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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. S51-S56, (2002)
© 2002 American Thoracic Society

Article

Transgenic and Knockout Models for Studying the Role of Lung Antioxidant Enzymes in Defense against Hyperoxia

Ye-Shih Ho

Institute of Environmental Health Sciences and Department of Biochemistry and Molecular Biology, Wayne State University, Detroit, Michigan

Correspondence and requests for reprints should be addressed to Ye-Shih Ho, Ph.D., Institute of Environmental Health Sciences, Wayne State University, 2727 Second Avenue, Detroit, MI 48201. E-mail: yho{at}wayne.edu

ABSTRACT

Although a role for antioxidant enzymes in preventing lung injury from hyperoxic exposure has been implicated in a number of early studies, a direct test for the hypothesis was not available. We intended to address this question using genetically modified mice in which the expression of a single antioxidant enzyme was either enhanced or diminished. We reasoned that if an antioxidant enzyme functions in protecting lung cells against oxidant-mediated injury, the level of its gene expression would correlate with the degree of tolerance to hyperoxia. Overexpression of functional human manganese superoxide dismutase (MnSOD) in lung alveolar type I and type II cells, fibroblasts, and capillary endothelial cells in strain B6C3 mice was achieved by incorporating a human ß-actin promoter-based MnSOD transgene into the mouse genome. However, MnSOD overexpression failed to prolong the survival of transgenic mice on exposure to greater than 99% oxygen compared with wild-type mice. In addition, mice deficient in copper–zinc superoxide dismutase or cellular glutathione peroxidase exhibited a marked sensitivity to numerous models of oxidant tissue injury but were not hypersensitive to hyperoxia. These data suggest that the role of these three antioxidant enzymes in preventing oxidant-mediated lung injury from hyperoxic exposure is negligible, and other cellular antioxidant enzymes and systems may be primarily used by the lungs in defense against hyperoxia.

Key Words: reactive oxygen species • superoxide dismutase • glutathione peroxidase • oxidant-mediated tissue injury

Reactive oxygen species (ROS), which are by-products of oxidative metabolism, are capable of causing oxidation of cellular macromolecules such as lipids, proteins, and DNA, leading to alterations of normal cellular metabolism, signaling, and function (1). To minimize the oxidative damage, all aerobic organisms from prokaryotes to eukaryotes are equipped with multiple enzymatic and nonenzymatic defense systems (2). The prototypic antioxidant enzymes include isoforms of superoxide dismutase for conversion of superoxide anion radical to hydrogen peroxide, and the enzymes catalase, isoforms of glutathione peroxidase (Gpx or GSHPx), and peroxiredoxins for detoxification of hydrogen peroxide and organic hydroperoxides (3). These antioxidant enzymes and other nonenzymatic mechanisms are generally adequate to prevent toxicity of ROS, thereby maintaining cellular homeostasis under normal physiologic conditions. However, increased production of ROS in both intracellular and extracellular spaces often occurs when cells or individuals are exposed to radiation, various toxicants, and certain therapeutic agents. Under these circumstances, the rate of ROS production may overwhelm the cellular capacity of antioxidant defense, resulting in oxidative tissue damage. An unbalanced production of ROS has been postulated to play a role in the pathogenesis of a large number of human clinical disorders such as acute respiratory distress syndrome, ischemia/reperfusion tissue injury, atherosclerosis, neurodegenerative diseases, and cancer (4).

The mechanism by which hyperoxia injures lungs has been studied extensively in adult rats (5). There are two distinct phases of lung injury during a lethal exposure of rats to hyperoxia (100% oxygen). Hyperoxia causes an increase in generation of superoxide anion radical and hydrogen peroxide in lung mitochondria and microsomes (6–8). Although the lung ultrastructure is apparently normal during the early phase (40 hours) of exposure, cell damage and death occur, ultimately leading to infiltration of phagocytic leukocytes and lymphocytes in the interstitial space in the later phase (60 hours) of exposure. The ROS generated by the inflammatory cells are believed to drastically exacerbate the oxidant injury resulting from the direct effect of hyperoxic exposure. Destruction of capillary endothelium appears to cause pulmonary edema and death.

