Departments of 1Environmental Medicine, 2Pathology and Laboratory Medicine, 3Pediatrics, and 4Radiation Oncology, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642
Submitted 3 November 2003 ; accepted in final form 4 January 2004
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ABSTRACT |
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DNA strand breaks; enhanced green fluorescent protein; lung; mice; oxidized guanine
Although it is evident that differential cell type sensitivities to in vivo hyperoxia exist, in vitro cell line studies have not identified potential mechanisms for these differences. For example, hyperoxia promotes necrosis of human A549 and mouse MLE15 adenocarcinoma cell lines, both of which are thought to be derived from alveolar type II cells (21, 27). It also promotes necrosis of cultured mouse bronchial epithelial cells (26). In contrast, hyperoxia stimulates apoptosis of MLE12 cells, another mouse type II-like cell line related to MLE15 (42). Likewise, hyperoxia promotes apoptosis of RAW 264.7 mouse macrophage cells (32). Two recent studies have suggested necrotic cell death may be mediated in part by activation of apoptotic pathways. One study showed that mouse embryonic fibroblasts derived from bax/bak-deficient mice were resistant to hyperoxia (9). Furthermore, hyperoxia-induced death of Rat1a cells could be blocked by overexpression of Bcl-XL. In another study, adenovirus-mediated expression of Bcl-XL in A549 cells was not found to protect against hyperoxia (36). Instead, overexpression of FLIP, a caspase-8 inhibitor, protected against hyperoxia through a Bax-independent process. These surprisingly distinct responses observed in cultured cell lines make it difficult to extrapolate in vitro findings to specific cells in vivo.
Damage to DNA is one of the most prominent toxic effects of elevated oxygen. The main reaction products of DNA oxidation are base modifications, base loss, and strand breaks. In cultured epithelial cell lines, hyperoxia induces DNA strand breaks and sister chromatid exchanges (14, 33, 41). Although hyperoxia-induced free radical-mediated DNA strand breaks have not been described in the intact lung, hyperoxia induces terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) staining. As pointed out in a recent review (3), it remains unclear whether TUNEL staining during hyperoxia represents apoptosis, free radical-mediated DNA strand breaks, or a combination of both. The observation that some TUNEL-positive cells in hyperoxic newborn rats also had perinuclear 8-hydroxy-2'-deoxyguanosine (8-oxoguanine) staining suggests that some TUNEL staining may reflect free radical-mediated damage and not solely apoptosis (5). Although positively stained cells were not identified, oxidized guanine is one of the most frequent oxidation products and may be detected in nuclear and mitochondrial DNA (6). During replication, oxidized guanine results in mutagenic GC-to-TA transversions.
The complex heterocellular nature of the lung has hampered efforts to identify DNA lesions within individual cell types in vivo. Moreover, it is difficult to localize DNA lesions to specific cell populations, because cell type-specific gene expression declines during exposure. For this reason, we generated a line of transgenic mice that express enhanced green fluorescence protein (EGFP) in type II cells (34). The intrinsic green fluorescence afforded by EGFP, which is not secreted, allows type II cells to be identified and isolated even when surfactant proteins are secreted and depleted during injury. These mice are used in the current study to investigate the genotoxic effects of in vivo exposure to hyperoxia on type II epithelial cells.
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MATERIALS AND METHODS |
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Immunohistochemistry. Lungs were inflation fixed through the trachea for 10 min with 10% neutral-buffered formalin (34). Lobes were dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. We stained paraffin sections (4 µm) for 8-oxoguanine using an OxyDNA assay kit (Calbiochem, San Diego, CA). Briefly, sections were deparaffinized and rehydrated through graded ethanol before being subjected to antigen retrieval by being boiled in 50 mM Tris (pH 9.5) to quench the endogenous EGFP signal. Sections were blocked and incubated overnight with FITC-conjugated 8-oxoguanine binding protein at 4°C. For detection of pro-SP-C, we incubated sections overnight with rabbit anti-pro-SP-C sera (Chemicon International, Temecula, CA) after blocking endogenous biotin with avidin/biotin (Vector Laboratories, Burlingame, CA). Pro-SP-C signal was amplified with a TSA Biotin kit (Perkin Elmer, Boston, MA) and detected with a streptavidin-conjugated Texas-red fluorophore. EGFP was detected by incubation overnight with goat anti-EGFP antibodies conjugated to FITC or Texas red dye (Novus Biologicals, Littleton, CO). We detected mitochondria by incubating sections overnight with an Alexa-594-conjugated mouse anticytochrome oxidase (COX) subunit 1 antibody (Molecular Probes, Eugene, OR). In situ DNA strand breaks were detected with a commercially available TUNEL assay kit according to the manufacturer's protocol (Chemicon). Sections were washed extensively, immersed in 4',6'-diamidino-2-phenylindole (DAPI), and visualized with a Nikon E800 fluorescence microscope (Nikon, Melville, NY). Images were captured with a SPOT-RT digital camera (Diagnostic Instruments, Sterling Heights, MI). For dual localization of 8-oxoguanine and mitochondria, sections were scanned with a Leica TCS SP Spectral Confocal microscope equipped with an upright DMRXE (Leica Microsystems, Mannheim, Germany).
