Divisions of 1 Pulmonary Medicine and 3 Neurology, Department of Medicine, and 2 Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Because carbon monoxide (CO) has been proposed to have anti-inflammatory properties, we sought protective effects of CO in pulmonary O2 toxicity, which leads rapidly to lung inflammation and respiratory failure. Based on published studies, we hypothesized that CO protects the lung against O2 by selectively increasing expression of antioxidant enzymes, thereby decreasing oxidative injury and inflammation. Rats exposed to O2 with or without CO [50-500 parts/million (ppm)] for 60 h were compared for lung wet-to-dry weight ratio (W/D), pleural fluid volume, myeloperoxidase (MPO) activity, histology, expression of heme oxygenase-1 (HO-1), and manganese superoxide dismutase (Mn SOD) proteins. The brains were evaluated for histological evidence of damage from CO. In O2-exposed animals, lung W/D increased from 4.8 in normal rats to 6.3; however, only CO at 200 and 500 ppm decreased W/D significantly (to 5.9) during O2 exposure. Large volumes of pleural fluid accumulated in all rats, with no significant CO treatment effect. Lung MPO values increased after O2 and were not attenuated by CO treatment. CO did not enhance lung expression of oxidant-responsive proteins Mn SOD and HO-1. Animals receiving O2 and CO at 200 or 500 ppm showed significant apoptotic cell death in the cortex and hippocampus by immunochemical staining. Thus significant protection by CO against O2-induced lung injury could not be confirmed in rats, even at CO concentrations associated with apoptosis in the brain.
oxidative stress; reactive oxygen species; oxygen toxicity; heme oxygenase
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INTRODUCTION |
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HYPEROXIA GENERATES reactive oxygen species (ROS), e.g., superoxide anion and hydrogen peroxide (H2O2), which can injure the lung (2, 9). Prolonged exposure to 100% O2 diffusely damages lung capillary epithelium and endothelium and causes extensive inflammatory cell infiltration and interstitial and intra-alveolar edema (5, 7, 8, 12). Rats exposed continuously to 100% O2 die of respiratory failure after 66-78 h (6).
The lung is protected against O2 by endogenous antioxidant enzymes such as superoxide dismutases (SOD) and catalase (9, 12, 23, 26). Lung mitochondria are particularly susceptible to oxidative damage (22), and the mitochondrial isoform of SOD [manganese SOD (Mn SOD)] is a major defense against oxidative lung injury (12). Mn SOD overexpression can ameliorate oxidative lung injury by helping restore cellular oxidant-antioxidant balance and preventing damage from excessive molecular oxidation. Pulmonary O2 tolerance produced by bacterial endotoxin (10) increases Mn SOD expression (6). Also, recombinant human Mn SOD given to baboons in hyperoxia preserves pulmonary gas exchange and decreases pulmonary edema (25).
O2 tolerance in rats develops during exposure to 80% O2 (4), which correlates with increased expression of Mn SOD in the lung. After several days under these conditions, rats survive indefinitely in 100% O2. O2-tolerant rats show less oxidative damage and increased expression of Mn SOD mRNA and protein, particularly in type II cells and interstitial fibroblasts (4, 7). These findings suggest that Mn SOD is crucial in protecting the lung against oxidative stress. Indeed, transgenic mice that overexpress Mn SOD are highly resistant to pulmonary O2 toxicity (30).
Hyperoxia also increases expression of the antioxidant enzyme heme oxygenase (HO)-1 (16), which converts heme to biliverdin and catalytically releases iron and carbon monoxide (CO; see Ref. 28). The HO-1 isoform (18) is a stress protein regulated by not only heme but by endotoxin, cytokines, metals, and glutathione depletion (1, 3, 14, 15). HO-1 protects cells in part because free heme is toxic and biliverdin is an antioxidant. Overexpression of HO-1 in the rat lung by enzyme induction or transfection can protect from O2 (19, 27); however, the protective mechanism is unknown and persists after enzyme inhibition (27).