A role for antioxidant enzymes, including cytosolic copper–zinc superoxide dismutase (CuZnSOD), mitochondrial manganese superoxide dismutase (MnSOD), and catalase, in attenuating hyperoxic lung injury has been implicated in a number of studies. Adult rats can survive under 100% oxygen for long periods if they are pre-exposed to a sublethal concentration (85%) of oxygen. This tolerance is associated with an increased activity of antioxidant enzymes in the lungs (5, 9–14). Protection for rats against hyperoxia can also be achieved by administration of a sublethal dose of endotoxin or cytokines, such as tumor necrosis factor and/or interleukin-1, through a variety of routes (15–24). In these models of tolerance to hyperoxia, an increase in lung activity of these antioxidant enzymes has also been observed. Lastly, intratracheal, intravenous, and intraperitoneal administration of CuZnSOD and/or catalase that are either encapsulated in liposomes or conjugated to polyethylene glycol can also ameliorate lung injury from hyperoxic exposure (25–27). These studies have suggested but have not directly demonstrated the protective role of antioxidant enzymes in hyperoxia-induced lung injury. For example, many other cellular changes probably occurred in addition to the observed induction of antioxidant enzymes in the lungs of rats after exposure to 85% oxygen and after administration of endotoxin and cytokines. These other cellular changes may play a crucial role in development of tolerance in adult rats to hyperoxia. In addition, most of the polymer-conjugated and liposome-entrapped antioxidant enzymes may remain in the extracellular space in the lungs, where they minimize the oxidative damage resulting from infiltrated phagocytic leukocytes, rather than protect lung cells after uptake. We therefore reasoned that additional animal models were needed to provide new insights into the mechanism of lung antioxidant defense against hyperoxia. We sought to address the question by using genetically modified mice in which the expression of a single antioxidant enzyme was altered by either transgenic or gene-targeting technology. We hypothesize that if an antioxidant enzyme plays a critical role in protecting lung cells against hyperoxia-induced injury, mice overexpressing this enzyme in the lungs will exhibit a resistant phenotype and mice deficient in this enzyme a sensitive phenotype.

MnSOD OVEREXPRESSION DOES NOT PROTECT B6C3 MICE AGAINST EXPOSURE TO GREATER THAN 99% OXYGEN

Because pulmonary capillary endothelial cells have been thought to be a primary target for hyperoxia-induced lung injury, which in turn leads to animal death, we initially planned to overexpress MnSOD in these cells. At that time there was no promoter that would direct a high level of gene expression specifically in lung capillary endothelial cells. We therefore constructed a MnSOD transgene driven by the human ß-actin promoter and 5' flanking sequence because ß-actin is abundantly expressed in all types of nonmuscle cells. Transgenic mice were subsequently generated using fertilized eggs harvested from breeding of B6C3 (C57BL/6 x C3H) F1 mice (28, 29). The transcriptional activity of the human ß-actin promoter-based transgene is very consistent in different lines of transgenic mice. The human MnSOD messenger RNA is highly expressed in brain, heart, lung, skeletal muscle, spleen, and tongue; and to a lesser extent in kidney and liver in line TgHMS66 transgenic mice that carry three copies of the transgene.

The biologic function and cell specificity of the expressed human MnSOD protein in mouse lungs were further characterized. We found an approximately 170% increase of MnSOD activity in total lung homogenates from hemizygous transgenic mice compared with nontransgenic littermates. Interestingly, the expressed human MnSOD in mouse lungs forms a tetramer as does the native human MnSOD protein, which can be separated from the native mouse MnSOD protein on a nondenaturing gel, and no heterotetramer between the human and mouse proteins can be detected. Immunocytochemistry study showed the overexpressed MnSOD in transgenic lungs to be localized to mitochondria of alveolar type I and type II cells, capillary endothelial cells, and fibroblasts. Furthermore, we found the labeling density of MnSOD in mitochondria of alveolar type II cells to correlate well with the increase in MnSOD activity. These studies established the function and cell distribution of the overexpressed MnSOD in lungs of transgenic mice (29).