Quantitative immunohistochemistry. Images of random, noncontiguous fields of parenchyma were acquired with a Nikon E-800 microscope and a SPOT RT camera. Regions were selected under DAPI fluorescence to prevent bias toward fields with FITC or Texas red signals. Five fields per lung were obtained from at least three separate animals for each treatment. Fields that contained a large airway or blood vessel were rejected. Different fluorescent filters were used to acquire images of each field displaying all nuclei (DAPI), pro-SP-C (Texas red), EGFP (Texas red), or 8-oxoguanine (FITC). Images were merged to identify cells that were both green and red. Quantification was performed using Metamorph software (Universal Imaging, Downingtown, PA). Metamorph was configured to measure total nuclei based on the average area of a nucleus. For 8-oxoguanine staining, a total of 30 fields containing 9091,368 nuclei (1,144.7 ± 119.8, mean ± SD) were counted under a x20 objective. Pro-SP-C-, EGFP-, and 8-oxoguanine-positive cells were counted manually with Metamorph to mark the counted cells. For each animal, the counts from all fields were summed, and the following ratios determined: 8-oxoguanine-positive/total cells; 8-oxoguanine-positive/pro-SP-C-positive; 8-oxoguanine-positive/EGFP-positive cells; and 8-oxoguanine-positive/total cells that were not EGFP positive. The ratios for all animals at each time point were averaged and graphed as means ± SE.
Isolation of type II cells by fluorescence-activated cell sorting. Epithelial cells were dissociated by instilling perfused lungs with Dispase followed by low-melt agarose (34). Cell suspensions were successively filtered through 100-µm and 40-µm cell strainers and finally through 25-µm nylon gauze. Single cell suspensions were then pelleted by centrifugation at 300 g for 10 min at 4°C. Cells obtained from transgenic mice (10 per experiment) were pooled and resuspended in 10 ml of DMEM with 0.5% FBS and 25 mM HEPES media. Green fluorescent (EGFP) type II cells from dissociated lung tissues were isolated with the B-D FACSVantage SE cell sorter (Becton Dickinson Immunocytometer Systems, Palo Alto, CA). Cellular EGFP was excited by an argon ion laser emitted at the wave-length of 488 nm, and the fluorescence was collected after a 530 ± 30-nm band pass filter. A two-parameter sorting window (forward light scattering and EGFP fluorescent intensity) was used to define the EGFP-positive cell populations. The cells were sorted through a flow chamber with a 80-µm nozzle tip under 12 psi of sheath fluid pressure. The sorted cells were collected into 15-ml conical tubes filled with sterile media for morphological and biochemical assays.
Cell viability. DNA content and cell viability were measured using propidium iodide (PI) to stain the DNA. Viability was measured in freshly isolated cells that were stained for 15 min with PI (2 µg/ml). Stained samples were then analyzed by flow cytometry for PI intensity. Highly positive cells were counted as dead because they had incorporated the dye, indicating that the cell membrane had been compromised. For DNA content measurement, samples were fixed overnight in 75% ethanol and treated with RNase (1 mg/ml) for 30 min. Samples were stained with PI (20 µg/ml) for 15 min before analysis by flow cytometry.
