Method for Assessing X-ray-Induced Hydroxyl Radical Scavenging Activity of Biological Compounds/Materials

A method for correctly assessing hydroxyl radical scavenging activity of antioxidative chemicals and/or biological compounds/materials was proposed. This method can simultaneously assess two factors, i.e. hydroxyl radical scavenging and 5,5-dimethyl-2-hydroxy-1-pyrrolidine-N-oxide (hydroxyl radical adduct of 5,5-dimethyl-1-pyrroline-N-oxide) reducing ability, as antioxidative properties. In this paper, some biologically common hydrophilic molecules, cell culture media, and rat plasma were tested. X-ray induced hydroxyl radical can be detected using the electron paramagnetic resonance spin trapping technique. Using X-ray irradiation of the reaction mixture as the hydroxyl radical source, the true hydroxyl radical scavenging ability of the subjected antioxidant can be assessed. In addition, the method simultaneously measures the reduction of 5,5-dimethyl-2-hydroxy-1-pyrrolidine-N-oxide, to estimate the reducing ability of the antioxidant. Biological materials, such as sugars and proteins, could abolish hydroxyl radical at the biological concentration. Ascorbic acid showed reducing ability at the biological concentration. The simultaneous assessment of hydroxyl radical scavenging and reducing ability of antioxidants can be an informative index for antioxidants

Trial of Brain Redox Imaging and Estimation of Radiation-Induced Redox Change in Mouse Brain

The in vivo T1-weighted contrasting abilities and signal decay behaviors of several nitroxyl contrast agents, which have been used as redox responsive contrast agents in several magnetic resonance-based imaging modalities, in mouse brain were compared. In addition, daily variations of redox behavior in mouse brain after irradiation of X-ray or carbon-ion beams (C-beam) were tried to estimate based on the in vivo reduction rate of amphiphilic nitroxyl contrast agents.Injection solutions of five types of five-membered-ring nitroxyl contrast agents, i.e. 3-carboxy-2,2,5,5-tetramethylpyrrolidine-N-oxyl (CxP), 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (CmP), 3-methoxy-carbonyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (MCP), acetoxymethyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-carboxylate (CxP-AM), and 4-(N-methylpiperidine)-2,2,5,5-tetramethylpyrroline-N-oxyl (23c), and a six-membered-ring nitroxyl contrast agent, i.e. 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), were prepared. The nitroxyl contrast agent was i.v. injected to a mouse through tail vein. Then, the distributions and pharmacokinetics of nitroxyl contrast agents were compared based on the time course of T1-weighted MRI. The MRI experiments using CMP or TEMPOL were repeated for mice irradiated by X-ray or C-beam to their head on several deferent timings, i.e. 1, 2, 4, 8 day(s) after irradiation. C-beam was irradiated at Heavy-Ion Medical Accelerator in Chiba (HIMAC, National Institute of Radiological Sciences/ National Institutes for Quantum and Radiological Science and Technology).The blood-brain-barrier (BBB)-impermeable CxP could not be distributed in the brain. The slightly lipophilic CmP showed slight distribution only in the ventricle, but not in the medulla and cortex. The amphiphilic MCP and TEMPOL had good initial uniform distribution in the brain and showed typical 2-phase signal decay profiles. A brain-seeking nitroxyl probe, CxP-AM, showed an accumulating phase, and then its accumulation was maintained in the medulla and ventricle regions, but not in the cortex. The lipophilic 23c was well distributed in the cortex and medulla, but slightly in the ventricle, and showed relatively rapid linear signal decay.Decay rates of MCP in mouse brain after irradiation of 8 Gy X-ray, 8 Gy C-beam or 16 Gy C-beams did not show marked clear changes, however relatively little decreasing were observed at day 1 and day 2 after irradiation. Decay rates of TEMPOL was increased 1 after irradiation then gradually recovered to the control level. MCP and TEMPOL showed opposite responses but the timing of redox change may be 1 or 2 days after irradiation.Nitroxyl contrast agents equipped with a suitable lipophilic substitution group could be BBB-permeable functional contrast agents. MR redox imaging, which can estimate not only the redox characteristics but also the detailed distribution of the contrast agents, is a good candidate for a theranostic tool. Irradiation of ionized radiation to head could cause alternation of redox status in the brain. Detail of redox mechanisms were still in progress.第7回国際放射線神経生物学会大

