1 Department of Human Nutrition, The Ohio State University, Columbus, Ohio 43210; and 2 Department of Molecular Biology, Cephalon, West Chester, Pennsylvania 19380
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ABSTRACT |
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Ceruloplasmin, metallothionein, and ferritin are metal-binding proteins with potential antioxidant activity. Despite evidence that they are upregulated in pulmonary tissue after oxidative stress, little is known regarding their influence on trace metal homeostasis. In this study, we have used copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) transgenic-overexpressing and gene knockout mice and hyperoxia to investigate the effects of chronic and acute oxidative stress on the expression of these metalloproteins and to identify their influence on copper, zinc, and iron homeostasis. We found that the oxidative stress-mediated induction of ceruloplasmin and metallothionein in the lung had no effect on tissue levels of copper, iron, or zinc. However, Cu/Zn SOD expression had a marked influence on hepatic copper and iron as well as circulating copper homeostasis. These results suggest that ceruloplasmin and metallothionein may function as antioxidants independent of their role in trace metal homeostasis and that Cu/Zn SOD functions in copper homeostasis via mechanisms distinct from its superoxide scavenging properties.
ceruloplasmin; metallothionein; ferritin; gene knockout mice; transgenic overexpressing mice; copper- and zinc-containing superoxide dismutase
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INTRODUCTION |
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THE BALANCE BETWEEN the antioxidant defenses and the rate of production of reactive oxygen species (ROS) is believed to be the critical factor that determines the extent of tissue injury in a number of pulmonary diseases (4, 23, 29, 42). For example, bronchopulmonary dysplasia (BPD) often develops in premature infants exposed to high levels of oxygen. Exposure to hyperoxia leads to increased production of ROS (19, 21) that can presumably overwhelm the immaturely developed antioxidant defense system and lead to oxidative tissue damage within the pulmonary tissue (8, 43, 44). The antioxidant defenses, which consist of a network of enzymes, proteins, and low molecular weight ROS scavengers, function to protect cellular structures against the damaging effects of ROS. Although antioxidant enzymes such as the superoxide dismutases [copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) and manganese superoxide dismutase (Mn SOD)], glutathione peroxidases (GSH-Px), and catalase are generally considered to play the major role in antioxidant defense, this view is being expanded as our knowledge of the cellular antioxidant defense response to different forms of oxidative insult continues to advance.
Metalloproteins, such as ceruloplasmin, metallothionein, and ferritin, are well known for their critical role in metal homeostasis and function as storage reservoirs and/or chaperones for essential trace metals, such as copper, zinc, and iron. Evidence indicates that these proteins are induced during the acute-phase response (27, 38) and under oxidative stress (14, 41). It has been speculated that they ameliorate the deleterious effects of ROS. The antioxidant properties of these proteins have been attributed primarily to their binding of the redox active metals copper and iron, thus minimizing their capacity to catalyze ROS production via the Fenton reaction. Yet, the contribution of these metalloproteins to the antioxidant defense network in vivo is still not clearly defined. Although many investigators have focused on identifying the antioxidant properties of the induced metalloproteins, limited attention has been given to their tissue-specific expression or their influence on metal homeostasis after exposure to acute or chronic oxidative stress. Disruption in trace element homeostasis may further exacerbate the pathogenesis of pulmonary diseases associated with oxidative stress, such as BPD and adult respiratory distress syndrome (10, 22, 34).