Ultrastructural morphology of isolated cells. Isolated cells were cytospun onto glass slides, fixed in a 2.5% phosphate-buffered glutaraldehyde, pH 7.4, and postfixed in 1.0% phosphate-buffered osmium tetroxide. The slides were dehydrated in ethanol to 100%, infiltrated with liquid Spurr epoxy resin, and embedded on the glass surface with inverted capsular molds containing fresh resin. After polymerization at 70°C, the hardened capsules were then "popped off" the glass slide by immersion into liquid nitrogen. The popped-off capsules were examined with a light microscope, and 70-nm-thin sections were prepared onto 200-mesh copper grids with a diamond knife. The grids were contrasted with aqueous uranyl acetate for 10 min, lead citrate for 15 min, examined, and photographed under a Hitachi 7100 transmission electron microscope.
Comet assay. After exposure to room air or hyperoxia, lungs from three mice per exposure per experiment were dissociated and flow sorted as described above. EGFP-positive cells were pelleted by centrifugation at 300 g for 10 min at 4°C. The cells were resuspended at 5 x 105 cells/ml in media, and 20 µl (10,000 cells) were diluted in 100 µl of 0.75% low-melt agarose. The mixture was gently vortexed and pipetted onto Superfrost plus micro slides (VWR, West Chester, PA) that had been precoated with 1.0% agarose. After polymerization, 150 µl of 1.0% agarose were overlaid onto the cells and polymerized for 15 min at room temperature. The slides were immersed for 1 h in ice-cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10, and 0.05% Triton X-100). Slides were then submerged in an electrophoresis tank containing fresh alkaline-EDTA (300 mM NaOH and 0.01 mM EDTA, pH 12.9) and electrophoresed for 40 min at 25 V (1.0 V/cm) at 4°C. We neutralized slides by washing them three times in 0.4 M Tris, pH 7.5, for 5 min each, rinsing them with distilled water, and staining them with 50 µl of 1x Sybr gold (Trevigen, Gaithersburg, MD). Individual cell DNA was visualized under a Nikon E800 fluorescence microscope. Images were captured with a SPOT-RT digital camera. Metamorph analysis software was used to measure tail moment {(TM) = tail length x [DNA in tail/(total DNA in head + tail)]} in 50 cells from each treatment group per experiment (30).
Statistical analysis. Values are typically expressed as means ± SD. Where appropriate, group means are expressed as means ± SE. Group means were compared by ANOVA using Fisher's procedure post hoc analysis, and P < 0.05 was considered significant.
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RESULTS |
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Because mitochondria are abundant in type II epithelial cells, we colocalized 8-oxoguanine to type II cells by staining sections with Texas red-conjugated anti-pro-SP-C sera. In room air-exposed lungs, green 8-oxoguanine occasionally overlapped with red pro-SP-C staining to create a faint yellow color (Fig. 2A). After 48 h of exposure, a strong yellow color was observed, caused by the intense green 8-oxoguanine staining overlapping with the red pro-SP-C (Fig. 2C). By 72 h of exposure, red pro-SP-C staining was markedly diminished, similar to that previously reported for pro-SP-B (Fig. 2E) (39). In contrast, green 8-oxoguanine stained cells were still readily detected. Because pro-SP-C staining declined at this time, it was not possible to localize 8-oxoguanine to type II cells with pro-SP-C antibody. However, type II cells could still be identified based on their transgenic expression of EGFP, which we identified by staining sections with a Texas red-conjugated anti-EGFP antibody. In lungs exposed to room air, green 8-oxoguanine was observed in some red EGFP cells (Fig. 2B). After 48 and 72 h of hyperoxia, 8-oxoguanine was detected in many EGFP labeled cells (Fig. 2, D and F).
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The proportion of 8-oxoguanine-positive cells in the alveoli was quantified by image analysis. Images that contained airway epithelium were excluded from this analysis. In room air-exposed lungs, 8-oxoguanine was detected in 4% of alveolar cells (Fig. 3A). Hyperoxia increased the proportion of all alveolar cells with 8-oxoguanine staining approximately threefold. Remarkably, nearly 30% of room air-exposed type II cells exhibited low but detectable levels of 8-oxoguanine that increased to >60% by 48 h of hyperoxia (Fig. 3B). Similarly, 8-oxoguanine was detected in 25% of EGFP-expressing type II cells and increased to >60% by 48 and 72 h of exposure (Fig. 3C). The proportion of 8-oxoguanine-positive cells that did not express EGFP was also determined. Relative to the total number of cells shown in Fig. 3A, 8-oxoguanine staining was mostly detected in non-EGFP-expressing alveolar cells and was increased by oxygen (Fig. 3D). This shows that 8-oxoguanine staining is not a function of transgenic expression of EGFP.
