Am. J. Respir. Cell Mol. Biol., Volume 26, Number 4, April, 2002 391-397

Blocking Neutrophil Influx Reduces DNA Damage in Hyperoxia-Exposed Newborn Rat Lung

Richard L. Auten, Mary H. Whorton, and S. Nicholas Mason

Neonatal-Perinatal Research Institute, Division of Neonatal Medicine, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina

Hyperoxia-induced neutrophil infux in neonatal rats may contribute to impaired lung development through oxidative DNA damage. To determine whether blocking neutrophil influx prevents DNA damage, we treated newborn rats with 95% O₂ beginning at birth, and at 3 and 4 d with nonimmune immunoglobulin G (IgG) (control) or anti-cytokine-induced neutrophil chemoattractant (CINC). At 8 d, lungs were inflation-fixed. Random sections were labeled using terminal transferase nick end-labeling (TUNEL), and DNA oxidation was measured using anti-8-OH-2'-deoxyguanosine (OHdG). To determine whether hyperoxia-induced TUNEL represented apoptosis, we labeled sections with anti-Bax (proapoptotic) and anti-Bcl-2 (antiapoptotic). We labled additional sections with anti-M30, directed against an epitope formed by caspase 6 digestion of cytokeratin 18 during apoptosis. Hyperoxia induced marked increases in TUNEL and OHdG signal in lung parenchymal cells, which was substantially prevented by treatment with anti-CINC. The large effects of hyperoxia on TUNEL were not accompanied by substantial effects on Bax, Bcl-2, or M30. We conclude that neutrophil influx during hyperoxia damages DNA by nicking and oxidation, and that blocking neutrophil influx can prevent this. Effects of 95% O₂ on TUNEL are not primarily due to apoptosis in this model. Neutrophil-mediated oxidative DNA damage may contribute to abnormal lung development in newborns subjected to significant oxidative stress.

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