Recently, CO has been proposed to have anti-inflammatory and/or antioxidant properties (20). CO has been reported to protect against pulmonary O2 toxicity in rats when given continuously during 100% O2 exposure (20). If so, lungs exposed to elevated O2 concentrations might be protected by inspired CO. Such effects could be derived from CO binding to reduced iron in cellular hemoproteins, e.g., in mitochondria, thereby altering electron flow and oxidative stress responses. Also, iron binding by CO could inhibit oxidant generation by preventing iron redox cycling.
This study was designed to investigate a possible protective mechanism of CO on O2 toxicity based on changes in Mn SOD expression in the lung. If CO interacts with reduced iron, e.g., in mitochondrial complex IV, ROS production could be altered, e.g., at complex I, perhaps inducing Mn SOD expression and enhancing resistance to further oxidative stress. We tested this hypothesis in rats exposed to O2 or O2 plus CO by comparing the extent of lung injury with Mn SOD and HO-1 expression. In addition, the brain, which is particularly sensitive to intracellular effects of CO, was examined for pathological effects of the exposures.
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MATERIALS AND METHODS |
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Materials. Adult male Sprague-Dawley rats (300-400 g) obtained from Charles River Laboratories (Wilmington, MA) were used in the experiments. The equipment included a Powergen homogenizer purchased from Omni International (Marietta, GA), a Branson sonifier purchased from Branson Ultrasonics (Danbury, CT), a model U2000 UV/Vis spectrophotometer made by Hitachi Instruments (Tokyo, Japan), a Minigel electrophoresis unit and power supply made by Hoefer Scientific Instruments (San Francisco, CA), a Bio-Rad gel densitometer (Hercules, CA), and an Optiphot-2 microscope, HFX-IIA photomicrographic attachment, and FX-35DX camera purchased from Nikon (Garden City, NY). For Western blots, polyvinylidene membranes were purchased from Millipore (Bedford, MA), and Hyperfilm was from Amersham Life Science (Arlington Heights, IL). The antibodies were rat Mn SOD and HO-1 (SPA 896) purchased from StressGen (Vancouver, BC) and horseradish peroxidase-labeled goat anti-rabbit secondary antibody purchased from Jackson ImmunoResearch (West Grove, PA). Anti-single-strand DNA (ssDNA) monoclonal antibody was obtained from Chemicon International (Temecula, CA). Chemicals included Tris · HCl, SDS, and polyacrylamide purchased from Bio-Rad, paraformaldehyde purchased from Stephens Scientific (Riverdale, NJ), halothane purchased from Halocarbon Laboratories (North Augusta, SC), and methanol purchased from EM Science (Gibbstown, NJ). All other chemicals were purchased from Sigma Chemical (St. Louis, MO).
Experimental design. The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee at Duke University. To determine the effects of CO on exposure to hyperoxia, rats were placed in gastight Plexiglas chambers and exposed continuously to 100% O2 or O2 plus one of four different concentrations [50, 100, 200, or 500 parts/million (ppm)] of CO for 60 h. A uniform 60-h O2 exposure was chosen because it produces severe, diffuse, and irreversible lung injury in rats before significant loss of life (7, 27). In the initial study, eight rats were exposed to O2 in each of the CO treatment groups. In six animals exposed to O2 and six exposed to O2 plus 500 ppm CO, arterial blood gases and pH were measured at 60 h. For each CO treatment group, two to eight control rats were exposed to 100% O2 at the same time. Based on results of the initial study, three groups of animals were exposed in a separate experiment to O2 or O2 plus 200 or 500 ppm CO for 84 h to determine if CO could produce O2 tolerance.
The rats were given free access to food and water throughout the exposures. The temperature in the cages was maintained at 22-23°C and relative humidity at 50-70%. One time per day, a collection shelf beneath the cages was opened briefly to replace the bedding. Rats for air control studies were maintained in cages nearby under similar conditions. Other rats exposed to CO in air (50 or 100 ppm) for 3 days as part of different experiments were used for reference measurements for the CO exposures in this study.Tissue collection.