The MnSOD transgenic and control mice were subsequently used in hyperoxic exposure studies to determine if a general overexpression of MnSOD in lungs provides protection against hyperoxia. There was no significant difference in the mean survival times of hemizygous MnSOD transgenic mice and control littermates when exposed to greater than 99% oxygen (4.5 ± 0.3 days and 4.6 ± 0.2 days, respectively). We also found, however, that the transgenic mice survived slightly longer than did control mice on exposure to 90% oxygen (6.3 ± 0.3 days and 5.3 ± 0.2 days, respectively; p = 0.012). These results are in contrast to those reported by Wispe and colleagues, where a line of MnSOD transgenic mice (strain FVB) with MnSOD overexpression specifically in alveolar type II cells and terminal bronchiolar epithelial cells, due to the use of a human surfactant protein C (SPC) promoter, are markedly resistant to hyperoxia (30). Because the levels of MnSOD overexpression in alveolar type II cells are equivalent in these two groups of transgenic mice generated with ß-actin promoter- or surfactant protein C promoter-based transgene, the discrepancy between our studies is likely the result of variance of mouse strains used in our studies.

Although our results show that MnSOD overexpression may not be capable of providing dramatic protection to the B6C3 hybrid strain of mice against hyperoxia-induced lung injury, the same transgenic mice have been shown to be more tolerant to several other models of oxidant-mediated tissue injuries that include acute adriamycin toxicity and ischemia/reperfusion injury in heart and kainic acid-induced hippocampal damage (31–33). These data have established the critical role of MnSOD in limiting tissue injury as a result of oxidative stress and suggested that the level of MnSOD expressed in certain tissues under normal physiologic conditions may not provide sufficient antioxidant defense under disease conditions.

Mice Deficient in CuZnSOD Develop Normally and Show No Increased Susceptibility to Hyperoxia
It is generally difficult to uniformly overexpress a gene in all types of cells within an organ of transgenic mice. Therefore, a negative result from the studies on different disease models is hardly conclusive as to the function of the enzyme in disease prevention. Mice that carry a targeted mutation will provide an alternate model to address the physiologic function of the gene of interest because the mutated gene is expressed in every single cell in the body. Toward this end, we have generated mice deficient in copper–zinc superoxide dismutase (CuZnSOD) by gene-targeting technology (34). The mouse Sod1 gene was mutated by replacing exon 5 including some intron 4 and 3' nontranslated sequences by a neomycin resistance cassette. This modification abolishes the expression of Sod1 messenger RNA with a concomitant depletion of CuZnSOD enzyme in all tissues examined. The diminished expression of CuZnSOD does not affect the expression of other antioxidant enzymes including MnSOD, catalase, and Gpx, and enzymes involved in the recycling of oxidized glutathione such as glutathione reductase and glucose-6-phosphate dehydrogenase. Mice deficient in CuZnSOD develop normally and are grossly healthy under routine animal husbandry. Furthermore, studies have shown that CuZnSOD-deficient mice do not exhibit an increased sensitivity to hyperoxia (34), suggesting the function of this enzyme in lung defense against the damage from an increased production of superoxide anion radical during hyperoxia is very limited. These results indicate that the protective role of CuZnSOD may depend on the subcellular site of production of superoxide anion radical. Because CuZnSOD is predominantly present in the cytosol, we conclude that the cytosol may not be a major site for superoxide overproduction in lung cells during hyperoxia. However, a cytosolic overproduction of superoxide anion radical is believed to participate in the pathogenesis of certain other models of oxidant tissue injury. Our data have shown that mice deficient in CuZnSOD are more sensitive to acute paraquat toxicity, myocardial ischemia/reperfsuion injury, and N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration (34–36).