The development of transgenic and knockout mice has provided researchers with powerful tools to manipulate the antioxidant defense status in vivo. We have used mice that express variable levels of the antioxidant enzyme Cu/Zn SOD and exposure to hyperoxia to systematically evaluate the in vivo consequences of chronic and acute oxidative stress on trace mineral metabolism. Cu/Zn SOD protects against oxidative stress by catalyzing the dismutation of superoxide generated under normal aerobic metabolism. Thus genetic deletion of this enzyme presumably leads to a chronic increase in endogenous levels of oxidative species, whereas overexpression of Cu/Zn SOD would have the opposite effect and would result in decreased levels of oxidative stress. In contrast, exposure to exogenous oxidative stresses, for example, exposure to a high level of oxygen, is known to generate an acute state of oxidative stress by elevating ROS production (19). Hence, in this study, we have used mice with variable Cu/Zn SOD expression and hyperoxic exposure to investigate the effects of chronic and acute oxidative stress on the expression of the antioxidant metalloproteins ceruloplasmin, metallothionein, and ferritin and to identify the influence of these metalloproteins on copper, zinc, and iron homeostasis.
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MATERIALS AND METHODS |
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Cu/Zn SOD-overexpressing and knockout mice.
Cu/Zn SOD overexpressor [Sod1(+++)] mice [TGN(SOD1)3Cje,
Jackson Laboratories, Bar Harbor, ME] contain multiple inserts of the
human Cu/Zn SOD gene and were derived from outbred matings between
CBYB/6 [(BALB/cxC57BL/6J)F1] and B6D/2
[(C57BL/6JxDBA)F1] mice (16). Mice
homozygous for the human Cu/Zn SOD insert were identified by SOD
activity gel (5) and pedigree analysis. Cu/Zn SOD knockout
mice [Sod1(/
)] were developed from CD-1 mice, an outbred strain, and inbred mice of the 129 strain (129/Sv and 129/Re
substrains) and were provided as a generous gift from Cephalon (West
Chester, PA). Construction of the Sod1(
/
) gene-targeted mice was performed by Cephalon, as previously described
(36). In our laboratory, heterozygotes
[Sod1(+/
)] were mated to produce knockouts,
heterozygotes, and wild-type controls [Sod1(+/+)]. Mouse
strain was similarly confirmed in the laboratory by SOD activity gel
(5) and pedigree analysis. All procedures involving animals were approved by the Ohio State University Institutional Laboratory Animal Care Committee.
SOD activity gel electrophoresis. Lung homogenate (~5-10 µg of protein) was electrophoresed through a 10% polyacrylamide gel for 1-2 h at 100 volts. Staining for SOD activity was done as described (5), except that gels were soaked for 10 min in nitroblue tetrazolium followed by a 15-min soak in phosphate buffer containing riboflavin. Clear bands appear in the gel that represent either human SOD, mouse SOD, or mouse-human SOD. The absence of a band is indicative of a Cu/Zn SOD knockout.
Oxygen exposure of mice.
Adult male mice (25-35 g) expressing either of the four different
levels of Cu/Zn SOD gene [Sod1(+++), Sod1(+/+),
Sod1(+/), and Sod1(
/
)] were used in all
experiments and were randomly assigned to either the control or
hyperoxia treatment group. All animals were placed in an airtight
Plexiglas chamber and exposed to either room air or 85% oxygen
continuously for 5 days (120 h). Oxygen (85%) was supplied to the
chamber at a flow rate of 3-4 l/min by mixing compressed air and
oxygen with a Bird 3800 Microblender (Bird Products, Palm Springs, CA).
The oxygen concentration of the chamber was continuously monitored
using a Servomex Oxygen Analyzer OA 570 (Servomex Limited, Crowborough,
Sussex, UK). Chamber temperature was maintained at 25-27°C, and
the relative humidity was 60-70%.
Tissue collection and preparation.
Tissue samples were collected after the mice had been exposed to either
air or 85% oxygen for 5 days. Mice were killed by decapitation, and
trunk blood was collected in heparinized tubes, centrifuged to obtain
plasma, and stored at 80°C until further use. The lung and liver
were removed, weighed, rinsed in ice-cold saline, immersed in liquid
nitrogen, and stored at
80°C. Before biochemical analysis, frozen
tissues were homogenized on ice with a PowerGen homogenizer (Fisher
Scientific, Pittsburgh, PA) in 4 vol of sucrose buffer (0.25 M sucrose
and 1.0 mM EDTA, pH 7.4) containing the following protease inhibitors
to prevent protein degradation during sample preparation: 0.5 µg/ml
aprotinin, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, and 40 µg/ml
phenylmethylsulfonyl fluoride (30). A fraction of the
homogenate (1 ml) was then removed for trace mineral analysis. The
remainder of the homogenate was centrifuged at 600 g for 10 min to remove the nuclei, and the supernatant was obtained from the
postnuclear supernatant by centrifugation at 10,000 g for 20 min.