After 60 h of exposure, the rats were anesthetized with halothane,
the chest and peritoneal cavities were opened carefully, the volume of
pleural fluid was measured, and blood was drawn from the left ventricle
to measure arterial carboxyhemoglobin (COHb) levels. The great vessels
were transected in the abdomen, and the lungs were removed en bloc and
drained of blood. The lungs were dissected from the hilar structures
and blotted gently on moist gauze. One lung was cut in half, weighed
immediately (wet weight), and placed in a vacuum oven. The remaining
lung tissue was snap-frozen in liquid nitrogen and stored at 80°C
for protein analysis, myeloperoxidase (MPO) assay, and Western blot analysis.
O2 and CO exposures. The concentrations of O2 and CO in the chambers were monitored continuously during the exposures. Control rats breathed either room air or 100% O2 continuously for 60 h. The gas flow through the cages was ~12 standard l/min, which was sufficient to allow complete gas exchange every 5 min. The atmosphere in the cages was sampled continuously near the chamber exhaust outlet, and the O2 concentration was monitored with a Servomex O2 analyzer and recorded on a strip chart. O2 levels were maintained above 98% at all times in the cages of the O2 control animals. For the treatments, 1% CO in 50% O2 (balance N2) was bled into the cages until the desired CO concentration was reached and a steady state was achieved. The CO concentration also was monitored continuously using a calibrated infrared CO detector (Snifit model 50; Bacharach, Pittsburgh, PA) and was recorded every 8 h. The CO concentration was maintained within ±10 ppm of the desired level. The O2 concentration in the cages of animals receiving CO was maintained at or above 98%, except at 500 ppm CO, where the O2 remained at ~97% throughout the exposures because of dilution of O2 with N2 from the CO carrier gas.
Arterial COHb, blood gases, and pH. COHb values were measured using a CO-oximeter calibrated with an algorithm for rat blood (IL model 482). Samples of arterial blood were drawn from all animals for these measurements at the end of the exposures. Arterial blood gases and pH also were measured in six anesthetized animals after 60 h of O2 and in six animals after 60 h of O2 plus 500 ppm CO (IL model 1304 blood gas/pH analyzer).
Wet-to-dry weight ratio. At the end of each experiment, total lung water was assessed using the lung wet-to-dry weight ratio. One-half of one lung from each animal prepared as described above was weighed immediately on preweighed foil to determine wet weight and was placed in a vacuum oven at 60°F for at least 72 h or until a stable dry weight was obtained. The wet and dry tissue values were used to determine the wet-to-dry weight ratio. No corrections for intravascular lung water were made.
Western blots.
Frozen lung tissue was homogenized at 4°C three times for 15 s
in lysis buffer [50 mM Tris, pH 7.6, 3% Nonidet P-40, 150 mM NaCl, 1 mM MgCl2, 5 mM EDTA, 2 mM 1,10-phenanthroline, 2 mM
3,4-dichloroisocoumarin, and 0.4 mM
trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane] and centrifuged at 10,000 g for 10 min at 4°C. The
supernatants were recovered, 100-µl aliquots were stored at 80°C
for protein assay, and the remainder was diluted 1:1 with Laemmli
sample buffer and
-mercaptoethanol (95:5) and frozen at
80°C.
The proteins were separated later using PAGE and a 12% resolving gel.
For all gels, 15 µg of protein were loaded in each lane, and
electrophoresis was run at 250 volts for 60-90 min. Gels were
transferred to polyvinylidene fluoride membranes using a Hoefer TE
series transfer apparatus. The membranes were blocked overnight at
4°C in a solution of 3% milk in 100 mM Tris, 1,000 mM NaCl, and 1%
Tween 20, pH 7.5 (TBS-T). The next day, membranes were washed for 30 min in five changes of TBS-T. The blots were placed in primary antibody
(1:5,000 Mn SOD or 1:1,000 HO-1) in a 3% milk/TBS-T solution
for 1 h at room temperature. The membranes were washed for 30 min
in five changes of TBS-T and were placed in a secondary antibody
solution of goat anti-rabbit IgG (1:10,000) in a 3% milk/TBS-T
solution for 1 h at room temperature. The membranes were washed
again for 30 min in TBS-T, and the blots were developed in enhanced
chemiluminescence solution and exposed to Hyperfilm to visualize the
proteins. The relative density of the bands on the gels was compared
using a scanning densitometry system (Bio-Rad).