One of the most intriguing observations we made in the Sod1 knockout mice is that homozygous female knockout mice are inferior in reproduction compared with wild-type and heterozygous knockout female mice (34). Although these mice ovulate and conceive normally, a majority of the embryos die before 12.5 days postcoitum. The mechanism by which CuZnSOD deficiency reduces female fertility remains to be determined.

MICE DEFICIENT IN CELLULAR GLUTATHIONE PEROXIDASE ARE HEALTHY UNDER NORMAL PHYSIOLOGIC CONDITIONS AND DO NOT EXHIBIT HYPERSENSITIVITY TO HYPEROXIA

Four isoforms of Gpx with different tissue specificity of expression have been found in mammals. To understand the function of Gpx in cellular antioxidant defense mechanism, we decided to inactivate the cellular glutathione peroxidase (Gpx1), the major isoform of Gpx expressed in all tissues. The mouse Gpx1 gene was interrupted by insertion of a neomycin resistance cassette in exon 2 (37). This modification resulted in expression of a high molecular weight messenger RNA presumably representing the fused transcripts between the mouse Gpx1 gene and the neomycin resistance gene. This aberrant transcript was very unstable and only very minute quantities could be detected in kidney and liver cells of homozygous knockout mice. No normal Gpx1 messenger RNA was expressed in any tissues of homozygous knockout mice. As expected, no or very low total Gpx activity could be detected in these tissues. The residual Gpx activity in tissues such as heart, kidney, liver, and lung may result from the expression of the Gpx genes coding for other isoforms. Furthermore, a deficiency or markedly reduced activity of Gpx seems to have no effect on the expression of some other known antioxidant enzymes such as CuZnSOD, MnSOD, and catalase. Mice deficient in Gpx1 are healthy and fertile and show no gross histologic changes in the major organs studied, with the exception of an earlier onset of cataract compared with that of wild-type mice (38). However, a deficiency in Gpx1 does not render mice more susceptible to hyperoxia, suggesting the limited role of Gpx1 in lung defense against cell injury resulting from hyperoxic exposure.

These results do not rule out the protective function of Gpx1 in mice in other models of oxidant tissue injury. Indeed, Gpx1-deficient mice are more susceptible to hydrogen peroxide-induced DNA strand breakage in lens epithelial cells (39), ischemia/reperfusion injury in heart and brain (40, 41), acute paraquat toxicity (42), Coxsackie virus B3-induced myocarditis (43), neutrophil-mediated hepatic parenchymal cell injury during endotoxemia (44), N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced loss of dopaminergic neurons (36), and noise-induced hearing loss than wild-type mice (45). It should be noted that the rates of detoxifying extracellular hydrogen peroxide ex vivo in tissue slices derived from wild-type and Gpx1-deficient mice are equivalent, suggesting that catalase is capable of removing hydrogen peroxide effectively without the help of Gpx1 in Gpx1-deficient tissues. We, therefore, speculate that the increased sensitivity of Gpx1 knockout mice in these models of oxidant tissue injury may result from an accumulation of organic hydroperoxides, against which catalase has very limited activity, in tissues after oxidative stress.