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•−

Multimodal Functional Imaging for Cancer/Tumor Microenvi-ronments based on MRI, EPRI, and PET

Radiation therapy is one of the main modalities to treat cancer/tumor. The response to radiation therapy, however, can be influenced by physiological and/or pathological conditions in the target tissues, especially by the low partial oxygen pressure and altered redox status in cancer/tumor tissues. Visualizing such cancer/tumor patho-physiological microenvironment would be a useful not only for planning radiotherapy but also to detect cancer/tumor in an earlier stage. Tumor hypoxia could be sensed by positron emission tomography (PET), electron paramagnetic resonance (EPR) oxygen mapping, and in vivo dynamic nuclear polarization (DNP) MRI. Tissue oxygenation could be visualized on a real-time basis by blood oxygen level dependent (BOLD) and/or tissue oxygen level dependent (TOLD) MRI signal. EPR imaging (EPRI) and/or T1-weighted MRI techniques can visualize tissue redox status non-invasively based on paramagnetic and dia-magnetic conversions of nitroxyl radical contrast agent. 13C-DNP MRI can visualize glycometabolism of tumor/cancer tissues. Accurate co-registration of those multimodal images could make mechanisms of drug and/or relation of resulted biological effects clear. A multimodal instrument, such as PET-MRI, may have another possibility to link multiple functions. Functional imaging techniques individually developed to date have being converged on the concept of theranostics

Estimation of the local concentration of the markedly dense hydroxyl radical generation induced by X-rays in water

The density of X-ray-induced markedly dense hydroxyl radicals (•OH) in water was estimated based on EPR spin-trapping measurement using DMPO as the spin-trapping agent. A lower (0.13 mM‒2.3 M) concentration series of DMPO water solutions and higher (1.7‒4.8 M) concentration series of DMPO water solutions plus neat DMPO liquid (8.8 M as DMPO) were irradiated with 32 Gy of X-rays. Then, the yield of DMPO-OH in DMPO water solutions and the total spin-adduct of DMPO in neat DMPO were quantified. Estimating the EPR signal peak area by the line fitting method is only available for well-separated single EPR lines. For the higher concentration DMPO series, the EPR signal peak area was estimated by double integration. The baseline correction of the integral spectrum is necessary for accurate estimation of the double integration of EPR spectrum. In addition, using the suitable standard sample corresponding to the EPR sensitivity, which was variable by the electric permittivity according to DMPO concentration, was quite important for quantification of DMPO-OH especially in the higher DMPO concentration beyond 2 mM. The local density of •OH generation in water by X-ray irradiation was estimated from the inflection point on the plot of the DMPO-OH yield versus DMPO density. The density of X-ray-induced markedly dense •OH was estimated as 1184 μm-1, which was converted to 0.84 nm as the intermolecular distance and 2.8 M as the local concentration

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

Expression of Ascorbate Peroxidase Derived from Cyanidioschyzon merolae in Mammalian Cells

Background: Ascorbate peroxidase (APX) derived from Cyanidioschyzon merolae, a primitive red alga living in high temperature and acidic environments, has a greater anti-oxidative capacity than similar peroxidases occurring in other plants. In the present study, we examined the ability of Cyanidioschyzon merolae-derived APX (cAPX) to increase anti-oxidative capacity when expressed in mammalian cells. Materials and Methods: The cAPX gene was introduced into the mouse fibroblast-like cell line C3H10T1/2. Production of reactive oxygen species (ROS) and/or cell viability was assessed after heat, H2O2 and acid stimulation. Results: Heat and H2O2 stimulation caused ROS production. cAPX-expressing cells were more tolerant to oxidative stress induced by heat, H2O2 and acid stimulations than control cells lacking cAPX. Conclusion: Introduction of cAPX increases anti-oxidative capacity in mammalian cells

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