanine (BPA) in Sorbitol Solution and the Treatment Outcome of BPA-Based Boron Neutron Capture Therapy on a Tumor-Bearing Mouse Model.

BACKGROUND AND OBJECTIVE: Boron neutron capture therapy (BNCT) is a binary cancer treatment that combines boron administration and neutron irradiation. The tumor cells take up the boron compound and the subsequent neutron irradiation results in a nuclear fission reaction caused by the neutron capture reaction of the boron nuclei. This produces highly cytocidal heavy particles, leading to the destruction of tumor cells. p-boronophenylalanine (BPA) is widely used in BNCT but is insoluble in water and requires reducing sugar or sugar alcohol as a dissolvent to create an aqueous solution for administration. The purpose of this study was to investigate the pharmacokinetics of 14C-radiolabeled BPA using sorbitol as a dissolvent, which has not been reported before, and confirm whether neutron irradiation with a sorbitol solution of BPA can produce an antitumor effect of BNCT. MATERIALS AND METHODS: In this study, we evaluated the sugar alcohol, sorbitol, as a novel dissolution aid and examined the consequent stability of the BPA for long-term storage. U-87 MG and SAS tumor cell lines were used for in vitro and in vivo experiments. We examined the pharmacokinetics of 14C-radiolabeled BPA in sorbitol solution, administered either intravenously or subcutaneously to a mouse tumor model. Neutron irradiation was performed in conjunction with the administration of BPA in sorbitol solution using the same tumor cell lines both in vitro and in vivo. RESULTS: We found that BPA in sorbitol solution maintains stability for longer than in fructose solution, and can therefore be stored for a longer period. Pharmacokinetic studies with 14C-radiolabeled BPA confirmed that the sorbitol solution of BPA distributed through tumors in much the same way as BPA in fructose. Neutron irradiation was found to produce dose-dependent antitumor effects, both in vitro and in vivo, after the administration of BPA in sorbitol solution. CONCLUSION: In this report, we demonstrate the efficacy of BPA in sorbitol solution as the boron source in BNCT.

VEGF affects mitochondrial ROS generation in glioma cells and acts as a radioresistance factor.

Vascular endothelial growth factor (VEGF) is closely related to angiogenesis. Anticancer therapy by inhibiting VEGF signaling is well established. However, the role of VEGF in cell-cell communication during the response to ionizing radiation is not well understood. Here, we examined the role of VEGF on radiosensitivity of cells. The addition of recombinant VEGF (rVEGF) on cultured rat C6 glioma cells showed a radioprotective effects on X-ray irradiation and reduced oxidative stress. These effects were also observed by endogenous VEGF in supernatant of C6 glioma cells. Reduction of oxidative stress by VEGF is suggested to underlie the radioprotective effects. The mechanism of VEGF-induced reduction of oxidative stress was indicated by a decreased oxygen consumption rate (OCR) in mitochondria. However, the number of DNA double-strand breaks (DSB) immediately after irradiation was not reduced by the treatment with VEGF. These results suggest that VEGF plays a role in cell survival after irradiation by controlling the oxidative condition through mitochondrial function that is independent of the efficiency of DSB induction.

27-Hydroxycholesterol regulates human SLC22A12 gene expression through estrogen receptor action.

The excretion and reabsorption of uric acid both to and from urine are tightly regulated by uric acid transporters. Metabolic syndrome conditions, such as obesity, hypercholesterolemia, and insulin resistance, are believed to regulate the expression of uric acid transporters and decrease the excretion of uric acid. However, the mechanisms driving cholesterol impacts on uric acid transporters have been unknown. Here, we show that cholesterol metabolite 27-hydroxycholesterol (27HC) upregulates the uric acid reabsorption transporter URAT1 encoded by SLC22A12 via estrogen receptors (ER). Transcriptional motif analysis showed that the SLC22A12 gene promoter has more estrogen response elements (EREs) than other uric acid reabsorption transporters such as SLC22A11 and SLC22A13, and 27HC-activated SLC22A12 gene promoter via ER through EREs. Furthermore, 27HC increased SLC22A12 gene expression in human kidney organoids. Our results suggest that in hypercholesterolemic conditions, elevated levels of 27HC derived from cholesterol induce URAT1/SLC22A12 expression to increase uric acid reabsorption, and thereby, could increase serum uric acid levels.