DISCUSSION

Our and other laboratories have taken the approach of using genetically modified mice to address the functional role of antioxidant enzymes in lung defense against hyperoxia. We believe that if an antioxidant enzyme plays a critical role in protecting lung cells from hyperoxia-induced injury, overexpression of this enzyme in the lungs of transgenic mice should result in a resistant phenotype and abolishment of enzyme expression in knockout mice a sensitive phenotype. This hypothesis has been validated in several models of oxidant-mediated tissue injury. For example, transgenic mice overexpressing CuZnSOD or Gpx1 are more resistant to ischemia/reperfusion injury in both brain and heart (46–49), whereas mice deficient in either of these enzymes are more susceptible to the injury (35, 40, 41, 50). These studies have not only established the role of these two antioxidant enzymes in cellular defense against injury from reperfusion of tissues after ischemia, but have also suggested the identities of the pathogenic species of ROS that are generated during reoxygenation. However, results from studies performed on these models of mice under hyperoxic conditions are not as straightforward as those from ischemia/reperfusion studies. Our data showed that mice deficient in CuZnSOD or Gpx1 did not exhibit a hypersensitive phenotype to hyperoxic exposure, suggesting that the role of these enzymes in defense against hyperoxia is negligible. This conclusion agrees with that from the studies reported by White and colleagues (51). Their studies showed that CuZnSOD transgenic mice were more resistant to hyperoxia at 630 mm Hg barometric pressure than control mice at a young age (2.5 months) but not at an older age (5.5 months). These results indicate that overexpression of CuZnSOD alone is not sufficient to prevent hyperoxic lung injury. The presence of other cellular responses that are diminished during the aging process is critical for the observed protection provided by CuZnSOD in young transgenic mice against hyperoxia.

A similar conclusion on the function of MnSOD in lung defense against hyperoxia may be drawn from the studies performed in our and Dr. Wispe's laboratories (29, 30). As indicated previously , the levels of MnSOD overexpression in lung alveolar type II cells are compatible in transgenic mice carrying a human ß-actin promoter-based or a human surfactant protein C promoter-based MnSOD transgene. However, protection of the lungs against hyperoxia is significantly less in mice with a B6C3 hybrid genetic background than in mice with an inbred FVB background. Again, these results suggest that a single overexpression of MnSOD may not be sufficient to ameliorate lung injury from hyperoxic exposure. Unfortunately, adult knockout mice homozygous for the mutated MnSOD gene are not available for hyperoxic study due to their limited life span (52, 53). Heterozygous MnSOD knockout mice are not more susceptible to hyperoxia, indicating that a 50% expression of MnSOD is as effective as a normal level of MnSOD in defense against hyperoxia (54, 55). However, a more severe lung structural damage in association with an increased mortality was found in neonatal homozygous knockout mice exposed to 50 or 80% of oxygen compared with that of wild-type mice (56). New models of knockout mice with tissue-specific or inducible disruption of MnSOD gene will be needed for extending this observation regarding the essential role of MnSOD as a lung antioxidant defense mechanism to adult mice.

To date, the only antioxidant enzyme that meets the criteria in our hypothesis on cellular protection against hyperoxia is extracellular superoxide dismutase. Extracellular superoxide dismutase is a copper–zinc-containing glycoprotein and is secreted by cells into the extracellular space. In mice, extracellular superoxide dismutase is expressed more highly in the lungs than in other organs (57). In human lungs, it is predominantly present in regions of extracellular matrix that contain high amounts of type I collagen, around the larger vessels and airways, and to a lesser extent in alveolar and capillary regions (58). Carlsson and colleagues have shown that knockout mice deficient in extracellular superoxide dismutase are more susceptible to hyperoxia than wild-type mice (57). In addition, transgenic mice overexpressing extracellular superoxide dismutase as a result of the carried surfactant protein C promoter-based transgene are more resistant to hyperoxia (59). Furthermore, this tolerance to hyperoxia in transgenic mice is associated with a decrease in infiltration of inflammatory cells, in particular polymorphonuclear leukocytes, into the lungs. These results not only indicate that tissue inflammation at the later phase of hyperoxic exposure plays a major role in causing lung injury but also suggest the potential of using antiinflammatory drugs in conjugation with antioxidant therapy for attenuating oxidant-mediated lung injury.

There is one concern that may complicate drawing straightforward conclusions from our studies. Changes in gene expression in the lungs in response to depletion of CuZnSOD or Gpx1 or overexpression of MnSOD may contribute to the observed phenotypes after hyperoxic exposure. Although no compensatory changes in expression of all the prototypic antioxidant enzymes have been found in these transgenic and knockout mice, studies have not ruled out the possibility that the expression of other cellular stress responsive and antioxidant enzymes is altered. Recent results have shown that MnSOD overexpression in two different tumor cell lines augments the expression of matrix metalloproteinase-1, proteinase inhibitor maspin, and DNA damage and repair gene GADD153 and downregulates the expression of several other genes (60–62). Future studies to uncover the altered gene expression in the lungs of these transgenic and knockout mice by microarray gene profiling and proteomics technologies should provide a more comprehensive understanding in the cellular and physiologic functions of antioxidant enzymes.