Measurement of enzyme activity. Total SOD activity was measured as the percent inhibition in the cytochrome c-xanthine-xanthine oxidase assay (11). SOD activity was measured in the presence of 10 µM KCN to remove interference resulting from cytochrome-c oxidase activity. To distinguish the contributions of Cu/Zn SOD and Mn SOD, Mn SOD activity was measured in the presence of 1 mM KCN, which inhibits Cu/Zn SOD activity. Cu/Zn SOD activity was then determined by subtracting Mn SOD activity from total SOD activity. One unit of SOD activity is defined as the amount of protein required to inhibit the rate of reduction of cytochrome c by 50%. GSH-Px activity was measured as described (33). Tissue homogenate (100 µl) was added to 900 µl of HEPES buffer (100 mM, pH 7.5) containing EDTA (0.1 mM), glutathione (1 mM), NADPH (0.11 mM), and glutathione reductase (0.6 units). The disappearance of NADPH was monitored at 340 nm for 5 min after the addition of 20 µl t-butyl hydroperoxide in methanol (0.25 mM). All concentrations were expressed as final molarity within the cuvette. Catalase activity was measured as the decomposition of H2O2 (9). Briefly, 333 µl of tissue homogenate were diluted in 1 volume of phosphate buffer (50 mM K2HPO4 and 50 mM KH2PO4, pH 7.0) and transferred immediately to a cuvette containing 1 volume of 30 mM H2O2, and the change in absorbance at 240 nm was recorded for 1 min. One unit of catalase activity is defined as 1 µmol of H2O2 consumed per minute per milligram of tissue protein.
Ceruloplasmin activity was measured using p-phenylenediamine (PPD) as a substrate (35). Serum samples (2 mg of protein) were added to cuvettes containing 0.6 ml of acetate buffer (0.1 M, pH = 6.0) and 0.3 ml of 0.25% PPD in acetate buffer. Acetate buffer and PPD solution were equilibrated at 37°C for 5 min before sample addition. The change in absorbance at 530 nm was monitored for 30 min after allowing for a 10-min lag phase. Blank samples containing serum (2 mg protein), 0.3 ml of acetate buffer, 0.3 ml of 0.25% PPD buffer, and 0.3 ml of 0.1% NaN in 0.1 M acetate buffer were prepared for each sample. Ceruloplasmin activity was determined using standard solutions of Bandrowski's base, where one enzyme unit is equivalent to 1 µmol of Bandroski's base formed per minute per liter of serum (37).Western blot analysis. Cu/Zn SOD, GSH-Px, and catalase as well as ceruloplasmin and ferritin proteins were separated by PAGE under standard conditions (26) using a Bio-Rad Mini Gel apparatus (Bio-Rad, Hercules, CA). For Cu/Zn SOD and catalase, samples containing 100 µg of protein were run on 10% polyacrylamide gels for 1.5-2 h at 100 volts. For GSH-Px, 100 µg of protein were separated over 2 h in a 12% polyacrylamide gel at 100 volts. Separation of ceruloplasmin and ferritin was accomplished by loading 100 µg of tissue sample protein on an 8% and 12% polyacrylamide gel, respectively, and running at 100 volts for 1.5-2 h. After proteins were separated, they were electrophoretically transferred overnight at 4°C and 7 V/cm2 to Hybond enhanced chemiluminescence nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ) using a Mini Trans-Blot Transfer Cell (Bio-Rad). Membranes were blocked with 5% nonfat dry milk in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4, pH 7.4) at room temperature for 4 h and then were washed four times in PBS containing 0.2% Tween 20 for 5 min. Membranes were then incubated with their respective primary antibodies [i.e., Cu/Zn SOD, GSH-Px, catalase, ceruloplasmin (sheep anti-human); ferritin (mouse anti-human)] at a 1:1,000 dilution at 37°C for 2 h in PBS containing 2% nonfat dried milk. All primary antibodies were supplied by Biodesign International (Kennebunk, ME). After incubation, each membrane was washed a second time as outlined above and then incubated with horseradish peroxidase-conjugated rabbit anti-sheep or rabbit anti-mouse (Sigma, St. Louis, MO) antisera at a dilution of 1:2,000 for 2 h at 37°C. Blots were developed in 3.4 mM 4-chloro-1-naphthol, 22% methanol, 0.018% H2O2, 15.6 mM Tris · HCl (pH 7.4), and 120.2 mM NaCl. Development was stopped by immersion of membranes in distilled water.
