DETECTION OF HYDROXYL RADICALS UPON INTERACTION OF OZONE WITH AQUEOUS-MEDIA OR EXTRACELLULAR SURFACTANT - THE ROLE OF TRACE IRON

As part of a study on mechanisms modulating ozone-induced surfactant p erturbations, we used the electron paramagnetic resonance (EPR) spin t rapping technique to determine the type and origin of radicals generat ed 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, con sistent with hydroxyl radical adduct formation (DMPO-OH), the amplitud es of which increased with the ozone concentration. No signals were ob tained from air-exposed samples. Similar four-line spectra were also p roduced following interaction of 3 ppm ozone with Kank's balanced salt solution (HBSS) alone or containing BAL; fractions. Addition of the h ydroxyl radical scavenger dimethyl sulfoxide (DMSO) to the incubation medium strongly inhibited formation of DMPO-OH adduct during ozone exp osure. As an alternate method of demonstrating the generation of hydro xyl radicals, aqueous solutions of 1 mM L-phenylalanine were exposed t o high concentrations of ozone and shown, using ion-exchange chromatog raphy, 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 o f DMSO which reacts with the hydroxyl radical resulting in the formati on of methyl radical. The methyl radical subsequently reacts with spin trap PBN, yielding PBN-methyl adduct. In the absence of DMSO there was no detectable formation of methyl radical adduct. EPR double distille d water containing DMPO showed a small amount of DMPO-OH adduct upon e xposure to ozone. Addition of 10 mu M 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 mu M def eroxamine or 8000 U catalase and abolished by higher concentrations of deferoxamine (1 mM). The signal was not influenced by 1000 U superoxi de dismutase. These results indicate that hydroxyl radicals are produc ed via iron-dependent reactions during the initial interaction of ozon e with aqueous media, including bronchoalveolar fluid. (C) 1995 Acndem ic Press, Inc.