Protein assay. Protein concentrations in lung tissue homogenates were determined using the assay of Lowry et al. (17). Protein loading for PAGE was checked after protein transfer by Coomassie blue staining.
Lung MPO content.
An MPO enzyme activity assay was used as reported to estimate the
neutrophil content of lung tissue. Cold PBS was added to frozen lung at
a ratio of 3 ml/600 mg tissue, and the tissue was homogenized at 4°C.
From each homogenate, a 250-µl aliquot was removed and diluted to 900 µl using 50 mM phosphate buffer at 4°C. One-half of the sample was
vigorously mixed with an equal volume of 1% hexadecyltrimethylammonium
bromide solution. Samples were frozen at 80°C and then thawed and
sonified for 10 pulses using a Branson ultrasonifier at 20% power. The
samples were frozen and thawed three times, homogenized again for one
15-s pulse each, and centrifuged at 15,000 g for 20 min at
4°C. For the assay, o-dianisidine was mixed in the dark
with 50 mM phosphate buffer to achieve a concentration of 0.167 mg/ml.
An aliquot of supernatant was mixed with 2.9 ml of
o-dianisidine solution and 5 µl of fresh 0.3%
H2O2 diluted from 10% stock. The samples were
read against a buffer blank for 5 min at 460 nm on a spectrophotometer.
MPO values were reported as change in optical density per minute per gram of wet lung.
Tissue histology. Inflation-fixed lung tissue was embedded in paraffin and cut into 6-µm sections for light microscopy. The sections were stained with hematoxylin and eosin (H&E) and examined for tissue damage. The perfusion-fixed brains were cut into 10-µm sections in the coronal plane and stained with H&E. The cortex, hippocampus, cerebellum, and basal ganglia were identified and examined for evidence of cellular injury. Regions of the brain that showed injury in animals receiving O2 plus 200 or 500 ppm CO were compared with sections of the same brain regions of air control and O2-exposed animals for evidence of apoptosis. An immunohistochemical method for detecting ssDNA was used as a sensitive and specific cellular marker for early apoptosis. The number of ssDNA-positive cells in the hippocampus and frontal parietal cortex were recorded per 1,000 nuclei. At least 1,000 nuclei were counted in each region.
Staining for ssDNA with monoclonal antibody differentiates more clearly than other immunochemical stains, e.g., TdT-UTP nick end labeling, between apoptotic and necrotic mechanisms of cell death and detects apoptosis before frank internucleosomal DNA fragmentation (11). The ssDNA stains were performed on deparaffinized sections of brain tissue incubated in 60°C hot (50%) formamide for 30 min, washed in PBS, and stained with monoclonal antibody against ssDNA at a dilution of 1:100. Cell nuclei containing ssDNA-positive material were identified by light microscopy and were counted in each of the two brain regions.Statistical analysis. The results of measurements in each group are expressed as mean values ± SE. Statistical analyses were performed with commercially available computer software (Statview, Calabasas, CA) using a factorial ANOVA and Fisher's post hoc comparison test. Statistical differences were considered significant for P values < 0.05.
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RESULTS |
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COHb and arterial blood gas measurements.
To assess the adequacy of the CO exposures, blood COHb levels were
measured at the end of each 60-h experiment in rats exposed to
hyperoxia alone (O2 controls) and rats exposed to
O2 plus CO at 50, 100, 200, or 500 ppm. These data are
presented in Fig. 1. Figure 1 shows a
mean COHb level for O2 control rats of 0.5 ± 0.1%.
In rats exposed to CO during hyperoxia, blood COHb increased in
proportion to the level of CO in the inspired gas. The relationship between the increase in COHb and level of CO exposure agreed with predicted calculations for a steady state using the Haldane
relationship and inspired PO2. These data
confirm uptake of CO by the animals in the exposure chambers and
provide evidence of a lack of hypoxemia at the end of the experiments.
Arterial blood gas measurements at 60 h showed that the rats had
adequate arterial oxygenation [PO2 in
O2 359 ± 64 vs. 372 ± 80 mmHg in
O2 + 500 ppm CO, P = not significant
(NS)] but had developed a respiratory acidosis (67 ± 7 vs.