Western blot analysis of metallothionein was accomplished using a modified procedure (28). Tissue homogenate (20 µg of total protein) was diluted 1:1 with sample buffer [10 mM Tris · HCl, 10 mM EDTA, 20% (vol/vol) glycerol, 1.0% (wt/vol) SDS, 0.005% (wt/vol) bromphenol blue, and 100 mM dithiothreitol] and then separated in an 18% polyacrylamide gel at 100 volts for 2 h. Membrane transfer was performed in 10 mM 3-cyclohexylamino-1-propane sulfonic acid, 2 mM CaCl2, and 10% methanol, pH 10.8, followed by membrane incubation in 2.5% glutaraldehyde in water for 1 h at room temperature. Glutaraldehyde-treated membranes were then washed three times for 10 min in phosphate buffer (8.1 mM Na2HPO4, 1.2 mM KH2PO4, and 2.7 mM KCl, pH 7.4); 50 mM monoethanolamine was added to the third wash to quench residual glutaraldehyde reactivity. Membranes were blocked in 3% BSA (Sigma). Application of primary (mouse anti-human; Zymed Laboratories, San Francisco, CA) and secondary antibodies (rabbit anti-mouse; Sigma) as well as all subsequent washing steps and color development was performed as outlined above, except that 2% BSA was substituted for 2% nonfat dried milk.Trace mineral analysis. Lung and liver copper, zinc, and iron levels were measured by atomic absorption spectrophotometry using a Varian Spectra AA-5 (Varian Techtron, Mulgrave, Australia). Briefly, 1 ml of homogenate was mixed with 4 ml of 2 N HCl and incubated with gentle agitation for 24 h. The suspension was then centrifuged at 7,000 g for 30 min, and the supernatant was used for direct measurement of each trace mineral. Standard reference material (SRM 1577; National Institute of Standards and Technology, Gaithersburg, MD) was analyzed simultaneously to ensure validity of the method.
Protein assay. Lung and liver homogenate protein concentrations were determined using the Bradford (6) method. Coomassie brilliant blue G-250 staining reagent (Bio-Rad) was used for protein analysis, with BSA (Sigma) prepared as standard.
Statistical methods. All results are expressed as means ± SE. Data were analyzed by the general linear models procedure to determine significant main effects and by the least significant means test to determine differences between means (40). A P value < 0.05 was taken as the level of significance for all analysis.
Results
Cu/Zn SOD protein levels in the lung (Fig. 1A) and liver (Fig. 1B) of transgenic mice with variable levels of expression [Sod1(+++), Sod1(+/+), Sod1(+/
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The effect of variable Sod1 expression and hyperoxia
exposure on the antioxidant enzyme levels of GSH-Px and catalase in the lung and liver are presented in Fig. 2.
