Hyperoxia, used therapeutically in the treatment of respiratory insufficiencies, can cause
lung injury, probably through the actions of
reactive oxygen species. The present studies were designed to test the hypothesis that oxidation of specific
proteins would provide useful
biomarkers of the onset of tissue injury, and thereby provide clues as to the mechanisms responsible. We exposed adult male Sprague-Dawley rats to room air or to greater than 95% O2 for 60 h and examined
proteins in
pleural effusion and broncho-alveolar lavage (BAL) fluids, and in lung tissue homogenates and subfractions. Oxidation of
protein thiols was assessed by derivatization with
monobromobimane, separation by electrophoresis, and visualization of the fluorescent
thioether derivatives. Derivatization with
2,4-dinitrophenylhydrazine (DNPH), electrophoresis, and western analysis was employed to assess a different class of oxidative modifications, frequently termed '
protein carbonyls'. In addition, we investigated the effects of the 21-aminosteroid
U-74389G, 10 mg/kg, given intraperitoneally every 12 h, on
biomarkers of
protein oxidation and on manifestations of
lung injury.
Hyperoxia caused
lung injury evidenced by
pleural effusions, increases in BAL
protein concentrations, and
pulmonary edema;
U-74389G attenuated the first two indices of
lung injury, but did not alter
edema.
Protein thiol status of the fractions studied were not affected notably by
hyperoxia, or by the aminosteroid. The formation of DNPH-reactive sites on a limited number of
proteins by
hyperoxia was observed, and some of these effects were attenuated in the animals given
U-74389G. Histological examination of lung tissues showed accumulation of intra-alveolar
protein exudates in hyperoxic rats, and a significant attenuation of this effect was observed in the animals treated with
U-74389G. In conclusion, studies of shifts in
protein thiol status that may be caused by
hyperoxia will require increasingly specific methods of analysis, and characterization of the specific DNPH-reactive
proteins formed in
hyperoxia may provide critical insights into the mechanisms of
lung injury. Administration of
U-74389G offers some degree of protection against
hyperoxia and attenuation of these
biomarkers of oxidation, but the precise mechanisms by which this protection is effected will require additional study.