DNA damage induced by hyperoxia: quantitation and correlation with lung injury

Abstract

Inspired oxygen, an essential therapy for cardiorespiratory disorders, has the potential to generate reactive oxygen species that damage cellular DNA. Although DNA damage is implicated in diverse pulmonary disorders, including neoplasia and acute lung injury, the type and magnitude of DNA lesion caused by oxygen in vivo is unclear. We used single-cell gel electrophoresis (SCGE) to quantitate two distinct forms of DNA damage, base adduction and disruption of the phosphodiester backbone, in the lungs of mice. Both lesions were induced by oxygen, but a marked difference between the two was found. With 40 h of oxygen exposure, oxidized base adducts increased 3- to 4-fold in the entire population of lung cells. This lesion displayed temporal characteristics (a progressive increase over the first 24 h) consistent with a direct effect of reactive oxygen species attack upon DNA. DNA strand breaks, on the other hand, occurred in < 10% of pulmonary cells, which acquired severe levels of the lesion; dividing cells were preferentially affected. Characteristics of these cells suggested that DNA strand breakage was secondary to cell death, rather than a primary effect of reactive oxygen species attack on DNA. By analysis of IL-6- and IL-11-overexpressing transgenic animals, which are resistant to hyperoxia, we found that DNA strand breaks, but not base damage, correlated with acute lung injury. Analysis of purified alveolar type 2 preparations from hyperoxic mice indicated that strand breaks preferentially affected this cell type.

Figure 1.

Native SCGE of lung suspensions from C57Bl6 mice treated with 94% oxygen for 3 d. (A) Representative low-power digital photomicrographs of SCGE gels from a control and an oxygen-exposed mouse. Intensity-map coloration was performed with Cometscore software. (B) Histogram analysis. Discontinuity of the y-axis is used to clarify low-frequency events. (C) Time course. Approximately 300 comets were quantitated for each mouse. Bars depict the mean %DNA in Tail of each group. *P < 0.0001 (by ANOVA) for the difference between the 84-h oxygen exposure group and all other groups. No other comparisons are significant.

Figure 2.

FPG-modified SCGE of lung suspensions from C57Bl6 mice treated with 94% oxygen. (A) Representative medium-power digital photomicrographs from two mice, colored by Autocomet software. (B) Histogram analysis. A 60 h duration of oxygen exposure was used. (C). Time course: for each time point, > 100 comets from each of three mice were quantitated under high-power microscopy (×400) and the mean value for each mouse extracted. Bars depict the average of the extracted means (i.e., each mouse equals one data point). *P = 0.019; ^#P = 0.039; **P = 0.0006; ^##P = 0.0001 (values are provided for comparison to the 0-h group). (D) SCGE and FPG-modified SCGE run in parallel, to compare the relative amounts of DNA strand breakage and base damage. A 40-h duration of oxygen exposure was used. Statistical analysis as in C. P value is shown for comparison of the room air– and oxygen-exposed lung samples.

Figure 3.

SCGE analysis of wild-type and IL-6–overexpressing transgenic mice exposed to 94% oxygen for 80 h. (A) Native SCGE analysis quantitated under low-power (×100) microscopy. More than 500 comets from each mouse were quantitated and the mean olive moment for each mouse extracted. Bars depict the average of the extracted mean olive moments for each group. *P < 0.01 versus WT, room air; ^#P < 0.05 versus WT, oxygen. (B) FPG-modified SCGE. More than 100 comets from each mouse were quantitated under high power with statistical analysis as in A. *P < 0.0001 versus room air; ^#P < 0.001 versus room air, NS versus WT. (C) Pictorial and graphic depiction of the distribution of cells from experiment shown in A. Lower panels: open bars, WT, room air; black bars, WT, 3 d 95% O₂; shaded bars, IL-6, 3 d 95% O₂.

Figure 4.

Effect of DNA replication on alkaline SCGE. Primary pulmonary microvascular endothelial cells were pulsed with 100 μm BrdU for 1 h before SCGE. BrdU-containing cells were detected as described in Materials and Methods. (A) Monochromatic images intensity-mapped by the image-analysis program Cometscore; these images are used for quantitation. Right panel, EtBr illumination; left panel, BrdU-specific illumination. (B) Quantitation of 100 BrdU-positive and 200 accompanying BrdU-negative cells.

Figure 5.

BrdU incorporation in vivo. Mice exposed to 95% oxygen for 3 d were injected with 1.5 mg of BrdU intraperitoneally 24 h before preparation of lung samples for SCGE followed by BrdU detection as described above. (A) Two representative sets of monochromatic images intensity mapped by Cometscore. Left panels are taken under BrdU-specific illumination; right panels show the same field under total DNA illumination. B and C show, respectively, mean levels of %DNA in Tail and histogram analysis. *P < 0.001 for comparison to BrdU-negative sample.

Figure 6.

Behavior of primary human cells exposed to 88% oxygen, 5% CO₂ atmosphere. (A) Native SCGE analysis: > 150 cells from each treatment condition were quantitated under medium power microscopy. *P < 0.001 for comparison to room air and 1 d O₂ samples. LF, lung fibroblasts; HPMEC, pulmonary microvascular endothelial cells; HUVEC, umbilical vein endothelial cells; SAE, small airway epithelial cells. (B) Light microscopy (×400) of Wright-stained cells exposed to humidified 88% oxygen, 5% carbon dioxide atmosphere for 3 d. (C) Fluorescent micrographs (×1,000) of Hoechst 33342–stained SAE cells.

Figure 7.

DNA strand breaks in alveolar cell preparations. (A) Top panels: modified Papanicolou staining. Brown material in positive fraction is the magnetic beads. Bottom panels: histogram analysis of SCGE results for > 100 cells from each fraction, quantitated under ×400 magnification. (B) Mean values from SCGE quantitation of the indicated fractions from three mice. More than 100 cells were quantitated under high power for each sample. Concordant results were obtained in three independent experiments.