8-hydroxyguanine and Hyperoxia

Recent studies indicate that the antiapoptotic Bcl-X(L), one of five isoforms expressed by the Bcl-X gene, protects a variety of cell lines exposed to hyperoxia. However, its role in lung development and protection against oxidative stress in vivo is not known. Here, we show Bcl-X(L) is the predominant isoform expressed in the lung, and the only isoform detected in respiratory epithelium. Because loss of Bcl-X(L) is embryonically lethal, Bcl-X(L) was ablated throughout the respiratory epithelium by mating mice with a floxed exon II of the Bcl-X gene with mice expressing Cre under control of the surfactant protein-C promoter. Interestingly, the loss of Bcl-X(L) in respiratory epithelium was perinatally lethal in approximately 50% of the expected offspring. However, some adult mice lacking the gene were obtained. The epithelial-specific ablation of Bcl-X(L) did not disrupt pulmonary function, the expression of epithelial cell-specific markers, or lung development. However, it shifted the lung toward a proapoptotic state, defined by a reduction in antiapoptotic Mcl-1, an increase in proapoptotic Bak, and increased sensitivity of the respiratory epithelium to hyperoxia. Intriguingly, increased 8-oxoguanine lesions seen during hyperoxia were also evident as lungs transitioned to room air at birth, a time when perinatal lethality in some mice lacking Bcl-X(L) was observed. These findings reveal that the epithelial-specific expression of Bcl-X(L) is not required for proper lung development, but functions to protect respiratory epithelial cells against oxygen-induced toxicity, such as during hyperoxia and the lung's first exposure to ambient air.

Topics: Animals; Apoptosis; bcl-X Protein; Blotting, Western; Guanine; Hyperoxia; Integrases; Lung; Mice; Mice, Knockout; Oxygen; Respiratory Mucosa; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Supplementary oxygen during resuscitation of the asphyxiated newborn is associated with long-term detrimental effects including increased risk of childhood cancer. It is suspected that the resuscitation procedure results in accumulated DNA damage and mutagenesis. Base excision repair (BER) is the major pathway for repair of premutagenic oxidative DNA lesions. This study addresses DNA base damage and BER in brain, lung, and liver in neonatal mice (P7) after hyperoxic resuscitation. Mice were randomized to 8% oxygen or room air for 60 min in a closed chamber and subsequent reoxygenation with 100% oxygen for 0 to 90 min. During this treatment, 8-oxoguanine accumulated in liver but not in lung or cerebellum. We observed a linear relation between 8-oxoguanine and reoxygenation time in liver DNA from hypoxic animals (n = 28; B = 0.011 [0.001, 0.020]; p = 0.037). BER activity was not significantly changed during resuscitation. Our data suggest that after hypoxia, the capacity for immediate repair in liver tissue is inadequate to meet increasing amounts of DNA damage. The duration of supplementary oxygen use during resuscitation should be kept as short as justifiable to minimize the risk of genetic instability.

Topics: Animals; Animals, Newborn; Cerebellum; DNA Damage; DNA Repair; Guanine; Hyperoxia; Liver; Lung; Mice; Mice, Inbred C57BL; Mutagenesis; Neoplasms; Oxygen; Oxygen Inhalation Therapy; Resuscitation; Risk

The effect of hyperoxia on levels of DNA damage and global DNA methylation was examined in lung epithelial-like A549 cells. DNA damage was assessed by the single-cell gel electrophoresis (comet assay) and DNA methylation status by the cytosine extension assays. Cells exposed to ionizing radiation (0, 1, 2, 4, or 8 Gy) showed increasing rates of percentage of DNA in the tail and tail length with increasing radiation dose. When cells were exposed to room air (normoxia) for 1 day and 95% O2 (hyperoxia) for 1, 2, 3, 4, and 5 days, data indicated that hyperoxia caused time-dependent increases in levels of (a) single strand breaks, (b) double strand breaks, and (c) 8-oxoguanine. Decreased DNA methylation also was observed at day 5 of hyperoxic exposure, suggesting that hyperoxia-induced DNA damage can influence patterns of DNA methylation in a lung-derived cell line.

Topics: Cell Line, Tumor; Comet Assay; DNA; DNA Damage; DNA Methylation; DNA Repair; DNA, Single-Stranded; Epithelial Cells; Guanine; Humans; Hyperoxia; Lung Neoplasms; Oxygen; Time Factors

It is well established that hyperoxia injures and kills alveolar endothelial and type I epithelial cells of the lung. Although type II epithelial cells remain morphologically intact, it remains unclear whether they are also damaged. DNA integrity was investigated in adult mice whose type II cells were identified by their endogenous expression of pro-surfactant protein C or transgenic expression of enhanced green fluorescent protein. In mice exposed to room air, punctate perinuclear 8-oxoguanine staining was detected in approximately 4% of all alveolar cells and in 30% of type II cells. After 48 or 72 h of hyperoxia, 8-oxoguanine was detected in 11% of all alveolar cells and in >60% of type II cells. 8-Oxoguanine colocalized by confocal microscopy with the mitochondrial transmembrane protein cytochrome oxidase subunit 1. Type II cells isolated from hyperoxic lungs exhibited nuclear DNA strand breaks by comet assay even though they were viable and morphologically indistinguishable from cells isolated from lungs exposed to room air. These data reveal that type II cells exposed to in vivo hyperoxia have oxidized and fragmented DNA. Because type II cells are essential for lung remodeling, our findings raise the possibility that they are proficient in DNA repair.

Topics: Air; Animals; DNA Damage; DNA, Mitochondrial; Green Fluorescent Proteins; Guanine; Humans; Hyperoxia; Luminescent Proteins; Mice; Mice, Transgenic; Pulmonary Alveoli; Pulmonary Surfactant-Associated Protein C; Recombinant Proteins; Respiratory Mucosa; Time Factors