PERSPECTIVE
DNA Oxidation or Apoptosis
Will the Real Culprit of DNA Damage in Hyperoxic Lung Injury Please Stand Up?

Kurt H. Albertine and Charles G. Plopper

Departments of Pediatrics, Medicine, and Neurobiology & Anatomy, University of Utah, Salt Lake City, Utah; and Department of Anatomy, Physiology, and Cell Biology, University of California Davis, Davis, California

Acute lung injury is induced by prolonged exposure to high concentrations of oxygen (1, 2). The injury is manifest as accumulation of inflammatory cells, particularly neutrophils, in the pulmonary circulation, interstitium, and air spaces, as well as by formation of interstitial and alveolar edema (3, 4). A common setting for hyperoxic acute lung injury is premature birth (5). The lungs of prematurely born humans and experimental animals are not sufficiently developed, however, to perform effective and efficient gas exchange. Therefore, oxygen therapy and ventilatory support are necessary. An undesired but anticipated consequence of both interventions is acute lung injury, which in neonates is called respiratory distress syndrome. If the preterm neonate does not recover from the acute lung injury (hyaline membrane disease), and therefore requires continuous oxygen and ventilatory support, the acute lung injury may give way to chronic lung injury of prematurity, also known as bronchopulmonary dysplasia (6, 7).

Mediation of pulmonary oxygen toxicity is thought to be via reactive oxygen species, particularly those generated by inflammatory cells (8). Among the first signs of oxygen toxicity in the lung are swelling of endothelial cells and altered appearance of mitochondria and microsomes (9). Subsequent changes include loss of cells, such as capillary endothelial cells, and proliferation of other cell types, including alveolar type II epithelial cells and interstitial cells (10).

A prevailing notion is that apoptosis plays a central role in DNA damage during the pathogenesis of hyperoxic lung injury. The cellular complexity of an organ like the lung, however, makes defining the mechanisms of cell injury and death, especially apoptotic events, a challenging task. A critical step is identifying the cell types that are primary or secondary targets of the injurious agent and the local microenvironment in which the susceptible cells reside. Once the responding cell population has been identified, careful delineation of the cellular events leading to cell injury and whether cell death signaling is initiated involves establishing the subcellular compartments that are affected and the temporal relationship of compartment changes to the injurious agent.

A wide variety of analytical approaches are now available for judging initiation and progression along the apoptotic cascade in situ and in vitro. The gold standards are light and electron microscopy. Among the characteristic morphologic changes are cell shrinkage, dense chromatin condensation in the nucleus, cellular budding and fragmentation, and phagocytosis (11). A universal biochemical marker is still lacking, however. DNA fragmentation, resulting in oligonucleosome-sized fragments, is detected as a ladder pattern of multiples of 185 bp by agarose gel electrophoresis (14). However, DNA fragmentation does not occur in all cell types undergoing cell death, even though morphologic features of apoptosis are expressed (15). DNA strand breaks are identified in situ and in vitro by end-labeling the breaks, using terminal transferase deoxyribonucleotidyl dUTP nuclear end labeling (TUNEL). The strand breaks precede the morphologic features of apoptosis and the staining pattern is generally accepted as indicating apoptosis (16). However, TUNEL results are not necessarily specific for apoptosis (17). Expression of apoptosis-related genes is also used to identify apoptosis. One of the essential genes is the caspase family of genes (reviewed in Ref. 20). Caspase-1 gene activation has been shown, for example, to occur in hyperoxic lung injury (21). Several other regulatory genes are members of the Bcl-2 gene family. For example, Bax is pro-apoptotic (22), whereas Bcl-2 is anti-apoptotic (23). During apoptosis, Bax expression is increased relative to Bcl-2 (24). Other genes also have been implicated in apoptosis, such as p53, Fas, and c-myc, but whether their role is obligatory or epiphenomenologic remains to be determined (25). A key point is that the number of involved and putatively involved genes demonstrates how complex the process of apoptosis is.