In addition to the studies on the function of these "prototypic" antioxidant enzymes, investigations in the potential role of other antioxidant/detoxification enzymes as well as DNA repair enzymes in lung defense have also been initiated. Cho and colleagues have shown that mice deficient in NF-E2–related factor 2, a Cap'n'Collar transcription factor functioning in transactivation of a number of phase II detoxification enzymes by binding to the antioxidant response elements in the promoters of the respective genes, are markedly more sensitive to hyperoxia than wild-type mice (63). As expected, the increased sensitivity of these knockout mice to hyperoxia is associated with decreased basal messenger RNA levels as well as hyperoxia-induced expression of many phase II genes including nicotinamide adenine dinucleotide (phosphate) reduced:quinone oxidoreductase 1, glutathione-S-transferase, and uridine diphosphate glycosyl transferase, etc. in the lungs. These studies have broadened our view to allow recognition of the function of other detoxification pathways additional to removal of ROS by the prototypic antioxidant enzymes in lung antioxidant mechanism. Another good example comes from the studies reported by Wu and colleagues (64). Because oxidative stress causes oxidation of DNA bases, they reasoned that an enhanced capacity in DNA repair would attenuate cellular oxidant damage. Indeed, overexpression of base excision repair enzymes, the human 8-oxoguanine DNA glycosylase, or the Escherichia coli formamidopyrimidine DNA glycosylase, renders cultured human lung carcinoma cell line A549 more resistant to hyperoxia and hydrogen peroxide. If the same approach can also provide protection to the lungs in a whole animal remains to be determined. Nonetheless, the conclusion that the efficiency of DNA repair after oxidative stress affects cell survival is supported by the studies performed by O'Reilly and colleagues (65). Their studies showed that knockout mice deficient in p21, a cyclin-dependent kinase inhibitor, were susceptible to hyperoxia. Apparently, the hyperoxia-induced inhibition of DNA replication is absent in the lungs of these mice. These results suggest that blockage of DNA replication in lung cells after hyperoxia may provide a critical time window at which repair of damaged DNA proceeds and that a failure in DNA repair is likely to exacerbate cell injury.

Lastly, manipulation of cellular antioxidant systems may not be the only approach to studying the mechanisms of protection of the lungs against hyperoxia. In mice, the injured lung cells exhibit features of cell death by both apoptotic and necrotic pathways at the late stage of hyperoxic exposure (66). Waxman and colleagues have shown that overexpression of interleukin-6 or interleukin-11 in transgenic mice carrying Clara cell 10-kD protein (CC10) promoter-based transgenes are markedly more resistant to hyperoxia than wild-type mice (67, 68). The mechanisms contributing to this tolerance are not understood. However, interleukin-6 overexpression in mouse lungs is associated with induction of antiapoptotic genes Bcl-2 and tissue inhibitor of metalloproteinase-1, and interleukin-11 overexpression with activation of an antiapoptotic gene, survivin (68, 69). Along the same line of research, Lu and colleagues have succeeded in prolonging survival of mice under hyperoxia by adenoviral gene transfer of a mutated gene coding for a constitutively active form of Akt, a general mediator of cellular survival signals (70). These studies strongly suggest that prevention of activation of cell apoptotic pathways in airway and alveolar epithelial cells can also dramatically delay or decrease cell death resulting from hyperoxic exposure. This further implies that modulation of cellular survival pathways may represent an alternate and effective therapy for treating oxidant-mediated lung diseases in humans.

Acknowledgments

The author thanks Dr. Homer A. Boushey for his editorial comments on this article.

Received in original form June 14, 2002; accepted in final form September 30, 2002

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