Uchiyama Hidefumi, Zhao Qing-Li, Hassan Mariame Ali, Andocs Gabor, Nojima Nobuyuki, Takeda Keigo, Ishikawa Kenji, Hori Masaru, Kondo Takashi
Tateyama Machine Co., Ltd., Toyama 930-1305, Japan.
Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan.
PLoS One. 2015 Aug 28;10(8):e0136956. doi: 10.1371/journal.pone.0136956. eCollection 2015.
Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH) radicals and hydrogen (H) atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H2O2, which is the recombination product of ·OH, and OCl- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and an intracellular milieu is discussed.
采用电子顺磁共振（EPR）自旋捕获和流式细胞术，与X射线辐射产生的自由基相比，鉴定氩冷大气等离子体（Ar-CAP）在水溶液和细胞内产生的自由基。使用具有低频激发的高压电源装置产生Ar-CAP。通过真空紫外吸收和发射光谱测量来评估Ar-CAP的特性。用自旋捕获剂5,5-二甲基-1-吡咯啉N-氧化物（DMPO）、3,3,5,5-四甲基-1-吡咯啉-N-氧化物（M4PO）和苯基N-叔丁基硝酮（PBN）鉴定水溶液中的羟基（·OH）自由基和氢（H）原子。在DNA成分、醋酸钠和L-丙氨酸的水溶液中，使用自旋捕获剂3,5-二溴-4-亚硝基苯磺酸盐（DBNBS）评估Ar-CAP诱导的热解的发生情况。使用对多种活性物质具有不同亲和力的五种荧光探针，用人淋巴瘤U937细胞研究细胞内氧化应激。EPR光谱的分析和定量显示，与X射线辐射相比，使用Ar-CAP会形成大量的·OH自由基。检测到极少量的H原子，未发现一氧化氮。·OH自由基的形成取决于所用稀有气体的类型，其产率与电离能呈反比，顺序为氪>氩=氖>氦。在暴露于Ar-CAP的水溶液中未检测到热解自由基。在细胞内，暴露于Ar-CAP后，·OH、·OH的重组产物H2O2和OCl-是最可能形成的活性氧物种。在细胞内，没有实际证据表明会形成NO，而只形成了极少量的超氧化物。尽管Ar-CAP在形成·OH自由基方面具有优势，但事实证明暴露于X射线更具致死性。本文讨论了水溶液和细胞内环境中自由基形成的机制。
