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Abstract
Introduction
Materials & Methods
Results
Discussion
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Recent studies have shown that homozygous Mn superoxide dismutase (Sod2) gene-knockout mice
(Sod2
/) die shortly after birth with extensive myocardial injury, whereas heterozygous mutants (Sod2+/)
are phenotypically normal in room air. In the current study, we showed that Sod2+/ mice with approximately 50% of normal pulmonary MnSOD activity and normal levels of lung CuZnSOD, catalase, and
glutathione peroxidase activities were not substantially more susceptible to 100% O2 toxicity than their
normal Sod2+/+ littermates. The mean (± SD) survival of Sod2+/ mice in 100% O2 was 101.4 ± 14.8 h (n = 20) versus 103.2 ± 11.3 h (n = 20) for Sod2+/+ littermates (P > 0.60). In addition, Sod2+/ mice with approximately 50% of normal heart MnSOD activity and Sod2+/+ mice did not develop any ultrastructural
abnormalities in the myocardium at 75 h or 90 h after 100% O2 exposure. These results suggest that in
mice, only 50% of MnSOD activity may be sufficient for normal resistance to 100% O2 toxicity.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Oxygen is an important therapeutic modality for patients
with severe hypoxemia. However, prolonged exposure to
a high partial pressure of O2 causes pulmonary injury (1-
3) and exacerbates the existing lung pathology (4). Considerable evidence suggests that reactive O2 species, such as
superoxide (·O2), H2O2, and hydroxyl radical (OH·) (5),
and more recently, peroxynitrite (ONOO) (6), play an
important role in hyperoxia-induced tissue injury.
Superoxide dismutase (SOD), a family of enzymes that
catalyze the dismutation of ·O2 to H2O2 and O2, reduces
the tissue concentration of ·O2 and prevents the production of OH· and ONOO (5). Thus, in conjunction with
catalase and glutathione (GSH) peroxidase, SOD may play
an important role in the host defense against O2 toxicity.
Manganese superoxide dismutase (MnSOD) is of particular interest because of its strategic location in mitochondria, a major site of ·O2 production under hyperoxic
conditions. However, despite recent investigation, the evidence supporting a role for MnSOD in the host defense
against O2 toxicity remains inconclusive (5).
The development of transgenic and gene-knockout
mice in which the MnSOD gene is either overexpressed or
inactivated, respectively, provides a powerful tool with
which to study the role of MnSOD in host defense against
O2 toxicity. However, contradictory results have been obtained with transgenic mice. Wispe and colleagues (7), using the human surfactant protein-C promoter, reported that overexpression of the human MnSOD gene in type II
alveolar epithelial and nonciliated epithelial cells of transgenic mice conferred protection against O2 toxicity. In
contrast, Ho (8), using the human 
-actin promoter, was
unable to demonstrate protection against O2 toxicity in
transgenic mice overexpressing the MnSOD gene in most
lung cells, including types I and II alveolar epithelial cells,
endothelial cells, and fibroblasts.
Recently, two laboratories have independently produced MnSOD gene-knockout mice: Li and associates (9) deleted exon 3 of the Sod2 gene (Sod2mlucsf), whereas Lebowitz and coworkers (10) deleted exons 1 and 2 of the Sod2 gene (Sod2mlbcm). On a CD-1 background, homozygous Sod2mlucsf-mutant mice, with no detectable MnSOD activity, all die within 10 d after birth with dilated cardiomyopathy, accumulation of lipid in the liver and skeletal muscle, and metabolic acidosis (9). Homozygous Sod2mlbcm-mutant mice of mixed genetic background, also without detectable MnSOD activity, survive for up to 18 d and exhibit severe anemia, degeneration of neurons in the basal ganglia and brain stem, and progressive motor disturbances characterized by weakness, rapid fatigue, and circling behavior. Mice surviving for more than 7 d exhibit extensive mitochondrial injury within degenerative neurons and cardiac myocytes. However, only 10% of them have markedly dilated cardiomyopathy (10). Heterozygous mutant mice, both Sod2mlucsf and Sod2mlbcm, with a reduced level of MnSOD (approximately 50% of normal) and normal CuZnSOD activities, are phenotypically normal and survive in room air without evidence of O2 toxicity (9, 10).