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The punctate and perinuclear fluorescence staining of 8-oxoguanine suggests that this lesion was present in mitochondria. This was confirmed by staining sections with an Alexa-594-conjugated mouse anti-COX subunit 1 antibody and the 8-oxoguanine binding protein. By confocal microscopy, green 8-oxoguanine fluorescence in type II cells colocalized with red COX fluorescence, indicating oxidation of mitochondrial DNA (Fig. 4).
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Isolation of type II cells from lungs. Dissociated lung cells obtained from 10 mice exposed to room air or hyperoxia were pooled, and type II cells were isolated on the basis of their intrinsic green fluorescence by fluorescence-activated cell sorting procedures. Approximately 20 million cells were sorted from each experiment, of which 1.3 ± 0.6% (mean ± SD, n = 11 sorts) were saved from room air-exposed lungs and 2.1 ± 0.5% (n = 5 sorts) were saved from hyperoxic lungs. Although unknown, the increased percentage of type II cells isolated from hyperoxic lungs (P = 0.04) might be attributed to enrichment as endothelial and type I cells died. Type II cells isolated from mice exposed to room air contained intact nuclear and cytoplasmic membranes along with abundant lamellar bodies characteristic of this cell type (Fig. 5). Type II cells isolated from mice exposed to hyperoxia contained intact nuclei that appeared indistinguishable from control cells. Darkly stained glycogen deposits in lamellar bodies and some swelling of mitochondria were the only morphological differences observed in cells exposed to hyperoxia.
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Flow cytometry was used to assess DNA content in the isolated type II cells. DNA histograms revealed that most type II cells isolated from mice exposed to room air or hyperoxia were in G1 with a small population in G2 (Fig. 6). Apoptosis, as defined by sub-G1 DNA content, was observed in 2.2% of cells isolated from room air-exposed lungs. Although hyperoxia increased the number of apoptotic cells to 5.8%, it was not statistically different from room air values (P = 0.09). Cell necrosis was assessed by exclusion of PI. The viability of type II cells isolated from hyperoxic lungs was 93.1 ± 0.8% (mean ± SD, n = 3), which was not significantly different from that of cells isolated from room air lungs (P = 0.17) (34). Thus most type II cells isolated from mice exposed to room air or hyperoxia are viable and display little evidence of apoptosis or necrosis.
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Hyperoxia induces DNA strand breaks in type II cells. DNA strand breaks were assessed by the comet assay, which detects alkaline-sensitive sites, single and double strand breaks, and incomplete base excision repair activity. Cells isolated from room air-exposed lungs exhibited intact nuclear DNA that failed to form a tail during electrophoresis (Fig. 7). In contrast, increasing tail length and intensity were observed in cells isolated from mice exposed to hyperoxia for 72 h. TM was quantified in 50 cells isolated from two different experiments and graphed. Because TM is dependent on electrophoresis times and agarose concentrations, data from two separate experiments are shown. Type II cells isolated from room air-exposed lungs exhibited a mean TM of 5, whereas cells isolated from hyperoxia-exposed lungs exhibited values of 35 and 65.
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TUNEL staining was used to confirm that hyperoxia causes DNA strand breaks in type II cells. TUNEL-positive cells were not readily detected in lungs exposed to room air (Fig. 8). In contrast, brown TUNEL-positive nuclei were abundantly detected in lungs exposed to hyperoxia. Stained sections were then immunostained with an FITC-conjugated anti-EGFP antibody. Under high power, green fluorescence was observed in hyperoxic cells with brown TUNEL staining. These dual positive cells did not exhibit pyknotic nuclei, characteristic of apoptosis. Thus the comet assay and TUNEL staining reveal that hyperoxia induces DNA strand breaks in a population of type II cells.
