Blocking neutrophil influx reduces DNA damage in hyperoxia-exposed newborn rat lung.
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.
Auten
Richard L
RL
Neonatal-Perinatal Research Institute, Division of Neonatal Medicine, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710, USA. auten@duke.edu
Whorton
Mary H
MH
Nicholas Mason
S
S
D000080242:8-Hydroxy-2'-Deoxyguanosine / Q000031:analogs & derivatives; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D053178:Caspase 6; D020169:Caspases / Q000378:metabolism; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000276:immunology; D004249:DNA Damage; D004848:Epithelium / Q000276:immunology / Q000378:metabolism; D006133:Growth Substances / Q000276:immunology; D006147:Guanine / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism; D018496:Hyperoxia / Q000473:pathology*; D007074:Immunoglobulin G / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D036341:Intercellular Signaling Peptides and Proteins; D007633:Keratins / Q000276:immunology / Q000378:metabolism; D008168:Lung / Q000473:pathology / Q000503:physiopathology*; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D018384:Oxidative Stress; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D006133:Growth Substances; D007074:Immunoglobulin G; D036341:Intercellular Signaling Peptides and Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D006147:Guanine; D007633:Keratins; C078307:8-oxo-7,8-dihydrodeoxyguanine; D000080242:8-Hydroxy-2'-Deoxyguanosine; C505518:Casp6 protein, rat; D053178:Caspase 6; D020169:Caspases