In the current study, we showed that heterozygous Sod2mlucsf-mutant mice with approximately 50% of pulmonary MnSOD activity and normal levels of CuZnSOD, catalase, and GSH peroxidase activities were not substantially more susceptible to 100% O2 toxicity than their normal littermates. In addition, neither heterozygous mutant mice nor their normal littermates developed ultrastructural abnormalities in the myocardium at 75 h or 90 h after 100% O2 exposure.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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MnSOD (Sod2) Gene-Knockout Mice
MnSOD gene-knockout mice (Sod2mlucsf) were obtained by
deletion of exon 3 of the Sod2 gene, using targeted homologous inactivation as described previously (9). Heterozygous male mutant mice (Sod2+/), backcrossed for six generations (N6) to C57BL/6 mice, were bred with normal
C57BL/6J female mice (Jackson Laboratory, Bar Harbor,
ME) at the animal research facility of Stratton VA Medical Center, Albany, NY. All cages, bedding, feed, and water were autoclaved, and the cages were placed in a ventilated rack.
Genotypes of mutant mice were determined according to Li and colleagues (9), with some modifications. Briefly, DNA was obtained by overnight digestion at 50°C of mouse tails with proteinase K (GIBCO BRL, Life Technologies, Gaithersburg, MD) and amplified through the polymerase chain reaction (PCR) of mutant and normal fragments, using one shared primer (5'-CGAGGGGCATCTAGTAGTGGAGAAG-3') and one primer for the normal (5'-TTAGGCTCAGGTTTGTCCAGAA-3') or mutant (5'-CACACATCGGGAAAATGGTTG-3') allele. PCR was performed with the following reaction conditions: pre-PCR incubation at 94°C for 10 min, followed by 35 cycles at 98°C for 30 s; 58°C for 30 s; and 72°C for 60 s, in 25 µl of reaction mixture containing DNA (20 to 100 ng), 10 mM Tris-HCl, pH 8.3, 50 mM KCl, MgCl2 (2 mM for normal allele, 1.5 mM for mutant allele), 200 µM deoxynucleotide triphosphates (dNTPs), primer set (1 µM for normal allele, 0.5 µM for mutant allele), and 0.725 U AmpliTaq Gold DNA polymerase (Perkin Elmer, Applied Biosystems Division, Foster City, CA), followed by agarose-gel electrophoresis. The normal allele produced a band of ~ 500 bp and the mutant allele produced a band of ~ 350 bp (9).
Adult heterozygous mutant (Sod2+/) mice and their
normal littermates (Sod2+/+), 2 to 4 mo old, were used in
the study.
Measurement of Pulmonary and Myocardial Antioxidant Enzyme Activities
Preparation of lung and heart extracts for enzyme assays was done as described previously (11, 12). Briefly, mice were killed by CO2 exposure and exsanguinated by transection of the inferior vena cava. Lungs and heart were removed, homogenized with a tissue homogenizer in 0.5 ml potassium phosphate buffer (0.05 M, pH 7.5) containing 1.0 mM ethylenediamine tetraacetic acid (EDTA), twice sonicated for 30 s, and then centrifuged at 15,000 × g for 10 min at 4°C. The supernate was then assayed for protein content, using bicinchoninic acid according to the method of Smith and coworkers (13), and for enzyme activities.
SOD activity was assayed through nondenaturing polyacrylamide gel electrophoresis (PAGE) according to Beauchamp and Fridovich (14), as described previously (15).
The assay was based on the inhibitory effect of SOD on
the reduction of tetrazolium by ·O2 generated by photochemically reduced riboflavin. This method allows simultaneous visualization and quantification of MnSOD and
CuZnSOD activities. The SOD activity gel is then quantified with a computing densitometer (Molecular Dynamics,
Sunnyvale, CA). In each assay, purified Escherichia coli
MnSOD (Sigma Chemical Co., St. Louis, MO), 4,000 U/mg
as determined according to McCord and Fridovich (16),
and bovine erythrocyte CuZnSOD (Sigma), 4,400 U/mg,
were used to obtain standard curves from which the lung-extract MnSOD and CuZnSOD activities, respectively, were derived.
The catalase activity was determined according to Bergmeyer (17), as modified previously (11), and the GSH peroxidase activity was determined according to Paglia and Valentine (18).
Exposure of Mice to Hyperoxia
Hyperoxic exposure of adult mice, 2 to 4 mo old, was done as described previously (11), by placing mice in groups of five in a Lucite chamber (45 × 40 × 25 cm3). The concentration of O2 in the chamber was maintained at > 99% at all times. Mice were given free access to water and diet. They were checked every 12 h, and their survival was recorded.
