title: Scavenging of reactive oxygen species induced by hyperthermia in biological fluid.
abstract: The scavenging activity of rat plasma against hyperthermia-induced reactive oxygen species was tested. The glutathione-dependent reduction of a nitroxyl radical, 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl, which was restricted by adding superoxide dismutase or by deoxygenating the reaction mixture, was applied to an index of superoxide (O2 (•-)) generation. A reaction mixture containing 0.1 mM 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl and 1 mM glutathione was prepared using 100 mM phosphate buffer containing 0.05 mM diethylenetriaminepentaacetic acid. The reaction mixture was kept in a screw-top vial and incubated in a water bath at 37 or 44°C. The time course of the electron paramagnetic resonance signal of 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl in the reaction mixture was measured by an X-band EPR spectrometer (JEOL, Tokyo, Japan). When the same experiment was performed using rat plasma instead of 100 mM PB, the glutathione-dependent reduction of 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl, i.e., generation of O2 (•-), was not obtained. Only the first-order decay reduction of 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl, which indicates direct reduction of 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl, was obtained in rat plasma. Adding 0.5% albumin to the phosphate buffer reaction mixture could almost completely inhibit O2 (•-) generation at 37°C. However, addition of 0.5% albumin could not inhibit O2 (•-) generation at 44°C, i.e., hyperthermic temperature. Ascorbic acid also showed inhibition of O2 (•-) generation by 0.01 mM at 37°C, but 0.02 mM or more could inhibit O2 (•-) generation at 44°C. A higher concentration of ascorbic acid showed first-order reduction, i.e., direct one-electron reduction, of 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl. Hyperthermia-induced O2 (•-) generation in rat plasma can be mostly inhibited by albumin and ascorbic acid in the plasma.
authors.affiliation: Radio-Redox-Response Research Team, Advanced Particle Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Ueno
authors.affiliation: Radio-Redox-Response Research Team, Advanced Particle Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan.
authors.forename: Minako
authors.initials: M
authors.lastname: Nyui
authors.affiliation: Radio-Redox-Response Research Team, Advanced Particle Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Nakanishi
authors.affiliation: Radio-Redox-Response Research Team, Advanced Particle Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan ; Department of Pharmaceutical Sciences, Nihon Pharmaceutical University, Kitaadachi-gun, Saitama 362-0806, Japan.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Anzai
authors.affiliation: Radio-Redox-Response Research Team, Advanced Particle Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan ; Yokohama College of Pharmacy, 61 Matano-cho, Totsuka-ku, Yokohama 245-0066, Japan.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Ozawa
authors.affiliation: Radio-Redox-Response Research Team, Advanced Particle Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan.
authors.forename: Ken-Ichiro
authors.initials: K
authors.lastname: Matsumoto
authors.affiliation: Department of Life System, Institute of Technorlogy and Science, Graduate School, The University of Tokushima, Tokushima 770-8506, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Uto
keywords: electron paramagnetic resonance; hyperthermia; nitroxyl redox probe; reactive oxygen species; superoxide