Inhibition of the ATR kinase enhances 5-FU sensitivity independently of nonhomologous end-joining and homologous recombination repair pathways.

The anticancer agent 5-fluorouracil (5-FU) is cytotoxic and often used to treat various cancers. 5-FU is thought to inhibit the enzyme thymidylate synthase, which plays a role in nucleotide synthesis and has been found to induce single- and double-strand DNA breaks. ATR Ser/Thr kinase (ATR) is a principal kinase in the DNA damage response and is activated in response to UV- and chemotherapeutic drug-induced DNA replication stress, but its role in cellular responses to 5-FU is unclear. In this study, we examined the effect of ATR inhibition on 5-FU sensitivity of mammalian cells. Using immunoblotting, we found that 5-FU treatment dose-dependently induced the phosphorylation of ATR at the autophosphorylation site Thr-1989 and thereby activated its kinase. Administration of 5-FU with a specific ATR inhibitor remarkably decreased cell survival, compared with 5-FU treatment combined with other major DNA repair kinase inhibitors. Of note, the ATR inhibition enhanced induction of DNA double-strand breaks and apoptosis in 5-FU-treated cells. Using gene expression analysis, we found that 5-FU induced the activation of the intra-S cell-cycle checkpoint. Cells lacking BRCA2 were sensitive to 5-FU in the presence of ATR inhibitor. Moreover, ATR inhibition enhanced the efficacy of the 5-FU treatment, independently of the nonhomologous end-joining and homologous recombination repair pathways. These findings suggest that ATR could be a potential therapeutic target in 5-FU-based chemotherapy.

Acquisition of radioresistance by IL-6 treatment is caused by suppression of oxidative stress derived from mitochondria after γ-irradiation.

Interleukin (IL)-6 is a multifunctional cytokine and is one of the radiation-induced bystander factors. This study aimed to clarify the mechanism of acquisition of radioresistance through the control of reactive oxygen species (ROS) by IL-6. We used a rat glioma cell line (C6) as tumor cells and a rat astrocyte cell line (RNB) as non-tumor cells. Our results showed that the surviving fraction of C6 cells after 6 Gy irradiation was increased by the addition of IL-6, but that this was not the case in RNB cells. In addition, the number of 53BP1 foci in C6 cells at 30 min after γ-irradiation were decreased by IL-6. Levels of ROS in whole C6 cells, and superoxide in the mitochondria of C6 cells immediately after γ-irradiation, were reduced by IL-6, but this was not observed in RNB cells. The mitochondrial membrane potential detected by JC-1 in C6 and RNB cells was inhibited by IL-6 alone. Therefore, it was concluded that IL-6 leads specifically to radioresistance in tumor cells by inhibition of increases in ROS after γ-irradiation.

Early and Delayed Induction of DSBs by Nontargeted Effects in ICR Mouse Lymphocytes after In Vivo X Irradiation.

The goal of this study was to determine whether in vivo X irradiation induces nontargeted effects, such as delayed effects and bystander effects in ICR mouse lymphocytes. We first examined the generation of DNA double-strand breaks (DSBs) in lymphocytes, isolated from ICR mice exposed to 1 Gy X irradiation, by enumeration of p53 binding protein 1 (53BP1) foci, and observed that the number of 53BP1 foci reached their maximum 3 days postirradiation and decreased to background level 30 days postirradiation. However, the number of 53BP1 foci was significantly increased in lymphocytes isolated from ICR mice 90-365 days postirradiation. This result indicates that in vivo X irradiation induced delayed DSBs in ICR mouse lymphocytes. We next counted the number of 53BP1 foci in lymphocytes isolated from sham-irradiated ICR mice that had been co-cultured with lymphocytes isolated from 1 Gy X-irradiated ICR mice, and observed a significant increase in the number of 53BP1 foci 1-7 days postirradiation. This result indicates that in vivo X irradiation induced bystander effects in ICR mouse lymphocytes. These findings suggest that in vivo X irradiation induces early and delayed nontargeted effects in ICR mouse lymphocytes.

Gemcitabine Induces Radiosensitization Through Inhibition of RAD51-dependent Repair for DNA Double-strand Breaks.

