Analysis of the Antioxidative Function of the Radioprotective Japanese Traditional (Kampo) Medicine, Hangeshashinto, in an Aqueous Phase

Oral mucositis (OM) is a common and painful complication of radiotherapy for head and neck cancer. Hangeshashinto (HST), a Japanese traditional medicine, is known to alleviate radiotherapy and/or chemotherapy-induced OM; however, the detailed mechanism has not yet been clarified. The aim of the present study is to clarify the details of the antioxidative functions of HST against reactive oxygen species (ROS) produced by radiation. The hydroxyl radical (•OH) scavenging ability and reduction ability was simultaneously measured using a modified electron paramagnetic resonance (EPR) spin trapping method. The superoxide (O2•−) scavenging ability was estimated by an EPR redox probing method. Water suspension of powdered HST and its seven constitutive crude drugs were tested. In addition, some of the main water soluble ingredients of the crude drugs were also tested. HST was found to scavenge both •OH and O2•−. Furthermore, HST was observed to reduce relatively stable nitroxyl radicals. Glycyrrhizae Radix (kanzo), Ginseng Radix (ninjin), Zizyphi Fructus (taiso), and glycyrrhizin (an ingredient of kanzo) were all found to be relatively good •OH scavengers. Scutellariae Radix (ogon) and Coptidis Rhizoma (oren) demonstrated reducing ability. In addition, acteoside and berberine chloride, which are water soluble ingredients of ogon and oren, respectively, also demonstrated reducing ability. Oren exhibited oxidative ability at higher concentrations, which may have a function to maintain catalytic redox action. The antioxidative function of HST probably worked in a balance of scavenging ROS, reducing stable free radicals and some minor oxidative effects

EPR based Estimation of Radiation-Induced Reactive Oxygen Species

Generation of reactive oxygen species (ROS) is considered as essential trigger of biological effects of ionizing radiations, and may be deeply linked with the radiation quality.Amounts of total oxidation reactions (i.e. oxidative free radical species, •OH and HO2•), H2O2 generations, Oxygen consumptions, and •OH generations induced by X-ray, 20 keV/μm carbon beam, and 80 keV/μm carbon beam were estimated using EPR based techniques.Total oxidation reactions were estimated as 3, 1.3, and 0.66 μmol/L/Gy, amount of H2O2 generations were 0.2, 0.57, and 0.35 μmol/L/Gy, oxygen consumptions were 0.4, 0.39, and 0.15 μmol/L/Gy for X-ray, 20 keV/μm carbon beam, and 80 keV/μm carbon beam, respectively. The ratio of H2O2 generation per oxygen consumption were increased with LET, and were 0.5, 1.46, 2.33 for X-ray, 20 keV/μm carbon beam, and 80 keV/μm carbon beam, respectively. The •OH generations expected to be localized on the track/range of the radiation beam/ray, and both sparse (≈ 3.3 mM) and very dense (> 1.7 M) •OH generations were suggested. Percentage of sparse •OH generation decreased with LET becoming higher.The SFRBM\u27s 23rd Annual Meeting, a joint meeting with the Society for Free Radical Research International (SFRBM/SFRRI 2016

Intestinal Bacteria as Powerful Trapping Lifeforms for the Elimination of Radioactive Cesium

In March 2011, an accident at the Fukushima Daiichi Nuclear Power Plant led to major problems, including the release of radionuclides such as Cesium (Cs)-137 into the environment. Ever since this accident, Cs-137 in foods has become a serious problem. In this study, we determined the concentration of Cs-137 in the feces, urine, and ruminal contents of cattle and demonstrated the possibility of its elimination from the body by intestinal bacteria. The results revealed a high Cs-137 concentration in the feces; in fact, this concentration was higher than that in skeletal muscles and other samples from several animals. Furthermore, intestinal bacteria were able to trap Cs-137, showing an uptake ratio within the range of 38–81% in vitro. This uptake appeared to be mediated through the sodium–potassium (Na+-K+) ion pump in the bacterial cell membrane. This inference was drawn based on the fact that the uptake ratio of Cs-137 was decreased in media with high potassium concentration. In addition, it was demonstrated that intestinal bacteria hindered the trapping of Cs-137 by the animal. Cattle feces showed high concentration of Cs-137 and intestinal bacteria trapped Cs-137. This study is the first report showing that intestinal bacteria contribute to the elimination of Cs-137 from the body

