Byvoet P, Balis J U, Shelley S A, Montgomery M R, Barber M J
Department of Pathology, College of Medicine, University of South Florida, Tampa, USA.
Arch Biochem Biophys. 1995 Jun 1;319(2):464-9. doi: 10.1006/abbi.1995.1318.
As part of a study on mechanisms modulating ozone-induced surfactant perturbations, we used the electron paramagnetic resonance (EPR) spin trapping technique to determine the type and origin of radicals generated following interaction of ozone with aqueous solutions and cell-free bronchoalveolar lavage fluid (BAL) fractions. All aqueous media were exposed to ozone at 25 degrees C with or without added chelator, 1 mM diethylenetriaminepentaacetic acid, and spintrap, 100 mM 5,5'-dimethyl-1-pyrroline-1-oxide (DMPO). Exposure of distilled water to 0.5, 1.0, 2.0, and 3.0 ppm ozone for 1 h yielded four-line spectra, 1:2:2:1, consistent with hydroxyl radical adduct formation (DMPO-OH), the amplitudes of which increased with the ozone concentration. No signals were obtained from air-exposed samples. Similar four-line spectra were also produced following interaction of 3 ppm ozone with Hank's balanced salt solution (HBSS) alone or containing BAL fractions. Addition of the hydroxyl radical scavenger dimethyl sulfoxide (DMSO) to the incubation medium strongly inhibited formation of DMPO-OH adduct during ozone exposure. As an alternate method of demonstrating the generation of hydroxyl radicals, aqueous solutions of 1 mM L-phenylalanine were exposed to high concentrations of ozone and shown, using ion-exchange chromatography, to contain small amounts of L-tyrosine. Production of hydroxyl radicals upon interaction of ozone and water was further substantiated using the spintrap PBN (phenyl-N-tert-butylnitrone) in the presence of DMSO which reacts with the hydroxyl radical resulting in the formation of methyl radical. The methyl radical subsequently reacts with spintrap PBN, yielding PBN-methyl adduct. In the absence of DMSO there was no detectable formation of methyl radical adduct. EPR double distilled water containing DMPO showed a small amount of DMPO-OH adduct upon exposure to ozone. Addition of 10 microM ferrous sulfate to this mixture produced a 10-fold increase of the signal, which was attenuated in the presence of 1500 U catalase, strongly attenuated with 50-500 microM deferoxamine or 8000 U catalase and abolished by higher concentration of deferoxamine (1 mM). The signal was not influenced by 1000 U superoxide dismutase. These results indicate that hydroxyl radicals are produced via iron-dependent reactions during the initial interaction of ozone with aqueous media, including bronchoalveolar fluid.
作为一项关于调节臭氧诱导表面活性剂扰动机制研究的一部分，我们使用电子顺磁共振（EPR）自旋捕获技术来确定臭氧与水溶液及无细胞支气管肺泡灌洗液（BAL）组分相互作用后产生的自由基的类型和来源。所有水性介质在25℃下暴露于臭氧，添加或不添加螯合剂1 mM二乙烯三胺五乙酸以及自旋捕获剂100 mM 5,5'-二甲基-1-吡咯啉-1-氧化物（DMPO）。将蒸馏水暴露于0.5、1.0、2.0和3.0 ppm的臭氧中1小时，产生了1:2:2:1的四线谱，这与羟基自由基加合物的形成（DMPO-OH）一致，其幅度随臭氧浓度增加。空气暴露的样品未获得信号。3 ppm臭氧与单独的汉克平衡盐溶液（HBSS）或含有BAL组分的溶液相互作用后也产生了类似的四线谱。向孵育介质中添加羟基自由基清除剂二甲基亚砜（DMSO）可强烈抑制臭氧暴露期间DMPO-OH加合物的形成。作为证明羟基自由基产生的另一种方法，将1 mM L-苯丙氨酸的水溶液暴露于高浓度臭氧中，并通过离子交换色谱法显示含有少量L-酪氨酸。在DMSO存在下使用自旋捕获剂PBN（苯基-N-叔丁基硝酮）进一步证实了臭氧与水相互作用时羟基自由基的产生，PBN与羟基自由基反应导致甲基自由基的形成。甲基自由基随后与自旋捕获剂PBN反应，产生PBN-甲基加合物。在没有DMSO的情况下，未检测到甲基自由基加合物的形成。含有DMPO的EPR双蒸水暴露于臭氧后显示出少量的DMPO-OH加合物。向该混合物中添加10 microM硫酸亚铁使信号增加了10倍，在存在1500 U过氧化氢酶的情况下信号减弱，在50 - 500 microM去铁胺或8000 U过氧化氢酶存在下强烈减弱，而在较高浓度的去铁胺（1 mM）存在下信号消失。该信号不受1000 U超氧化物歧化酶的影响。这些结果表明，在臭氧与包括支气管肺泡液在内的水性介质的初始相互作用过程中，羟基自由基是通过铁依赖性反应产生的。