BACKGROUND/AIM: Gemcitabine (GEM) is used in clinical chemo-radiotherapy; however, the mechanism that contributes to enhanced radiosensitivity by GEM is not fully-understood. We evaluated the effect of GEM on radiosensitization in pancreatic cancer cell lines. MATERIALS AND METHODS: Pancreatic cell lines PK-59 and PK-45p were used. A total of 5 µM GEM for 4 h were administered pre- or post-gamma irradiation. RESULTS: Enhanced cell killing effects by GEM in radiotherapy were observed for pre-treatment but not post-treatment GEM. We focused on the dynamics of RAD51 and phospho-H2AX foci after irradiation. Significantly higher numbers of phospho-H2AX foci were observed in GEM pre-treated cells than in untreated cells after irradiation. We also found inhibition of the formation and degradation of RAD51 foci by GEM pre-treatment. The radiosensitizing effect of GEM was suppressed by knockdown of RAD51. CONCLUSION: RAD51-dependent homologous recombination is one of the key targets in the GEM-induced radiosensitizing effect.

Relief of delayed oxidative stress by ascorbic acid can suppress radiation-induced cellular senescence in mammalian fibroblast cells.

Ionizing radiation-induced cellular senescence is thought to be caused by nuclear DNA damage that cannot be repaired. However, here we found that radiation induces delayed increase of intracellular oxidative stress after irradiation. We investigated whether the relief of delayed oxidative stress by ascorbic acid would suppress the radiation-induced cellular senescence in Syrian golden hamster embryo (SHE) cells. We observed that the level of oxidative stress was drastically increased soon after irradiation, then declined to the level in non-irradiated cells, and increased again with a peak on day 3 after irradiation. We found that the inductions of cellular senescence after X-irradiation were reduced along with suppression of the delayed induction of oxidative stress by treatment with ascorbic acid, but not when oxidative stress occurred immediately after irradiation. Moreover, treatment of ascorbic acid inhibited p53 accumulation at 3 days after irradiation. Our data suggested a delayed increase of intracellular oxidative stress levels plays an important role in the process of radiation-induced cellular senescence by p53 accumulation.

Ionizing radiation-induced cell death is partly caused by increase of mitochondrial reactive oxygen species in normal human fibroblast cells.

Radiation-induced cell death is thought to be caused by nuclear DNA damage that cannot be repaired. However, in this study we found that a delayed increase of mitochondrial reactive oxygen species (ROS) is responsible for some of the radiation-induced cell death in normal human fibroblast cells. We have previously reported that there is a delayed increase of mitochondrial (·)O2(-), measured using MitoSOX™ Red reagent, due to gamma irradiation. This is dependent on Drp1 localization to mitochondria. Here, we show that knockdown of Drp1 expression reduces the level of DNA double-strand breaks (DSBs) remaining 3 days after 6 Gy irradiation. Furthermore, cells with knockdown of Drp1 expression are more resistant to gamma radiation. We then tested whether the delayed increase of ROS causes DNA damage. The antioxidant, 2-glucopyranoside ascorbic acid (AA-2G), was applied before or after irradiation to inhibit ROS production during irradiation or to inhibit delayed ROS production from mitochondria. Interestingly, 1 h after exposure, the AA-2G treatment reduced the level of DSBs remaining 3 days after 6 Gy irradiation. In addition, irradiated AA-2G-treated cells were more resistant to radiation than the untreated cells. These results indicate that delayed mitochondrial ROS production may cause some of the cell death after irradiation.

Comparison of the biological effects of (18)F at different intracellular levels.

We herein examined the biological effects of cells treated with (18)F labeled drugs for positron emission tomography (PET). The relationship between the intracellular distribution of (18)F and levels of damaged DNA has yet to be clarified in detail. We used culture cells (Chinese Hamster Ovary cells) treated with two types of (18)F labeled drugs, fluorodeoxyglucose (FDG) and fluorine ion (HF). FDG efficiently accumulated in cells, whereas HF did not. To examine the induction of DNA double strand breaks (DSB), we measured the number of foci for 53BP1 that formed at the site of DNA DSB. The results revealed that although radioactivity levels were the same, the induction of 53BP1 foci was stronger in cells treated with (18)F-FDG than in those treated with (18)F-HF. The clonogenic survival of cells was significantly lower with (18)F-FDG than with (18)F-HF. We concluded that the efficient accumulation of (18)F in cells led to stronger biological effects due to more severe cellular lethality via the induction of DNA DSB.