52 ± 9 mmHg, P = NS). Although this difference in PCO2 between O2- and O2
plus CO-exposed animals did not achieve statistical significance, a
trend toward higher pH in the animals treated with CO (7.20 ± 0.05 vs. 7.33 ± 0.05, P = 0.08) raises the
possibility that CO had stimulated pulmonary ventilation during hyperoxia.
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Lung Mn SOD and HO-1 expression.
Western blot analyses were performed on whole lung homogenates to
determine whether CO exposure has an effect on the
O2-induced expression of either antioxidant enzyme. Figure
2 shows a representative Western blot of
Mn SOD expression in lung tissue of CO-treated and untreated
rats (0-500 ppm) after 60 h. The results of densitometry indicate that O2 exposure significantly increased Mn
SOD expression relative to air control animals (P < 0.05). Mn SOD induction was still detectable at 50 ppm CO
but was no longer statistically significant in the lungs of rats
treated with 100, 200, or 500 ppm CO during hyperoxia. Figure
3 shows a representative blot of HO-1
protein expression in air control rats and rats exposed to
O2 or O2 at different concentrations of CO. By
densitometry, lung HO-1 expression increased in O2 controls
compared with air control rats (P < 0.05). This
increase was sustained at 50 and 100 ppm CO but not at 200 or 500 ppm.
Thus addition of CO at 50-500 ppm did not significantly increase
either Mn SOD or HO-1 expression relative to that in
O2 control rats.
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Pleural fluid volume and lung edema.
To evaluate the extent of O2-induced lung damage, pleural
fluid volume and the wet-to-dry weight ratio were measured after 60 h of exposure to O2 or O2 plus CO.
Pleural fluid volume and the wet-to-dry ratio increased significantly
in rats exposed to O2 or O2 plus CO. These
effects were the result of hyperoxic injury because 50 and 100 ppm CO
in air for 3 days does not cause pleural effusion or increase the lung
wet-to-dry weight ratio (data not shown). Measurements of pleural fluid
volume and the wet-to-dry weight ratio in hyperoxia are summarized in
Fig. 4. Figure 4A shows that
exposure to O2 alone produced a mean pleural fluid volume
of 9.2 ml ± 0.7, whereas O2 in combination with CO
treatment at 50, 100, 200, or 500 ppm produced pleural fluid volumes of 7.2 ± 1.4, 8.1 ± 1.0, 10.8 ± 0.6, and 9.9 ± 0.7, respectively. The pleural fluid volumes for CO-exposed animals
were not significantly different from those of the O2
control rats, indicating that CO did not attenuate the effusion. Figure
4B indicates that the wet-to-dry weight ratio increased to
6.3 ± 0.1 in O2-exposed rats compared with ~4.8 in
air control animals. The wet-to-dry weight ratio remained significantly
elevated at all levels of CO; however, the values at 200 and 500 ppm
were statistically lower than in O2 control rats
(P < 0.05). The wet-to-dry weight ratios in these two
groups, however, remained significantly elevated and well above the
wet-to-dry weight of normal air control animals. That the wet-to-dry
weight ratio at 200 and 500 ppm CO remained close to 6 (5.9 ± 0.2 and 5.9 ± 0.1, respectively) was taken as evidence that CO did
not provide a physiologically important reduction in total lung water.
Taken together, the pleural fluid volume and wet-to-dry weight ratio
findings provide consistent evidence that CO does not have a
physiologically important effect on preventing O2-induced
alveolar capillary injury in rats. This conclusion was supported by an
O2 tolerance study in which rats were exposed to either
O2 or O2 plus 200 or 500 ppm CO for 84 h
(Fig. 5). As shown in Fig. 5, CO did not
alter O2 tolerance significantly; survival time in
CO-exposed rats did not show a dose effect and was not statistically
significant by contingency testing.
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CO exposure and O2-induced inflammation in the lung.
To determine whether CO exposure decreased hyperoxic pulmonary
neutrophil accumulation compared with O2 alone, MPO
activity was assayed in the lungs. The MPO assay data (see Fig.