Morphologic Studies
Morphologic studies were performed as described previously (11, 12). At 75 h or 90 h after hyperoxic or normoxic (room air) exposure, mice were killed by CO2 exposure and exsanguinated. Lungs were then fixed in the inflated state, using 10% buffered formalin for histologic studies or 4% formaldehyde and 1% glutaraldehyde in Millonig's phosphate buffer, pH 7.3, for ultrastructural studies. Heart tissue was fixed with 10% formalin for histology or 4% formaldehyde plus 1% glutaraldehyde in Millonig's phosphate buffer for ultrastructural study.
For histologic studies, sections of lung and heart were taken after 24 to 48 h of fixation, embedded in paraffin, stained with hematoxylin and eosin (H&E), and examined with a light microscope. For ultrastructural studies, lung and heart tissues were postfixed in 2% OsO4 in Millonig's phosphate buffer, dehydrated, and embedded in Polybed 812 (Polysciences, Warrington, PA). Thin sections were stained with uranyl acetate and lead citrate, and were examined with a Hitachi H-7000 scanning/transmission electron microscope. Two to four mice in each group and at each time point were studied to ensure the reliability of the observations.
Statistical Analysis
Power analysis was performed according to Cohen's method (19) for independent-sample t tests. Data from two groups were compared through a two-tailed t test. Differences were considered significant at P < 0.05.
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Materials & Methods
Results
Discussion
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Levels of Pulmonary and Myocardial Antioxidant Enzyme
Activities in Sod2+/ and Sod2+/+ Mice
The activities of SOD were measured with activity gels that allowed simultaneous visualization and quantification of MnSOD and CuZnSOD activities. As shown in Figure 1 (lung) and Figure 2 (heart), mouse MnSOD and CuZnSOD could be readily separated through PAGE. Note that we used purified E. coli MnSOD and bovine erythrocyte CuZnSOD, which migrated to different positions from those of mouse enzymes, to construct standard curves for the purpose of densitometric quantification of SOD activities.
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For the quantification of lung MnSOD activities, 200 µg
protein per lane was used in the presence of 5 mM KCN to
partly inhibit the activities of CuZnSOD (Figure 1), since
lung CuZnSOD activity was much higher than that of MnSOD. For lung CuZnSOD activities, a separate gel was
used, containing 12.5 µg protein per lane (not shown). For
the quantification of heart MnSOD and CuZnSOD activities, 50 µg protein per lane was used (Figure 2), since normal littermates (Sod2+/+) had comparable myocardial MnSOD and CuZnSOD activities. Figures 1 and 2 show that
Sod2+/ mice had reduced lung and heart MnSOD activities, whereas CuZnSOD activities were comparable with
those of Sod2+/+ littermates.
As shown in Figure 3, MnSOD activities of the lungs
and heart in Sod2+/ mice were approximately half those
of Sod2+/+ littermates (e.g., 56% and 43%, respectively).
In contrast, there was no statistically significant difference
between Sod2+/ and Sod2+/+ mice in the activities of lung
CuZnSOD, catalase, and GSH peroxidase, or of heart catalase. However, heart CuZnSOD and GSH peroxidase in
Sod2+/ mice were slightly, but significantly, higher than in
Sod2+/+ littermates. It should be pointed out that MnSOD
activity in the heart was much higher than that in lungs,
whereas CuZnSOD activity was higher in the lungs than in
the heart.
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Survival of Sod2+/ and Sod2+/+ Mice in 100% O2
Preliminary studies were done to determine the survival of mice in 100% O2, using commercially available C57BL/6J mice (Jackson Laboratory). As shown in Figure 4C, the mean (± SD) survival was 101.4 ± 8.2 h (n = 20, all male). Power analysis based on these data revealed that a total of 40 animals, at 20 per group, would provide ample power to detect a difference of as little as 10% in mean survival time between the two groups (two-tailed alpha of 0.05 and power of 0.97, assuming common variance).
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Sod2+/+ and Sod2+/ mice in two groups of 20 each
were then exposed to 100% O2. As shown in Figures 4A
and 4B, no significant difference in survival was noted; the
mean (± SD) survival for Sod2+/+ mice was 103.2 ± 11.3 h
(n = 20, 11 male and nine female), and that for Sod2+/
mice was 101.4 ± 14.8 h (n = 20, nine male and 11 female),
P > 0.60. These survival data were comparable with the
survival of the commercially obtained C57BL/6J mice described previously (Figure 4C).