Evaluation of the bubble point test of a 0.22-µm membrane filter used for the sterilizing filtration of PET radiopharmaceuticals.

OBJECTIVE: We developed a bubble point test kit and investigated the bubble point test of a 0.22-µm membrane filter used for the sterilizing filtration of [(18)F]FDG, [(11)C]MET and [(11)C]PIB. The bubble point test of the Millex-GS vented filter was often difficult due to air leakage from the vented portion of this filter. Therefore, to close the vented portion of this filter simply and reliably, we investigated the use of various materials. METHODS: The bubble point test of the Millex-GS vented filter was performed by closing the vented portion of this filter with various materials, such as vinyl tape, plastic paraffin film (parafilm), urethane elastomer adhesive mat and polyethylene foam cushion tape. Gradually, the plunger inside a syringe filled with air was pushed down to increase the pressure on the pressure gauge and the bubble point test kit. Simultaneously, the pressure when a continuous stream of air bubbles that appeared out of the 0.22-µm membrane filter was measured as the product-wetted bubble point value. Then, the plunger inside a syringe filled with 10 mL of water was pushed down to wash the 0.22-µm membrane filter. As in the case in the above-mentioned method of measuring the product-wetted bubble point, the water-wetted bubble point value was measured. RESULTS: The use of the polyethylene foam cushion tape and a double clip could easily and reliably prevent air leakage from the vented portion of the Millex-GS vented filter. In the bubble point test of [(18)F]FDG, [(11)C]MET and [(11)C]PIB, the product-wetted bubble point values were 382.7 ± 6.9 kPa, 385.4 ± 6.2 kPa and 351.6 ± 7.6 kPa, respectively. The bubble point ratio was used to determine the minimum product-wetted bubble point value. All results of the product-wetted bubble point test were beyond the minimum product-wetted bubble point value (334.4 kPa ([(18)F]FDG), 334.4 kPa ([(11)C]MET) and 310.3 kPa ([(11)C]PIB)). Then, the water-wetted bubble point values were 396.5 ± 8.3 kPa, 395.8 ± 8.3 kPa and 390.3 ± 7.6 kPa, respectively. All results of the water-wetted bubble point test were beyond the filter manufacturer's minimum bubble point specification (344.8 kPa). CONCLUSIONS: The bubble point test technique using the bubble point test kit was practical for routine quality control tests of PET radiopharmaceuticals.

Effect of bevacizumab treatment on p-boronophenylalanine distribution in murine tumor.

Previous studies have demonstrated that angiogenesis inhibitors can enhance tumor inhibitory effects of chemo- and radiotherapy via their action on tumor vessels. Here, we studied the effect of the angiogenesis inhibitor, bevacizumab (Avastin), on boron distribution in a murine tumor model. The human head and neck squamous cell carcinoma cell line was used for inoculation into mice. Boron-10 concentrations in tissues were measured by prompt γ-ray spectrometry (PGA). Hoechst 33342 perfusion and p-boronophenylalanine (BPA) distribution were determined by immunofluorescence staining. Our results revealed enhanced tumor blood perfusion and BPA accumulation in tumors after Avastin treatment, suggesting that combination of angiogenesis inhibition with treatment with boron compound administration may improve the efficacy of boron neutron capture therapy (BNCT) by modifying tumor vessels. In addition, our results also demonstrated the usefulness of immunofluorescence staining for investigating boron compound distribution at the cellular level.

Protective roles of ascorbic acid in oxidative stress induced by depletion of superoxide dismutase in vertebrate cells.

Superoxide dismutases (SODs) are antioxidant proteins that convert superoxide to hydrogen peroxide. In vertebrate cells, SOD1 is mainly present in the cytoplasm, with small levels also found in the nucleus and mitochondrial intermembrane space, and SOD2 is present in the mitochondrial matrix. Previously, the authors conditionally disrupted the SOD1 or SOD2 gene in DT40 cells and found that depletion of SOD1 caused lethality, while depletion of SOD2 led to growth retardation. The observations from previous work showed that the lethality observed in SOD1-depleted cells was completely rescued by ascorbic acid. Ascorbic acid is a water-soluble antioxidant present in biological fluids; however, the exact target for its antioxidant effects is not known. In this study, the authors demonstrated that ascorbic acid offset growth defects observed in SOD2-depleted cells and also lowered mitochondrial superoxide to physiological levels in both SOD1- or SOD2-depleted cells. Moreover, depletion of SOD1 or SOD2 resulted in the accumulation of intracellular oxidative stress, and this increased oxidative stress was reduced by ascorbic acid. Taken together, this study suggests that ascorbic acid can be applied as a nontoxic antioxidant that mimics the functions of cytoplasmic and mitochondrial SODs.