6) revealed high levels of activity in
all groups of O2-exposed rats, indicating the presence of
increased polymorphonuclear inflammatory cells. No significant
difference in lung MPO activity was found between the O2
control (0 ppm CO) animals and CO-treated animals except at 500 ppm CO,
where MPO values were significantly higher than in the O2
alone group (P < 0.05). This indicates that treatment with CO did not impede pulmonary neutrophil influx during hyperoxia. Of
note, exposure to 50 and 100 ppm CO in air for 7 days, which produces
COHb values similar to those produced by 200 and 500 ppm CO in
O2, does not increase lung MPO content (data not shown).
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Brain injury after CO exposure. To determine whether exposure to CO during hyperoxia caused neuronal injury, selected regions of perfusion-fixed brains (cortex, hippocampus, cerebellum, and basal ganglia) from CO-exposed rats (200 and 500 ppm) were compared with those of O2 control and normal animals. H&E-stained sections of the hippocampus and cortex showed definite cellular abnormalities in all four CO-exposed animals. In coronal sections through the hippocampus of normal rats and animals exposed to O2 for 60 h, hippocampal neurons appeared normal; dark cells with deeply stained pyknotic nuclei suggesting damage were rare. In contrast, coronal sections of hippocampus in rats exposed to hyperoxia and CO at 200 or 500 ppm often showed dark cells, particularly in the vulnerable CA1 subfield. Many such cells showed pyknotic nuclei, suggesting apoptosis. Sections from the frontal parietal cortex of CO-exposed rats also revealed significant injury compared with O2-exposed animals. The cortical injury primarily involved neuronal cell layer 4 of the cortex, although scattered damage was seen in other layers as well. By comparison, the basal ganglia and cerebellum showed relatively few damaged cells after hyperoxia and CO.
The results of immunohistochemical staining for ssDNA of sections of hippocampus and cortex showed remarkable effects of treatment with CO during hyperoxia. These two brain regions of rats exposed to O2 plus CO at 200 or 500 ppm for 60 h contained increased numbers of cells positive for ssDNA, a specific and sensitive marker for apoptosis. In air control animals, the number of ssDNA-positive cells in the hippocampus and frontal parietal cortex averaged ~1 per 1,000, where after 60 h of O2, ssDNA-positive cells averaged 1.3 and 1.5 per 1,000 in hippocampus and cortex (P = NS). After exposure to O2 and 200 ppm CO, however, the number of ssDNA-positive cells increased in the hippocampus to 10.9 per 1,000 and in cortex to 8.1 per 1,000 cells (P < 0.05 relative to O2 alone). In animals exposed to 500 ppm CO during hyperoxia, nuclear ssDNA counts of 13.3 and 11.6 were obtained in the hippocampus and cortex, respectively. The results of representative immunohistochemical stains of sections of hippocampus and cortex for ssDNA are shown in Fig. 8. Figure 8, A-C, shows typical sections of hippocampus stained for ssDNA for air control, O2-, and O2 plus 200 ppm CO-exposed animals. In Fig. 8, D-F, typical sections of frontal parietal cortex stained for ssDNA are shown for air control, O2-, and O2 plus 500 ppm CO-exposed animals. These findings indicate that continuous CO administration at 200 or 500 ppm during simultaneous exposure to 100% O2 caused cellular injury in the rat brain, with features of apoptosis most likely involving the neuronal cell types of the hippocampus and cortex.
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DISCUSSION |
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To evaluate the role of CO as a protective molecule in pulmonary O2 toxicity, we examined parameters of lung injury, inflammation, and expression of two antioxidant enzymes, Mn SOD and HO-1, in rats exposed to O2 and O2 plus CO at 50-500 ppm. Because Mn SOD and HO-1 are regulated by oxidative stress, we reasoned that CO might increase expression of these enzymes if it protected against O2 toxicity by influencing oxidative reactions. As expected, O2 induced both Mn SOD and HO-1 protein in the lung, but CO did not further augment the expression of either enzyme. In fact, O2 induction of both proteins was no longer statistically significant at 200 and 500 ppm CO. These results indicate that CO in the tested dose range has complex effects but does not stimulate either of these two major antioxidant enzymes in the hyperoxic rat lung in vivo.