Nano-Geometry of Hydroxyl Radical Generated by Low-LET Irradiation in Water

For suppressing the side effect of radiation therapy, the regulation of hydroxyl radical (OH) is essential, particularly in low-LET radiation such as X-ray or gamma-ray. Primary generation of reactive species, called "spur," attributed to radiation-induced ionization and/or excitation of water can be characterized as cluster of radicals. In this study, the density of OH generation in spur was evaluated by electron paramagnetic resonance (EPR) spin-trapping technique. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was used as EPR spin trapping agent. Several concentrations of DMPO (0.5-1680 mM) were prepared and then irradiated with 32 Gy X-rays (3.2, 4.8, or 6.4 Gy/min) or gamma-ray (7.9 Gy/min). The amount of OH adduct of DMPO (DMPO-OH) in the irradiated sample was measured by X-band EPR. The relationship between molecular density of DMPO in the reaction mixture and DMPO-OH generated was analyzed, where molecular distance was defined as reciprocal of molecule-to-molecule distance of DMPO. The amount of DMPO-OH was increased in proportion to the molecular density of DMPO and reached plateau. The DMPO concentration at inflection point was 3.3 mM. Again increasing DMPO density, another linear trend that passed through the origin was observed when the DMPO concentrations were above 490 mM. Two different OH density, i.e., sparse (around 3 mM; molecule-to-molecule distance is 8 nm) and dense (much higher than 1700 mM; molecule-to-molecule distance is less than 1 nm), were predicted as in sub-spur structure.第1回QST国際シンポジウム「量子生命科学 -Quantum Life Science-

LET Dependent Hydroxyl Radical Generation in Water by Heavy-Ion Beam Irradiation

Nano-geometry of hydroxyl radical (OH) generation in water caused by heavy-ion bream irradiation was evaluated using a fluorescent probe, disodium terephthalate (TPA-Na). Since OH is recognized as a primary product in biological effects of radiation, elimination of OH may be important to regulate the side effect of radiation therapy. Recently, using the electron paramagnetic resonance (EPR) spin-trapping technique, we reported that two different types of OH generations by ionizing radiation. In this study, linear energy transfer (LET) dependence on the density of localized OH generation was evaluated using TPA-Na. The irradiation of heavy-ion beams was performed at the Heavy-Ion Medical Accelerator in Chiba (HIMAC, National Institute of Radiological Sciences, Japan). Several concentrations (0.5-208 mM) of TPA-Na in aqueous solutions were irradiated with 32 Gy of a heavy-ion beam (C400, Si490 or Ffe500) at several different LET. The amount of OH adduct of TPA-Na (hTPA) thus generated was measured by fluorophotometer (Ex. 310 nm/Em. 425 nm). The relationship between molecular density of TPA-Na and hTPA generated was analyzed. The amount of hTPA generated by low-LET heavy-ion beam was increased in proportion to the molecular density of TPA-Na and reached a plateau through an unclear inflection point. In the case of high-LET heavy-ion beam, however, another linear trend that passed through the origin was again observed when TPA-Na density was extremely high ([TPA-Na] = 156-208 mM). The linear increasing trend observed at high TPA-Na density suggests an extremely dense OH generation

Localized hydroxyl radical generation at mmol/l and mol/l levels in water by photon irradiation