Increased susceptibility to delayed genetic effects of low dose X-irradiation in DNA repair deficient cells.

PURPOSE: To examine whether the levels of micronuclei induction, as a marker for genomic instability in the progeny of X-irradiated cells, correlates with DNA repair function. MATERIALS AND METHODS: Two repair deficient cell lines (X-ray repair cross-complementing 1 [XRCC1] deficient cell line [EM9] and X-ray repair cross complementing 5 [XRCC5; Ku80] deficient X-ray sensitive Chinese hamster ovary [CHO] cell line [xrs5]) were used in addition to wild-type CHO cells. These cells were irradiated with low doses of X-rays (up to 1 Gy). Seven days after irradiation, micronuclei formed in binucleated cells were counted. To assess the contribution of the bystander effect micronuclei induction was measured in progeny of non-irradiated cells co-cultured with cells that had been irradiated with 1Gy. RESULTS: The delayed induction of micronuclei in 1 Gy-irradiated cells was observed in normal CHO and EM9 but not in xrs5. In the clone analysis, progenies of xrs5 under bystander conditions showed significantly higher levels of micronuclei, while CHO and EM9 did not. CONCLUSION: Genomic instability induced by X-irradiation is associated with DSB (double-strand break) repair, even at low doses. It is also suggested that bystander signals, which lead to genomic instability, may be enhanced when DSB repair is compromised.

Micronuclei formation induced by X-ray irradiation does not always result from DNA double-strand breaks.

X-ray induced formation of micronuclei is generally thought to result from DNA double-strand breaks (DSBs). However, DNA DSBs inhibit the cell cycle progression that is required for micronucleus formation. In order to reconcile this apparent discrepancy, we investigated whether DNA DSBs induced during the G1 phase could lead to micronucleus formation. We irradiated human embryonic (HE17) cells that had been treated with a radical scavenger, either DMSO or ascorbic acid (AsA), and determined the level of suppression of DNA DSBs or micronuclei. When DNA DSBs were evaluated using 53BP1 foci, treatment with 5 mM AsA did not inhibit the numbers of foci at various intervals after X-ray irradiation; however, treatment with 5 mM or 256 mM DMSO did have a significant inhibitory effect. By contrast, an assay of micronucleus numbers showed that treatment with 5 mM or 256 mM DMSO before X-ray irradiation resulted in almost no inhibition of micronucleus formation, but treatment with 5 mM AsA did have a significant inhibitory effect. These results clearly showed that AsA could suppress micronucleus formation, although it was not effective for suppression of DNA DSBs. Therefore, we conclude that DNA DSBs induced in the G1 phase do not directly lead to micronucleus formation.

The bystander effect is a novel mechanism of UVA-induced melanogenesis.

We successfully identified the bystander effect in B16 murine melanoma cells exposed to UVA irradiation. The effect was identified based on melanogenesis following the medium transfer of the B16 cells, which had been cultured for 24 h after being exposed to UVA irradiation, to nonirradiated cells (bystander cells). Our confirmation study of the functional mechanism of bystander cells confirmed the reduced levels of mitochondrial membrane potential 1-4 h after the medium transfer. In addition, we observed increased levels of intracellular oxidation after 9-12 h, and the generation of melanin radicals, including long-lived radicals, 24 h after medium transfer. Further analysis of bystander factors revealed that the administration of EGTA treatment at the time of medium transfer led to an inhibition of melanogenesis and to neutralization of the mitochondrial membrane potential level, as well as to the restoration of intracellular oxidation levels to those of controls. The results demonstrated that the UVA irradiation bystander effect in B16 cells, as indicated by melanogenesis, was induced by the increase in intracellular oxidation due to the mitochondrial activity of calcium ions, which were among the bystander factors involved in the increase.

Dysregulation of gene expression in the artificial human trisomy cells of chromosome 8 associated with transformed cell phenotypes.