Effect of Hyperoxia on Lung and Heart Morphology
The lung is the primary target organ for normobaric hyperoxia-induced injury. Mice in the present study developed mild respiratory distress after approximately 72 h of
exposure to 100% O2. Therefore, we examined the histology and ultrastructure of lungs to determine whether there
were differences in O2-induced lung injury between Sod2+/+
and Sod2+/ mice. Since homozygous mutant mice (Sod2/)
died of dilated cardiomyopathy with extensive myocardial
changes (9, 10), we also examined heart morphology to determine whether the heart of Sod2+/mice was more susceptible to O2 toxicity than that of Sod2+/+ mice.
As shown in Figure 5, at 75 h after O2 exposure, only
scattered focal edema and inflammatory-cell infiltration in
interstitial, alveolar, and perivascular spaces were noted (Figure 5B, as compared with Figure 5A, showing a normoxia-exposed control). However, at 90 h after O2 exposure,
more diffuse and extensive edema with fibrin exudation
was noted (Figures 5C and 5D). There was no difference in O2-induced lung pathology between Sod2+/+ and Sod2+/
mice at either 75 h or 90 h after O2 exposure. Ultrastructural studies revealed that at 90 h after O2 exposure, there
were severe endothelial-cell damage as reflected by focal
thinning, breaks and detachment of endothelial cells from
the basement membrane, and alveolar epithelial-cell damage as reflected by destruction and disruption of alveolar
epithelium with cellular debris and fibrin in the alveolar
space (Figure 6B as compared with Figure 6A showing a
normoxia-exposed control). Again, similar ultrastructural abnormalities were seen in Sod2+/+ and Sod2+/ mice.
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No histologic or ultrastructural abnormalities in the
heart were noted in Sod2+/+ or Sod2+/ mice either at 75 h
or at 90 h after O2 exposure (not shown). In particular,
there were no mitochondrial abnormalities even after 90 h
of 100% O2 exposure in either Sod2+/+ or Sod2+/ mice.
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Discussion |
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Materials & Methods
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The results presented in the current study showed that
Sod2+/-mutant mice with levels of lung and heart MnSOD activities approximately 50% of those of Sod2+/+
mice: (1) developed and survived normally in room air
with no demonstrable histologic or ultrastructural abnormalities in the lung or heart; (2) survived in a 100% O2 environment to a similar extent as Sod2+/+ littermates; (3)
showed similar hyperoxia-induced lung injuries at both 75 h
and 90 h after O2 exposure to those of Sod2+/+ littermates;
and (4) showed no myocardial ultrastructural abnormalities even at 90 h after O2 exposure. Thus, Sod2+/ mice
were not substantially more susceptible to 100% O2 toxicity than their Sod2+/+ littermates, suggesting that in mice,
only 50% of MnSOD activity might be sufficient for normal resistance to 100% O2 toxicity.
We used a sufficient number of mice (20 each group) to
detect a 10% difference in survival between Sod2+/+ and
Sod2+/ mice. The high level of power (0.97) to detect an
effect of this magnitude corresponds to a low likelihood of
false-negative results (type II statistical error probability
of 0.03). However, we cannot rule out the possibility that
our expectation of differences in the population effect size
may have been overestimated. For example, the chances
of falsely rejecting the null hypothesis increase if the true
population difference in survival between these two groups
is 5%, rather than 10%. To detect a 5% difference would have required a total of 84 animals (42 in each group) to
achieve a power of 0.80 (an alpha of 0.05).
Homozygous mutant mice (Sod2/) with no MnSOD
activity on a CD-1 background have been shown to develop extensive myocardial damage and die of dilated cardiomyopathy shortly after birth even in room air (9). We
found that Sod2/ mice on a C57BL/6 background survived for only 1 d after birth. On the other hand, Sod2+/
mice with a 50% reduction in myocardial MnSOD activity
did not develop any myocardial injury in a 100% O2 environment. It is possible that we overlooked biochemical
evidence of myocardial O2 toxicity (e.g., reduced mitochondrial enzyme activities [aconitase and succinate dehydrogenase]), since we looked only for morphologic changes.