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DISCUSSION |
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Although direct exposure of DNA to oxygen is not genotoxic (12), DNA within the cell is damaged through production of cytotoxic ROS. An important new observation in the current study is that punctate cytoplasmic oxidized guanine staining was detected in virtually all airway epithelial and nearly 70% of alveolar type II epithelial cells. Oxidized guanine colocalized to mitochondria, whose DNA is three times more susceptible to oxidation than nuclear DNA (25). The lack of staining in nuclei may reflect less damage or less accessibility to the binding protein used to visualize lesions. Our findings are consistent with a previous report showing hyperoxia increased punctate 8-oxoguanine immunostaining in newborn rats (5). A recent study showing that in vivo hyperoxia promotes cytochrome c release in type II cells supports the concept that mitochondria become damaged during exposure (31). Significant damage to mitochondria will compromise cellular respiration, resulting in elevated levels of ROS that cause further damage to mitochondria and nuclear DNA. Indeed, the comet assay and TUNEL staining revealed that hyperoxia induces nuclear DNA strand breaks in a population of type II cells. Continuous attack on the DNA helix, such as during several days of hyperoxia, may cause double strand breaks. In cell lines, clastogenicity is one of the most prominent genotoxic effects of hyperoxia and has been argued to be a major contributor to cytotoxicity (13, 15). To our knowledge, this is the first study to show that in vivo hyperoxia causes mitochondrial and nuclear DNA damage in a specific lung cell population.
Another important finding in this study is that type II cells freshly isolated from hyperoxic lungs exhibit DNA damage without loss of cell viability. It does not appear that the flow sorting procedure preferentially selected for viable cells, because more type II cells were actually obtained from lungs exposed to hyperoxia compared with room air. Although morphological signs of necrotic endothelial and type I epithelial cells have been documented in monkeys, rats, or mice exposed to hyperoxia for the same length of time, type II cell necrosis or apoptosis was not observed (2, 11, 20). Similarly, apoptosis was not observed in type II cells freshly isolated from rats exposed to hyperoxia (7). However, these cells were damaged because they became apoptotic when cultured ex vivo for 48 h, whereas cells isolated and cultured from rats exposed to room air did not. Treating the cultured cells with keratinocyte growth factor or plating on Matrigel enhanced survival and reduced expression of the tumor suppressor protein p53, which increases when DNA is damaged. Matrix-dependent survival of injured type II cells also required ERK activation (8). These findings suggest that integrin-dependent interactions with extracellular matrix may dictate whether type II cells repair oxygen-induced DNA strand breaks or activate the apoptotic machinery. This ability to effect repair or apoptosis may explain why hyperoxia killed SV40-transformed MLE12 mouse type II cells by apoptosis, whereas MLE15 cells, another SV40-transformed line of mouse type II cells, died primarily by necrosis (27, 42). Although unproven, the transformed nature of these individual lines may have altered their interactions with extracellular matrix or affected other signal transduction pathways. The observation that ERK activation inhibited apoptosis of freshly isolated rat type II cells while stimulating apoptosis of MLE12 cells is consistent with this hypothesis (8, 42). ERK activation is, however, not a universal response to hyperoxia, because it was not detected in A549 cells but was in mouse LC4 cells (19). Because type II cells proliferate and differentiate into type I cells during recovery in room air (1), we speculate that they activate signal transduction pathways that promote repair over apoptosis. If true, type II cells may induce a series of programmed cell life genes such as those patterned by the antioxidant response element (22).
Unlike type II cells that survive, endothelial and type I cells die during hyperoxia. These cells are exposed to the same oxidant stress as type II cells but succumb to the cumulative damage. One hypothesis is that endothelial and type I epithelial cells may express less antioxidant enzymes and therefore accumulate more oxidant damage over time than type II cells. At this time, we do not have any evidence that endothelial cells accumulate more 8-oxoguanine lesions during hyperoxia, because these cells are difficult to quantify within intact tissues by methods described in the current study to count type II cells. Although expression of antioxidants may contribute to cell type resistance, transgenic knockout mice are at best modestly sensitive to hyperoxia (17). This suggests that no single antioxidant by itself is sufficient to protect lungs. An alternative hypothesis is that dying cells develop the same amount of damage as type II cells but initiate programmed cell death. The observation that antiapoptotic Bcl-2 expression is higher in IL-6 transgenic mice, which are resistant to hyperoxia, supports the latter hypothesis (37). Similarly, this hypothesis is supported by studies showing that Bax/Bak-deficient or Bid-deficient mice are slightly more resistant to hyperoxia (9, 36). Together, these studies suggest that antioxidant enzymes, apoptotic processes, and cell survival/repair pathways all contribute to the differential cell type sensitivity to hyperoxia.