GSH-Px protein levels increased as Cu/Zn SOD protein levels decreased
in the lung. Hyperoxia did not further induce GSH-Px protein levels, as
expected (Fig. 2A). The inverse relationship between GSH-Px
and Cu/Zn SOD protein level was not as apparent in the liver (Fig.
2C). Figure 2, B and D, demonstrates
that Cu/Zn SOD protein levels did not affect catalase expression in
either the lung or liver tissue. Unexpectedly, hyperoxia significantly
reduced catalase protein levels in all Sod1 strains of mice
in the lung (Fig. 2B) but not in the liver (Fig.
2D).
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The activity of GSH-Px and catalase was then analyzed (Fig.
3) to determine if variable Cu/Zn SOD
expression affected other antioxidant enzymes during hyperoxic
exposure. Notably, GSH-Px activity in the lung (Fig.
3A) did not reflect protein levels (Fig. 2A). As
a result, the previously noted inverse relationship between
Sod1 gene expression and GSH-Px protein levels did not exist
between Cu/Zn SOD gene expression (Fig. 1A) and GSH-Px
activity (Fig. 3A). Moreover, GSH-Px activity in
Sod1(+/+) and Sod1(+/) mice was increased after
exposure to hyperoxia without a corresponding increase in GSH-Px
protein levels. In the liver, neither Sod1 genotype nor
hyperoxic exposure influenced the activity of GSH-Px (Fig.
3C). In contrast to GSH-Px activity, the activity of
catalase was significantly reduced after exposure to hyperoxia in the
lung of all Sod1 genotypes (Fig. 3B), a result
that mirrored the effects of hyperoxia on catalase protein expression
(Fig. 2B). In the liver, catalase activity was not affected
by hyperoxic exposure. However, hepatic catalase activity in
Sod1(+++) mice was reduced by >50% (P < 0.05) compared with all Sod1 genotypes (Fig. 3D) despite similar levels of enzyme protein in all other strains (Fig.
2D).
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Figure 4 shows an increase in
ceruloplasmin protein in the lung (Fig. 4A) but not the
liver (Fig. 4C) of Sod1 mice after exposure to
hyperoxia. Notably, ceruloplasmin protein level was not influenced by
Cu/Zn SOD gene expression, as there were no observable differences in
protein level based on Sod1 genotype in the lung or liver. However, ceruloplasmin protein levels were found to be responsive to
the oxidative challenge of hyperoxia in the target tissue, as they were
elevated in the lung but remained unchanged in the liver after 5 days
of hyperoxia exposure. The significant increases in ceruloplasmin
protein levels in the lung did not, however, affect copper
concentrations (Fig. 4B). In contrast to the lung, liver
copper concentrations exhibited substantial variability (Fig.
4D), yet ceruloplasmin levels remained constant across all treatments (Fig. 4B). Notably, copper levels were found to
correlate with Cu/Zn SOD expression rather than ceruloplasmin
protein levels, i.e., copper levels were highest in
Sod1(+++) mice, intermediate in Sod1(+/+)
and Sod1(+/) mice, and lowest in Sod1(
/
)
mice.
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In contrast to ceruloplasmin, metallothionein synthesis was found to be
responsive to the oxidative challenge of hyperoxia in both the target
and nontarget tissue of all Sod1 strains, with statistically
significant increases observed in the lungs of Sod1(+++) and
Sod1(/
) and the liver of Sod1(
/
) mice
(Fig. 5, A and C). The increases in metallothionein protein levels in the lung and liver
were not, however, correlated with increases in zinc concentrations (Fig. 5, B and D). In the lung, zinc
concentrations were slightly reduced by hyperoxia in all
Sod1 genotypes, whereas, in the liver, zinc levels were
increased by hyperoxia in Sod1(+/+) and
Sod1(
/
) mice. Liver zinc concentrations were also
affected by Cu/Zn SOD expression, i.e., zinc levels were highest in
Sod1(+++) mice, intermediate in Sod1(+/+) and
Sod1(+/
) mice, and lowest in Sod1(
/
) mice
(Fig. 5D).
