Temperature-dependent free radical reaction in water

Temperature-dependent free radical reactions were investigated using nitroxyl radicals as redox probes. Reactions of two types of nitroxyl radicals, TEMPOL (4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl) and carbamoyl-PROXYL (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl), were tested in this paper. Heating a solution containing a nitroxyl radical and a reduced form of glutathione (GSH) caused temperature-dependent decay of electron paramagnetic resonance (EPR) signal of the nitroxyl radical. Heating a solution of the corresponding hydroxylamine form of the nitroxyl radical showed EPR signal recovery. The GSH-dependent reduction of nitroxyl radicals at 70°C was suppressed by antioxidants, spin trapping agents, and/or bubbling N2 gas, although heating carbamoyl-PROXYL with GSH showed temporarily enhanced signal decay by bubbling N2 gas. Since SOD could restrict the GSH-dependent EPR signal decay of TEMPOL, O2•− is related with this reaction. O2•− was probably generated from dissolved oxygen in the reaction mixture. Oxidation of the hydroxylamines at 70°C was also suppressed by bubbling N2 gas. Heating a solution of spin trapping agent, DMPO (5,5-dimethyl-1-pyrroline-N-oxide) showed a temperature-dependent increase of the EPR signal of the hydroxyl radical adduct of DMPO. Synthesis of hydroxyl radical adduct of DMPO at 70°C was suppressed by antioxidants and/or bubbling N2 gas. The results suggested that heating an aqueous solution containing oxygen can generate O2•−

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

Generation of hydrogen peroxide in water irradiated by carbon-ion beam; Effects of dissolved oxygen and LET

The oxygen independent formation of hydrogen peroxide (H2O2) in an aqueous solution during carbon-ion beam irradiation was demonstrated. The radiation-induced hydroxyl radical (•OH) generation in an aqueous solution was reported to occur in two different localization densities, which were at the milli-molar (relatively sparse) and/or molar (super-dense) levels. In the milli-molar-level •OH generation atmosphere, •OH generated at a molecular distance of 4.3‒6.6 nm are likely unable to interact each other. However, in the molar-level •OH generation atmosphere, several •OH were generated with a molecular distance of 1 nm or less, and two •OH can react to directly make H2O2.An aliquot of ultra-pure water was irradiated by 290 MeV/nucleon carbon-ion beam at the Heavy-Ion Medical Accelerator in Chiba (HIMAC, NIRS/QST, Chiba, Japan). Irradiation experiments were performed under air or hypoxic (100 keV/μm). H2O2 generations in irradiated samples were estimated by three methods below. 1) A red quinoid dye (absorbance at 505 nm) formed by a reaction of 4-aminoantipyrine and phenol and H2O2 under coexisting peroxidase were measured using the spectrophotometer. 2) The •OH synthesized from H2O2 under the presence of Fe2+ was spin-trapped with DMPO, and the •OH adduct of DMPO (DMPO-OH) was then measured as an index of H2O2 using an X-band EPR. 3) The •OH synthesized from H2O2 under the UVB irradiation was spin-trapped with DMPO, and the DMPO-OH was then measured by an X-band EPR. Amounts of H2O2 generation per dose was estimated.H2O2 generation under air condition, i.e. total H2O2 generation, decreased with LET increasing. H2O2 generation under hypoxic condition, i.e. oxygen independent H2O2 generation, increased with LET increasing. The oxygen dependent H2O2 generation, i.e. subtraction of H2O2 generation under hypoxic condition from H2O2 generation under air condition, decreased with LET increasing. Those results suggest that the super-dense •OH generation was increased with LET increasing. High LET beam could make H2O2 oxygen independently.20th Biennial Meeting of SFRR International (SFRR-I 2021

Oxygen independent generation of hydrogen peroxide in water irradiated by carbon-ion beam