One previous study reported dose-dependent protection of the rat lung in hyperoxia by CO at levels up to 500 ppm (20). In that study, CO decreased mortality and reduced acute lung injury as measured by pleural effusion and recovery of BAL neutrophils. In the current study, we used a timed, near-lethal exposure as our primary endpoint for the injury. After 60 h of hyperoxia, we detected small decreases in the wet-to-dry weight ratio in animals exposed to 200 or 500 ppm CO but were unable to confirm significant protection by accumulation of pleural fluid at any tested CO concentration. We were also unable to demonstrate that CO at 200 or 500 ppm significantly improved O2 tolerance during an 84-h exposure. In addition, lung MPO activity in CO-treated rats exposed to hyperoxia remained elevated in accordance with evidence of persistent neutrophil influx by histological examination. The failure of CO to reduce inflammation is significant, since the inflammatory response is a key amplification mechanism of tissue damage in pulmonary O2 toxicity (7, 8, 11).
An important difference between our study and that of Otterbein et al. (20) that reported protection can be found in the apparent uptake of CO based on COHb levels. The COHb levels that we measured in animals exposed to 500 ppm CO in O2 were <10%. The Otterbein et al. study, however, reported baseline blood COHb values of 6.6%, which increased to 11.3% at 250 ppm CO. A ready explanation for these unexpectedly high COHb levels is not apparent, and values were not provided for the 500 ppm CO exposures. Endogenous CO production from heme turnover normally produces basal COHb levels of ~1%. According to the Haldane relationship, an inspired CO of 500 ppm in O2, which provides a PCO2-to-PO2 ratio of ~1:2,000, should produce an equilibrium value of ~10% COHb at an affinity constant of 200 (29). The lack of concordance of Otterbein et al.'s (20) COHb values with equilibrium calculations could indicate, for example, that the measurements were not correct, the dose of CO was higher or inspired and/or blood PO2 values were less than ours during their exposures. We found no significant differences in preterminal arterial blood gas values in animals exposed to O2 or O2 plus 500 ppm CO for 60 h. The animals still had elevated arterial O2 tensions, which indicated that the measured COHb were reasonable. The elevated arterial PCO2 values also suggest that failure of ventilation and not hypoxemia was the impending cause of death.
CO poisoning is a well-known cause of both acute and delayed brain injury because of its 200-fold greater affinity than O2 for hemoglobin. This results in tissue hypoxia and a range of related cellular effects, which can lead to both necrosis and apoptosis (21). Consequently, we examined the brain to assess possible neuronal injury even though the highest concentration of CO in the study was moderate in terms of potential toxicity, approximately one-tenth of a lethal dose for the rat. Because COHb levels in our animals did not exceed 10%, it is unlikely that cerebral hypoxia or ischemia was a significant factor in the findings. This premise is supported by the relative lack of injury to the basal ganglia, which in CO poisoning is associated with an insufficient cerebral blood flow response (13). However, immunochemical evidence of apoptotic cell death in hippocampus and cortex at levels of 200 and 500 ppm CO indicates that combined exposure to O2 and CO had a significant adverse effect on the brain. Indeed, CO exposures that produce COHb levels of 15-20% correspond to the threshold for cerebrovascular injury and neurobehavioral decrements in normal air-breathing animals and humans (13). As a result, further studies to determine if higher levels of CO can provide significant lung protection are not likely to yield evidence of useful therapeutic benefit.
In summary, we have not been able to demonstrate a significant benefit of CO treatment on acute lung injury in rats during near-lethal O2 exposure. The lack of augmentation of key antioxidant enzyme defenses by CO and failure of a physiologically meaningful improvement in lung water and cellular inflammation during O2 toxicity imply that CO does not ameliorate the effects of accelerated ROS generation in the lung. Administration of CO at 200 and 500 ppm during hyperoxia is also associated with pathological evidence of neurotoxicity at modest COHb levels. Although the mechanisms of this toxicity are unknown, dual exposures to high levels of O2 and CO for prolonged periods of time increase cell death in the brain of the rat.
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. A. Piantadosi, PO Box 3315, Dept. of Medicine, Duke Univ. Medical Center, Durham, NC 27710 (E-mail: piant001{at}mc.duke.edu).
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.
Received 8 January 2001; accepted in final form 30 May 2001.
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