|
Ferritin protein levels and iron concentrations in the lung and liver
of each strain of Sod1 mice after exposure to hyperoxia are
depicted in Fig. 6. Lung and liver
ferritin protein levels (Fig. 6, A and C)
exhibited little fluctuation in response to either Cu/Zn SOD gene
expression or hyperoxic exposure. In the lung, there is a trend toward
a decrease in iron levels after exposure to hyperoxia, with significant
reductions observed in Sod1(+/) and Sod1(
/
)
mice (Fig. 6C). Although liver iron concentrations were not
affected by exposure to hyperoxia, iron concentration was influenced by
Sod1 genotype, as Sod1 overexpressors had
significantly lower iron levels (Fig. 6D). Significantly
lower iron levels were also observed in the lung of
Sod1(+++) mice (Fig. 6B).
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Figure 7 depicts circulating trace
mineral levels and serum ceruloplasmin activity in each strain of
Sod1 mice after exposure to hyperoxia. Circulating iron
levels were significantly elevated in Sod1(+/+),
Sod1(+/), and Sod1(
/
) mice
after hyperoxia (Fig. 7A). Hyperoxia did not, however, have
any effect on circulating copper (Fig. 7B) or zinc (Fig.
7C) levels, nor did it influence serum ceruloplasmin
activity (Fig. 7D). Notably, both circulating copper levels
and serum ceruloplasmin activity were markedly influenced by
overexpression of Cu/Zn SOD, as each was significantly reduced in
Sod1(+++) mice compared with all other genotypes.
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DISCUSSION |
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In this study, we have used genetically altered transgenic mice and exposure to a high but nonlethal level of oxygen as an animal model that is relevant clinically to the precarious situation experienced by premature infants. By using Cu/Zn SOD overexpressor and knockout transgenic mice, we have incorporated animals that disproportionately express a component of the endogenous antioxidant defense system. These animals are therefore chronically exposed to graded levels of oxidative stress in a manner analogous to premature infants that invariably inherit a compromised antioxidant defense system. We have also employed hyperoxic exposure, an exogenous oxidative stress that is relatively acute but nonlethal, to parallel its clinical use as a therapeutic treatment for preterm infants. Hence, by employing both Cu/Zn SOD transgenic mice and hyperoxia, our laboratory has been provided with a unique opportunity to examine the effects of both chronic and acute oxidative stress within a physiologically relevant condition. In particular, this model has allowed us to systematically examine the impact of oxidative stress on tissue-specific responses of metalloproteins as part of the cellular defense system and to identify the role of these metalloproteins in trace element homeostasis.
Results from this study provide in vivo evidence that, under acute oxidative stress, the metalloproteins ceruloplasmin and metallothionein may function as antioxidants without affecting tissue concentrations of copper or zinc. Specifically, our results demonstrate that ceruloplasmin may function as an important antioxidant in vivo, as lung tissue levels were markedly induced after acute exposure to hyperoxia (Fig. 4A), without significant changes in tissue copper levels (Fig. 4B). In the liver, the nontarget organ of hyperoxic exposure, ceruloplasmin levels were not affected by hyperoxia (Fig. 4C). Our results extend earlier observations of others that showed tissue-specific induction of ceruloplasmin mRNA in rat lung tissue during acute inflammation and hyperoxia (17). Furthermore, it is noted that tissue ceruloplasmin expression was induced by acute stress only and was not responsive to exposure to chronic oxidative stress imposed by Cu/Zn SOD knockout.
Similarly, metallothionein protein was induced in the lung (Fig.