However, it is noteworthy that Sod2+/ fibroblasts exposed in vitro to paraquat, a generator of ·O2, are not
more sensitive than are Sod2+/+ cells (20).
Li and colleagues (9) reported that at 4 to 5 d after
birth, lung-water content of Sod2/ mice was similar to
that of Sod2+/+ littermates, but no detailed study of lungs
in Sod2/ mice at later time points has been reported.
Therefore, whether Sod2/ mice develop pulmonary O2
toxicity in room air remains unclear. However, it has been
observed that clonal bone-marrow precursors derived from
Sod2/ mice, but not from Sod2+/ mice, are extremely
sensitive to 20% O2, and grow much better in 5% O2 (21).
CuZnSOD (Sod1)- and extracellular SOD (EC-SOD,
Sod3)-gene-knockout mice have also been produced (22,
23). In contrast to Sod2/ mice, homozygous mutant
mice, Sod1/ and Sod3/, with no CuZnSOD or EC-SOD activity, respectively, develop and survive normally
for months in room air with no apparent abnormalities (22, 23). Thus, despite the fact that MnSOD constitutes
only a minor fraction of total tissue SOD activity, it is the
most important SOD, being essential for the survival of
animals in room air. It has been shown that Sod3/ mice,
when exposed to a lethal dose of O2, develop severe lung injury earlier and have a shorter survival than their wild-type (Sod3+/+) littermates (23). Thus, EC-SOD contributes to the host defense against hyperoxia in mice. On the
other hand, Ho and colleagues (24) recently reported that
Sod1/ mutant mice were not more susceptible to 100%
O2 toxicity than their wild-type (Sod1+/+) littermates. These
observations suggest that the specific locations of various
SODs are critical in these enzymes' role against pulmonary O2 toxicity.
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Footnotes |
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Abbreviations: copper/zinc superoxide dismutase, CuZnSOD; deoxynucleotide triphosphate, dNTP; glutathione, GSH; manganese superoxide dismutase, MnSOD; polymerase chain reaction, PCR.
(Received in original form June 23, 1997 and in revised form September 29, 1997).
Address correspondence to: Min-Fu Tsan, M.D., Ph.D., Research Serv ice (151), Stratton VA Medical Center, 113 Holland Avenue, Albany, NY 12208. E-mail: TSAN.MIN-FU{at}ALBANY.VA.GOV
Acknowledgments: The authors thank Alexander Durant for excellent animal care, Xiaomin Cai for technical assistance, Elaine J. Carlson for advice, and Rhoda Drumm for secretarial work. This work was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs, and by Grant AG-08938 from the National Institute of Aging.
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Y.-S. Ho Transgenic and Knockout Models for Studying the Role of Lung Antioxidant Enzymes in Defense against Hyperoxia Am. J. Respir. Crit. Care Med., December 15, 2002; 166(12): S51 - 56. [Abstract] [Full Text] [PDF]
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 H.-Y. Cho, A. E. Jedlicka, S. P. M. Reddy, T. W. Kensler, M. Yamamoto, L.-Y. Zhang, and S. R. Kleeberger Role of NRF2 in Protection Against Hyperoxic Lung Injury in Mice Am. J. Respir. Cell Mol. Biol., February 1, 2002; 26(2): 175 - 182. [Abstract] [Full Text] [PDF]
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H. Kobayashi, R. Hataishi, H. Mitsufuji, M. Tanaka, M. Jacobson, T. Tomita, W. M. Zapol, and R. C. Jones Antiinflammatory Properties of Inducible Nitric Oxide Synthase in Acute Hyperoxic Lung Injury Am. J. Respir. Cell Mol. Biol., April 1, 2001; 24(4): 390 - 397. [Abstract] [Full Text]
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 J.-C. COPIN, Y. GASCHE, Y. LI, and P. H. CHAN Prolonged hypoxia during cell development protects mature manganese superoxide dismutase-deficient astrocytes from damage by oxidative stress FASEB J, February 1, 2001; 15(2): 525 - 534. [Abstract] [Full Text] [PDF]
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M.-F. Tsan, X. Cao, J. E. White, J. Sacco, and C. Y. Lee Pertussis Toxin-Induced Lung Edema . Role of Manganese Superoxide Dismutase and Protein Kinase C Am. J. Respir. Cell Mol. Biol., March 1, 1999; 20(3): 465 - 473. [Abstract] [Full Text]
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