Previous studies in mice and rats revealed that hyperoxia increases TUNEL staining in the bronchiolar epithelium and nearly 60% of alveolar cells (24, 29, 38). To date, TUNEL-positive cells have never been formally identified with molecular markers to specific cell types. Because TUNEL requires the presence of a free 3'-hydroxyl, it can stain DNA undergoing fragmentation due to damage, apoptosis, necrosis, repair, or replication (4, 16). The current finding that viable EGFP-positive type II cells exhibited DNA strand breaks by comet assay and TUNEL staining is consistent with the hypothesis that some TUNEL staining reflects oxidative DNA strand breaks. Attempts to colocalize pro-SP-C to TUNEL-positive cells were unsuccessful, in part because the antigen retrieval method used to detect pro-SP-C increased background staining in room air control tissues (data not shown). Like type II cells, airway epithelial cells exhibit 8-oxoguanine and TUNEL staining during hyperoxia. Although unproven, airway epithelial cells are likely to be damaged rather than undergo cell death because significant proliferation during recovery in room air is not observed (35). As pointed out recently (3), our findings suggest that TUNEL staining by itself is not an accurate indicator of apoptosis but can be used to confirm the presence of DNA strand breaks associated with damage or apoptosis.
There are several limitations to this study. Although hyperoxia increased 8-oxoguanine staining in airway and type II epithelial cells, it remains unclear whether increased signal in these cells is due to mitochondrial content or more oxidation per mitochondria. Although type II cells are rich in mitochondria, the observation that 8-oxoguanine was detected in non-type II alveolar cells suggests that staining is not solely reflective of mitochondrial content. Because DNA becomes oxidized during extraction, it would be difficult to quantify 8-oxoguanine lesions between different cell types by currently available methods (6). Another limitation of the study is that DNA strand breaks were measured in those type II cells that also expressed EGFP. It is unlikely that EGFP is expressed by a unique subpopulation of type II cells because they express SP-A, SP-B, and SP-C, as well as exhibit morphological characteristics of type II cells (34). Moreover, hyperoxia decreased pro-SP-C staining in both EGFP-positive and -negative type II cells. It remains unclear why hyperoxia does not decrease EGFP expression, when the GATA-6 and thyroid transcription factor-1 regulatory elements that control SP-C expression are contained with the 3.7-kb promoter used to target EGFP to type II cells (23). Because EGFP is not secreted, we speculate that it may be retained longer in type II cells than pro-SP-C. This is currently being investigated. The finding that 8-oxoguanine increased in 70% of type II cells as characterized by their expression of pro-SP-C or EGFP suggests that type II cells expressing EGFP are not uniquely responsive to hyperoxia. Conversely, DNA damage was not detected in all pro-SP-C- or EGFP-positive cells. Thus type II cells exhibit a range of damage that does not appear to be dependent on their ability to express pro-SP-C or EGFP. Finally, our findings are consistent with Buckley et al. (7), who showed that type II cells freshly isolated from hyperoxic rats are viable. Together, the genotoxic effects of hyperoxia on type II cells expressing EGFP appear to be representative of those occurring in all type II epithelial cells.
In summary, this study demonstrates that type II cells exposed to hyperoxia in vivo exhibit oxidized guanine and DNA strand breaks. Oxidized and fragmented DNA, unless repaired, is potentially mutagenic or lethal. It is highly likely that type II cells repair oxygen-induced lesions, because hyperoxia is not mutagenic, and overt death of type II cells before mortality remains to be documented. Our findings suggest that a better understanding of how damaged DNA is recognized and repaired may have therapeutic potential for reducing oxidant lung injury.
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ACKNOWLEDGMENTS |
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The University of Rochester's Electron Microscopy Core performed the ultrastructural analyses on isolated type II cells.
GRANTS
This work was funded in part by National Heart, Lung, and Blood Institute Grants HL-58774 and HL-67392 (M. A. O'Reilly). National Institutes of Health training grant ES-07026 and HL-66988 supported J. M. Roper. The Flow cytometry, Pathology/Morphology and Imaging, and Animal Inhalation facilities are supported in part by National Institute of Environmental Health Sciences Center Grant ES-01247.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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