A change in chromosome number, known as aneuploidy, is a common characteristic of cancer. Aneuploidy disrupts gene expression in human cancer cells and immortalized human epithelial cells, but not in normal human cells. However, the relationship between aneuploidy and cancer remains unclear. To study the effects of aneuploidy in normal human cells, we generated artificial cells of human primary fibroblast having three chromosome 8 (trisomy 8 cells) by using microcell-mediated chromosome transfer technique. In addition to decreased proliferation, the trisomy 8 cells lost contact inhibition and reproliferated after exhibiting senescence-like characteristics that are typical of transformed cells. Furthermore, the trisomy 8 cells exhibited chromosome instability, and the overall gene expression profile based on microarray analyses was significantly different from that of diploid human primary fibroblasts. Our data suggest that aneuploidy, even a single chromosome gain, can be introduced into normal human cells and causes, in some cases, a partial cancer phenotype due to a disruption in overall gene expression.

DNA double-strand break induction in Ku80-deficient CHO cells following boron neutron capture reaction.

BACKGROUND: Boron neutron capture reaction (BNCR) is based on irradiation of tumors after accumulation of boron compound. 10B captures neutrons and produces an alpha (4He) particle and a recoiled lithium nucleus (7Li). These particles have the characteristics of high linear energy transfer (LET) radiation and have marked biological effects. The purpose of this study is to verify that BNCR will increase cell killing and slow disappearance of repair protein-related foci to a greater extent in DNA repair-deficient cells than in wild-type cells. METHODS: Chinese hamster ovary (CHO-K1) cells and a DNA double-strand break (DSB) repair deficient mutant derivative, xrs-5 (Ku80 deficient CHO mutant cells), were irradiated by thermal neutrons. The quantity of DNA-DSBs following BNCR was evaluated by measuring the phosphorylation of histone protein H2AX (gamma-H2AX) and 53BP1 foci using immunofluorescence intensity. RESULTS: Two hours after neutron irradiation, the number of gamma-H2AX and 53BP1 foci in the CHO-K1 cells was decreased to 36.5-42.8% of the levels seen 30 min after irradiation. In contrast, two hours after irradiation, foci levels in the xrs-5 cells were 58.4-69.5% of those observed 30 min after irradiation. The number of gamma-H2AX foci in xrs-5 cells at 60-120 min after BNCT correlated with the cell killing effect of BNCR. However, in CHO-K1 cells, the RBE (relative biological effectiveness) estimated by the number of foci following BNCR was increased depending on the repair time and was not always correlated with the RBE of cytotoxicity. CONCLUSION: Mutant xrs-5 cells show extreme sensitivity to ionizing radiation, because xrs-5 cells lack functional Ku-protein. Our results suggest that the DNA-DSBs induced by BNCR were not well repaired in the Ku80 deficient cells. The RBE following BNCR of radio-sensitive mutant cells was not increased but was lower than that of radio-resistant cells. These results suggest that gamma-ray resistant cells have an advantage over gamma-ray sensitive cells in BNCR.

An alternative mechanism for radioprotection by dimethyl sulfoxide; possible facilitation of DNA double-strand break repair.

The radioprotective effects of dimethyl sulfoxide (DMSO) have been known for many years, and the suppression of hydroxyl (OH) radicals induced by ionizing radiation has been thought to be the main cause of this effect. However, the DMSO concentration used was very high, and might be toxic, in earlier studies. In the present study, we administered a lower, non-toxic concentration (0.5%, i.e., 64 mM) of DMSO before irradiation and examined its radioprotective effects. Colony formation assay and micronucleus assay showed significant radioprotective effects in CHO, but not in xrs5, which is defective in the repair function of DNA double-strand breaks. The levels of phosphorylated H2AX and the formation of 53BP1 foci 15 minutes after irradiation, which might reflect initial DNA double-strand breaks, in DMSO-treated CHO cells were similar to those in non-treated cells, suggesting that the radioprotective effects were not attributable to the suppression of general indirect action in the lower concentration of DMSO. On the other hand, 2 hours after irradiation, the average number of 53BP1 foci, which might reflect residual DNA double-strand breaks, was significantly decreased in DMSO-treated CHO cells compared to non-treated cells. The results indicated that low concentration of DMSO exerts radioprotective effects through the facilitation of DNA double-strand break repair rather than through the suppression of indirect action.