5A) after hyperoxia without significantly affecting zinc concentrations (Fig. 5B). Previous studies have shown that
metallothionein mRNA levels (metallothionein I and metallothionein II)
are increased 10- to 12-fold in the liver of Sod1(/
)
mice. This induction was specific to the liver, as no significant
induction was observed in the kidney, spleen, heart, or lung of these
mice (20). Our study extends these findings by
demonstrating that, under hyperoxic exposure, metallothionein protein
levels are significantly induced not only in the liver but also in the
lung of each Sod1 strain of mice. In particular, induction
of metallothionein expression was highest in the lung and liver of
Sod1(
/
) mice compared with the other Sod1
strains (Fig. 5, A and C). Furthermore,
metallothionein appears to be more responsive to oxidative stress than
ceruloplasmin, as metallothionein was elevated in both the lung and
liver under hyperoxic conditions, whereas ceruloplasmin was elevated in
the lung only. In addition, liver metallothionein was also induced in
response to chronic oxidative stress imposed by diminished Cu/Zn SOD
expression (Fig. 5C). Together, these results demonstrate that, under acute oxidative stress, ceruloplasmin and metallothionein may function as antioxidant metalloproteins independent of their role
in trace metal homeostasis.
The mechanism by which the induction of these metalloproteins protects against acute oxidative stress is still unknown. It is interesting to speculate on the functional role of ceruloplasmin in lung tissue. Notably, the lung has been recently identified as a major site of extrahepatic ceruloplasmin synthesis (46). Ceruloplasmin protein has been shown in vitro to exhibit GSH-Px-like activity by removing H2O2 and lipid hydroperoxides (32). Moreover, Atanasiu et al. (2) have demonstrated in vitro that ceruloplasmin was an effective peroxyl radical scavenger and an effective chain-breaking antioxidant independent of its iron regulatory ferroxidase activity. Collectively, the characterization of GSH-Px-like activity and radical scavenging abilities of ceruloplasmin, as well as the hyperoxia-induced decline in pulmonary catalase protein (Fig. 2B) and activity (Fig. 3B) observed in our experiments, clearly suggest a critical role for ceruloplasmin as a compensatory antioxidant protein that is induced in the lung during acute oxidative stress. Indeed, the absence of hyperoxia-induced increases in ceruloplasmin in the liver or circulation may point to the tissue specificity of ceruloplasmin synthesis during oxidative stress. This proposed antioxidant function of ceruloplasmin under acute oxidative stress occurs independent of its function in binding copper and preventing Fenton-catalyzed ROS generation. In fact, our experiments demonstrate that the hyperoxia-induced increase in ceruloplasmin protein levels did not significantly alter lung tissue copper levels.
Similar to ceruloplasmin, the mechanism by which metallothionein exerts its antioxidant function is not clear. In vitro evidence reveals that cysteine-rich metallothionein reacts directly with ROS (1, 45). Metallothionein may also function as an antioxidant indirectly by releasing zinc, which may then inhibit metabolic activity, protect free thiols from oxidation, and stabilize biomembranes (7). Unfortunately, we were not able to quantify the release of zinc from metallothionein in vivo. Nevertheless, because the significant induction in metallothionein protein levels in the lung and liver under acute oxidative stress were not accompanied by an increase in zinc levels, our results are consistent with an antioxidant function of metallothionein distinct from its role in trace metal homeostasis. It is well documented that metallothionein is a redox-sensitive gene that is elevated during various forms of oxidative stress, including the administration of endotoxin, heavy metals, cytokines, and glucocorticoids (20). Metallothionein antioxidant properties may be indirectly executed through redox signaling, but the precise mechanisms remain unknown. Nevertheless, metallothionein protection against oxidative injury has been explored extensively using metallothionein-overexpressing transgenic mouse models. For example, under both acute and chronic conditions of oxidative stress, cardiac tissue of metallothionein-overexpressing mice displayed a marked resistance to oxidative injury compared with wild-type control animals (24).