New and important insights into the relative role of DNA oxidation versus apoptosis in the DNA damage that is associated with hyperoxic lung injury in neonates are provided by the paper authored by Auten and colleagues in this issue of the AJRCMB (26). Auten and colleagues took advantage of the knowledge that accumulation of neutrophils in the lung requires cellular signaling to initiate and amplify neutrophil transmigration. These investigators used an anti-cytokine-induced neutrophil chemoattractant (CINC) antibody to treat neonatal rat pups that were reared in 95% oxygen for 8 d. Anti-CINC antibody was administered intraperitoneally on Days 3 and 4, at a dosage that the same group of investigators have shown is optimal to block neutrophil influx in the lung (27).

Among the strengths of the study by Auten and coworkers are the use of morphologic methods in situ and the stepwise progression of analyses. Use of morphologic methods is a strength because the lung is composed of more than 40 cell types (reviewed in Ref. 28). As noted above, oxygen toxicity affects many cell types in the lung, making it difficult to discriminate between molecular changes that occur among the affected cell types. Morphologic methods provide an approach to identify the affected cell types in situ. On the other hand, a limitation of morphologic methods, particularly histochemical and immunohistochemical methods, is that results are qualitative or semiquantitative. However, morphologic methods were used for the original description (11) and continue today to be a preferred analytical approach. There are few other choices when organs are studied. The other strength of this study is use of progressive steps of analysis. The primary analysis used TUNEL and 8-OH-2'-deoxyguanosine (OHdG) immunohistochemistry. The results show that DNA nicking (TUNEL) and oxidation (OHdG immunohistochemistry) were widespread in the lung and that blocking neutrophil accumulation in the lung significantly reduced both DNA nicking and oxidation. This result indicates that reactive oxygen species released from neutrophils contribute significantly to nucleic acid damage, even when the ambient concentration of oxygen is high. Stemming from this result is the intriguing implication that an approach to minimize oxidant stress in neonates who require supplemental oxygen may be to pharmacologically inhibit release of neutrophil chemoattractants in the lung.

As previously discussed, TUNEL results are used as a histological marker for apoptosis, including apoptosis in the lung, but TUNEL is not specific for apoptosis in the lung (29). In the present study, TUNEL results were comparable to those for DNA oxidation, an observation that led the authors to suggest that nucleic acid oxidation was predominant. Supporting this suggestion was the purpose of the second and third analyses performed by Auten and coworkers. Accordingly, immunohistochemical expression was assessed for two proteins involved in the apoptotic cascade. Bax, which is pro-apoptotic, is increased relative to Bcl-2, which is anti-apoptotic, during apoptosis (24). However, Auten and colleagues did not detect increased expression of Bax or decreased expression of Bcl-2 in situ in the lungs of the rat pups that breathed 95% oxygen continuously for 8 d, in the presence or absence of anti-CINC antibody. The third analysis complemented the second by assessing the expression of M30, a molecule that becomes evident only when caspase 6 has sufficiently degraded cytokeratin 18, an intermediate filament in epithelial cells. A key point is that cytokeratin digestion immediately precedes internucleosomal DNA fragmentation, which is revealed by TUNEL. Fewer cells were positive for M30 than for TUNEL, OHdG, or Bax in the hyperoxic-exposed lungs. Thus, two independent methods provided support for the conclusion that nucleic acid oxidation prevailed more than apoptosis.

Whether the authors went far enough to support their conclusion might be debatable. They did not use transmission electron microscopy, for example, to demonstrate ultrastructural changes of the nucleus. Nor did they examine DNA ladder appearance by gel electrophoresis or measure ceramide (30) or other death effector molecules along the apoptotic cascade (reviewed in Ref. 20). However, the analyses that were used built upon each other such that a reasonable conclusion could be reached, one that is consistent with another study (31). Whether the results can be extended to chronic lung disease of prematurity, as speculated by the authors, remains to be seen because the experimental model used by the authors is not a model of bronchopulmonary dysplasia. Nonetheless, Auten and coworkers' study provides new insight into cellular mechanisms that participate in the cycle of acute lung injury and repair following 8 d of continuous exposure of neonatal rat pups to hyperoxia. In this model of acute lung injury, DNA oxidation appears to be the principal culprit of DNA damage.

	Footnotes

Address correspondence to: Kurt H. Albertine, Ph.D., Department of Pediatrics, University of Utah, 2A129 School of Medicine, 30 North 1900 East, Salt Lake City, UT 84143-2202. E-mail: kurt.albertine{at}hsc.utah.edu

(Received in original form February 22, 2002).

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