The generation of localized hydroxyl radical (•OH) in aqueous samples by low linear energy transfer (LET) irradiation was investigated. Several concentrations of 5,5-dimethyl-1-pyrroline-N-oxid (DMPO) solution (from 0.5 to 1680 mmol/l) were prepared and irradiated with an identical dose of X-ray or γ-ray. The density of •OH generation in aqueous solution was evaluated by the electron paramagnetic resonance (EPR) spin-trapping technique using DMPO as an EPR spin-trapping agent. The relationship between the molecular density of DMPO in the samples and the concentration of DMPO-OH generated in the irradiated samples was analyzed. Two different characteristic linear trends were observed in the DMPO-OH/DMPO plots, which suggested •OH generation in two fashions, i.e., mmol/l- and mol/l-level local concentrations. The dose, dose rate, and/or the energy of photon irradiation did not affect the shapes of the DMPO-OH/DMPO plots. Moreover, the addition of 5 mmol/l caffeine could cancel the contribution of mmol/l-level •OH generation, leaving a trace of mol/l-level •OH generation. Thus, the localized mmol/l- and mol/l-level generations of •OH, which were independent of experimental parameters such as dose, dose rate, and/or the energy of photon of low LET radiation, were established

Chemopreventive Agent Sulforaphane Enhances Radiosensitivity in Human Tumor Cells

Sulforaphane (SFN), an isothiocyanate derived from broccoli and other cruciferous vegetables, is a positive regulator of Phase II detoxification enzymes, and is highly effective in protection against chemically induced cancers by inducing apoptosis and cell cycle arrest. Here, we report that SFN also enhances radiosensitivity in human tumor cells. Cell survival in HeLa human cervix carcinoma cells pre-treated with SFN was significantly lower than in cells treated with radiation only. Constant field gel-electrophoresis and a gamma-H2AX foci assay showed marked inhibition of DSB repair in irradiated cells treated with SFN, while little inhibition was observed in cells with DMSO (control). In addition, immunofluorescence experiments revealed a significant delay in Rad51 (a key protein for homologous recombination repair (HRR) ) foci formation and disappearance in irradiated cells treated with SFN when compared to the cells with X-irradiation alone. The de-phosphorylation of DNA-PKcs (a critical non-homologous end joining (NHEJ) protein) was also markedly delayed by SFN pre-treatment in irradiated cells. These DSB repair inhibition data partially support the high apoptotic frequency of irradiated cells pretreated with SFN. Furthermore, the combined treatment of X-rays and SFN (i.p. 300 umol/kg) in the xenograft model with HeLa cells showed efficient inhibition of in vivo tumor growth. To the best of our knowledge, this study is the first report showing SFN enhanced radiosensitivity of tumor cells in vitro and in vivo, which opens the door for a multitude of clinical applications for chemo-radiotherapy using SFN. in pres

Preparation of an experimental mouse model lacking selenium-dependent glutathione peroxidase activities by feeding a selenium-deficient diet

Relatively young (4-week-old) selenium deficient (SeD) mice, which lack the activity of selenium-dependent glutathione peroxidase (GSH-Px) isomers, were prepared using torula yeast-based SeD diet.Mice were fed the torula yeast-based SeD diet and ultra-pure water. Several different timings for starting the SeD diet were assessed. The weekly time course of liver comprehensive GSH-Px activity after weaning was monitored. Protein expression levels of GPx1 and 4 in the liver were measured by Western blot analysis. Gene expression levels of GPx1, 2, 3, 4, and 7 in the liver were measured by quantitative real-time PCR. Apoptotic activity of thymocytes after H2O2 exposure was compared. Thirty-day survival rates after whole-body X-ray irradiation were estimated.Pre-birth or right-after-birth starting of the SeD diet in dams was unable to lead to creation of SeD mice due to neonatal death. This suggests that Se is necessary for normal birth and healthy growing of mouse pups. Starting the mother on the SeD diet from 2 weeks after giving birth (SeD-trial-2w group) resulted in a usable SeD mouse model. The liver GSH-Px activity of the SeD-trial-2w group was almost none from 4 week olds, but the mice survived for more than 63 weeks. Protein and gene expression of GPx1 was suppressed in the SeD-trial-2w group, but that of GPx4 was not. The thymocytes of the SeD-trial-2w group were sensitive to H2O2-induced apoptosis. The SeD-trial-2w group was sensitive to whole-body X-ray irradiation compared with control mice. The SeD-trial-2w model may be a useful animal model for H2O2/hydroperoxide-induced oxidative stress