The oxygen independent formation of hydrogen peroxide (H2O2) in an aqueous solution during carbon-ion beam irradiation was demonstrated. The radiation-induced hydroxyl radical (•OH) generation in an aqueous solution was reported to occur in two different localization densities, which were at the milli-molar (relatively sparse) and/or molar (super-dense) levels. In the milli-molar-level •OH generation atmosphere, •OH generated at a molecular distance of 4.3‒6.6 nm are likely unable to interact each other. However, in the molar-level •OH generation atmosphere, several •OH were generated with a molecular distance of 1 nm or less, and two •OH can react to directly make H2O2.An aliquot of ultra-pure water was irradiated by 290 MeV/nucleon carbon-ion beam at the Heavy-Ion Medical Accelerator in Chiba (HIMAC, NIRS/QST, Chiba, Japan). Irradiation experiments were performed under air or hypoxic (100 keV/μm). H2O2 generations in irradiated samples were estimated by two methods below. 1) The •OH synthesized from H2O2 by UVB irradiation was spin-trapped with DMPO, and the •OH adduct of DMPO (DMPO-OH) was then measured as an index of H2O2 using an X-band EPR. 2) A red quinoid dye (absorbance at 505 nm) formed by a reaction of 4-aminoantipyrine and phenol and H2O2 under coexisting peroxidase were measured using the spectrophotometer. Amounts of H2O2 generation per dose was estimated. H2O2 generation under air condition, i.e. total H2O2 generation, decreased with LET H2O2 generation under air condition, i.e. total H2O2 generation, decreased with LET increasing. H2O2 generation under hypoxic condition, i.e. oxygen independent H2O2 generation, increased with LET increasing. The oxygen dependent H2O2 generation, i.e. subtraction of H2O2 generation under hypoxic condition from H2O2 generation under air condition, decreased with LET increasing. Those results suggest that the super-dense •OH generation was increased with LET increasing. High LET beam could make H2O2 oxygen independently.27th Annual Conference of the Society for Redox Biology & Medicine (SfRBM 2020

Availability of thiol compounds to generate superoxide by reducing molecular oxygen

The availability of thiol compounds to generate superoxide by reducing molecular oxygen at hyperthermal temperature was investigated. The reaction properties of cysteine (Cys), N-acetyl-L-cysteine (NAC), reduced form glutathione (GSH), and homocysteine (HCS) were compared. A sulfur-containing amino acid, methionine (Met), biological hydrogen-donor, reduced beta-nicotinamide adenine dinucleotide (NADH), and reduced beta-nicotinamide adenine dinucleotide phosphate (NADPH) were also tested for comparison. An oxidative atmosphere, i.e. superoxide generation by the hypoxanthine-xanthine oxidase reaction, hydroxyl radical generation by X-ray irradiation, or direct one-electron oxidation by ferricyanide, was prepared in a reaction mixture containing 0.1 mM TEMPOL and 1 mM of the test compound, and the EPR signal decay profile of TEMPOL was observed. A reaction mixture containing 0.1 mM TEMPOL and 1 mM thiol compound, Cys, NAC, GSH, or HCS was incubated at 44DegreeC, and the EPR signal decay profile of TEMPOL was observed. Thiols can work as H-donors to the oxoammonium cation form and produce the hydroxyl amine form of TEMPOL in an oxidative atmosphere. Thiols can also irreversibly react with the oxoammonium cation. NAD(P)H could work only as an H-donor. Met did not show any reaction with TEMPOL. GSH and Cys can reduce molecular oxygen to form hydroperoxyl radical/superoxide at hyperthermal temperatures, but HCS and NAC cannot reduce molecular oxygen. GSH and Cys may possibly cause reductive stress, whereas NAC is a simple tractable antioxidant.Oxygen Radicals, Biology and Pathobiology of Oxygen Radicals from Cell Signaling and Detection to Therap

LET Dependent Generations of Reactive Oxygen Species and RedOx Reactions in an Aqueous Sample Irradiated by X-ray or Carbon-Ion Beam

Hydroxyl radicals (*OH), hydrogen peroxide (H{sub}2{/sub}O{sub}2{/sub}), and superoxide (O{sub}2{/sub}{sup}*-{/sup}) are the important reactive oxygen species (ROS) for considering radio-biological effects. ROS generations in aqueous samples by X-ray or carbon-ion beams were quantified.Amount and distribution of *OH, H{sub}2{/sub}O{sub}2{/sub}, and total oxidation reactions were estimated by an electron paramagnetic resonance (EPR) spin-trapping, a modified spin-trapping, and spin-probing method, respectively.*OH generation was expected to be localized on the track/range of the radiation, and mM and M levels of *OH generation were observed. Total *OH generation was expected to be identical at the same dose even for X-ray or carbon-ion beam. Sparse *OH generation decreased with increasing LET. Carbon-ion beam produced denser H{sub}2{/sub}O{sub}2{/sub} compared with X-ray irradiation. H{sub}2{/sub}O{sub}2{/sub} generation decreased with the LET increasing, although the ratio of H{sub}2{/sub}O{sub}2{/sub} generation per oxygen consumed increased with LET. Total oxidation reactions decreased with increasing LET. Total oxidation reactions caused by low LET conditions decreased when the experiment was performed at low O{sub}2{/sub} concentration, while no difference was observed by high LET conditions.ROS generation was not uniform and was LET dependent at the molecular level.9th Biennial Meeting for the Society for Free Radical Research Asi