Iron and oxidized beta-casein in the lavages of hyperoxic Fischer-344 rats

We reported previously that Fischer-344 (F344) rats were more susceptible to hyperoxic lung injury than were Sprague Dawley (SD) rats. In the present study we exposed adult male F344 and SD rats to >95% oxygen for up to 48 h, and measured lung wet-to-dry weight ratios and lavage protein concentrations as indices of lung injury. In addition, we measured nonheme iron contents in the lung subcellular fractions and in bronchoalveolar lavages (BAL), and we derivatized samples from the subcellular compartments and lavages with 2,4-dinitrophenylhydrazine (DNPH), separated the proteins electrophoretically, and detected DNPH-derivatized proteins by western blotting. After 48 h of hyperoxia, BAL protein and nonheme iron concentrations were higher in F344 rats than in SD rats (2.17+/-0.77 versus 0.17+/-0.17 mg/ml, and 1.61+/-0.45 versus 0.45+/-0.18 nmol/ml, respectively, both P<0.05). In addition, two DNPH-reactive proteins of about 40 and 120 kDa were identified in the lavage fluids of hyperoxic F-344 rats that were not observed similarly in hyperoxic SD rats or in air-breathing rats of either strain. N-terminal amino acid sequences of the two DNPH-reactive proteins 100% identical over 16 residues to rat beta-casein, which is a potent neutrophil chemotaxin, and has been reported to be a product of cytotoxic T-lymphocytes. There were no significant alterations in iron contents in lung subcellular fractions in either strain of rat as a consequence of hyperoxia-exposure, nor were there any significant alterations in DNPH-reactive carbonyls, as determined by western blotting. These data suggest that increased iron concentrations in the airspaces reflect altered iron homeostasis, which may contribute to the greater susceptibility of F344 rats than SD rats to hyperoxic lung injury. The identification of oxidized beta-casein in the BAL of the hyperoxic F344 rats suggests a role for cytotoxic T-lymphocytes in hyperoxic lung inflammation and injury, although the nature of this possible involvement is not known at this time.