The third metalloprotein we examined, ferritin, is characterized primarily as an iron storage protein (3). There is a growing body of evidence that the role of this protein may extend beyond metal storage and include, among other functions, a direct role in free radical defense. For example, mRNA encoding the ferritin light chain, which is involved in long-term iron storage, was increased severalfold in the lung tissue of rats exposed to hyperoxia (39). Ferritin protein levels were also found to increase in mice after exposure to 95% oxygen for 3 days (13). Our results, however, do not support these findings, since we did not detect any increase in lung or liver ferritin levels either in response to hyperoxia or Sod1 gene dosage (Fig. 6, A and C).
It is generally recognized that SOD, GSH-Px, and catalase form a
network that provides the first line of cellular defense against
oxidative stress, and there is evidence that manipulating the
expression of one enzyme may lead to an imbalance in antioxidant defense (15, 31). Therefore, we measured the effects of
Cu/Zn SOD on the expression and the activity of GSH-Px and catalase in
both lung and liver tissue. First, we observed that Cu/Zn SOD protein
levels were not responsive to acute oxidative stress, since neither
lung nor liver Cu/Zn SOD levels were significantly affected by exposure
to hyperoxia (Fig. 1, A and B). Others have reported similar observations (18, 25). Subsequently, we
observed that, in the lung, GSH-Px protein levels were increased as
Cu/Zn SOD expression decreased, yet enzyme analysis revealed that
GSH-Px activity did not reflect GSH-Px protein levels (Fig.
3A). However, we did observe increased GSH-Px activity after
hyperoxia in Sod1(+/+) and Sod1(+/) animals,
findings that are consistent with previous research (25).
Finally, we measured the activity and expression of catalase, an enzyme located primarily within peroxisomes. The results demonstrated, quite unexpectedly, that, in the lung, both the protein level and the activity of this enzyme were suppressed by >50% under the acute oxidative stress of hyperoxia (Figs. 2B and 3B). To our knowledge, this is the first time that this phenomenon has been reported. In the liver, the nontarget organ, catalase activity was reduced by 50% in mice that overexpress Cu/Zn SOD. The reason for this decline is not readily apparent and requires further study. However, it is interesting to note that the activity of catalase, a heme-containing enzyme, is lowest in the liver of Sod1(+++) mice, a result that coincides with significantly lower tissue iron levels in this same group (Fig. 6D). Nevertheless, our studies did not demonstrate a linkage between the expression and activity of the three primary antioxidant enzymes Cu/Zn SOD, GSH-Px, and catalase.
Copper is an integral component of Cu/Zn SOD and ceruloplasmin. It is generally recognized that ceruloplasmin is the primary extracellular copper transport protein, particularly when excess copper is present. Our study demonstrated that liver Cu/Zn SOD plays a critical role in copper homeostasis. This is indicated by the parallel increase in hepatic copper levels and hepatic Cu/Zn SOD expression (Figs. 4D and 1B). Moreover, the increased expression of Cu/Zn SOD in Sod1(+++) mice coincides with a significant reduction in circulating ceruloplasmin and copper levels (Fig. 6, D and B). Hence, we speculate that the demand for increased liver copper brought about by elevated Cu/Zn SOD protein levels was met, at least in part, by the supply of copper from circulating ceruloplasmin. Hence, these findings are consistent with previous work demonstrating that, in vitro, ceruloplasmin donates copper to Cu/Zn SOD (12) and may provide in vivo evidence that ceruloplasmin functions as a reservoir that supplies copper for Cu/Zn SOD synthesis.
In this study, we have employed transgenic animals in an in vivo model to describe the homeostatic relationship between the induction of antioxidant metalloproteins and trace mineral metabolism under chronic and acute oxidative stress. Understanding the regulatory link between the antioxidant defense system and trace mineral homeostasis under normal and pathophysiological conditions may provide insights for future therapeutic strategies, for example, in premature infants exposed to hyperoxia and other forms of respiratory diseases associated with excessive oxidative stress.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. M. Bray, College of Human Ecology, 350D Campbell Hall, 1787 Neil Ave., The Ohio State Univ., Columbus, OH 43210 (E-mail: bray.21{at}osu.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 9 January 2001; accepted in final form 15 February 2001.
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