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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Reactive oxygen species (ROS) are implicated as agents of cellular damage in pulmonary oxygen toxicity. Glutathione (GSH) and GSH-dependent antioxidant enzymes protect against damage by ROS, and recycling of glutathione disulfide (GSSG) to GSH by glutathione reductase (GR) is essential for the optimum functioning of this system. Exposure to hyperoxia inhibits lung development in newborn animals and humans, and attenuates cell growth in proliferating cell cultures. Considerable evidence supports a role for ROS as growth-altering molecules. Previously, we have observed that gene transfer of GR to mitochondria in H441 cells, using a vector containing a mitochondrial leader sequence (LGR), protected these cells against t-BuOOH-induced cytotoxicity. The present studies tested the hypothesis that gene transfer of LGR would attenuate the cytostatic effects of hyperoxia exposure in H441 cells. H441 cells (0.9 × 106 cells/plate) transfected with adenovirus containing LGR or the complementary DNA (cDNA) for manganese superoxide dismutase in reverse orientation (DOS) as a control construct, and untransfected cells (CON) were maintained in 21% oxygen (normoxia) or 95% oxygen (hyperoxia) for 48 h, and cell growth was assessed by cell counts and by reduction of the tetrazolium dye 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan. Cells maintained in normoxia achieved normal growth (CON, 1.98; DOS, 1.91; LGR, 2.0 × 106 cells/plate). Hyperoxia inhibited cell growth and the reduction of MTT; however, cells transfected with LGR had greater mitochondrial GR activities (CON, 16 ± 2; DOS, 19 ± 3; LGR, 322 ± 18 mU/mg of protein), sustained more normal growth patterns (CON, 1.25 ± 0.12; DOS, 1.24 ± 0.21, LGR, 1.8 ± 0.25 × 106 cells/plate), and had less inhibition of MTT reduction (CON, 29; DOS, 27; LGR, 16% inhibition, P < 0.01) after exposure to hyperoxia for 48 h than was observed in cells transfected with DOS or in control cells not infected with virus. In addition, resistant cells had higher mitochondrial GSH levels and maintained mitochondrial GSH/GSSG ratios in hyperoxia, suggesting that maintaining mitochondrial GSH homeostasis determined critical aspects of cell division in these studies. The mechanisms for sustaining cell growth during hyperoxia in H441 cells with enhanced mitochondrial GR activities are unknown, but similar effects in infants exposed to supplemental oxygen could be highly beneficial.
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Introduction
Materials and Methods
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Discussion
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Exposure to high concentrations of oxygen is often necessary in the management of critically ill newborn infants. However, the benefits of such therapy are achieved with a substantial risk of oxygen-induced lung damage (1). The association between lung injury and high oxygen concentrations has been well established, but the precise cellular mechanisms involved remain unclear. Reactive oxygen species (ROS) are implicated as agents of cellular damage in many disease states, including pulmonary oxygen toxicity (4, 5). Evidence also indicates that these by-products of cellular respiration are important components of normal cell metabolism and may play roles in cell cycle regulation (6, 7). A dynamic range of redox interactions exist, governed by a complex network of cellular antioxidant systems, which, if unbalanced by excessive production of ROS, may exceed the capacities of cellular defenses and result in oxidative damage to proteins, lipids, and DNA, which can cause cell dysfunction or death (8).
Intracellular glutathione (GSH) and GSH-dependent enzymes provide cellular protection against oxidants by using selenium-dependent and -independent peroxidases to reduce hydrogen peroxide to water or lipid peroxides to the respective alcohols, with the concurrent oxidation of GSH to glutathione disulfide (GSSG) (13). The flavoenzyme glutathione reductase (GR) catalyzes the reduction of most intracellular GSSG back to GSH, at the expense of nicotinamide adenine dinucleotide phosphate (NADPH) oxidation. Increased steady-state concentrations of GSSG have been implicated as contributing to mechanisms through which oxidants injure cells, and the protective role of GR is attributed to preservation of protein thiol status by limiting the concentrations of GSSG and sustaining GSH availability (14). Several studies have indicated that cells with diminished GR activities have increased susceptibilities to oxidative stresses, and we have observed enhanced resistance to oxidant injury in cells with increased GR activities (17).
Mitochondria are major sources of ROS production, and mitochondrial GSH is critical to cell viability (4, 23). We have observed that stable Chinese hamster ovary (CHO) cell lines expressing a construct in which a functional mitochondrial targeting sequence was ligated to the 5' end of the human GR complementary DNA (cDNA) (LGR) showed highly selective enhancement of mitochondrial GR activities and were more resistant to oxidant stresses than were CHO cells stably transfected with GR lacking mitochondrial targeting (hGR) (21). In other studies, we observed that H441 cells, which are derived from lung Clara cells, were protected from t-BuOOH-induced cell death by adenovirus-mediated gene transfer of GR similarly targeted for expression in the mitochondria.
Exposure to hyperoxia inhibits lung development in newborn animals and humans, and attenuates cell growth in proliferating cell cultures (3, 24, 25). In premature infants, hyperoxia exposure during a critical period of pulmonary maturation may inhibit lung alveolarization and result in a permanent reduction in alveolar number associated with long-term pulmonary morbidity. Considerable evidence supports a role for ROS as mediators of inhibition of cell growth and development (6, 7). Although the mechanisms of action are largely unknown, experimental data suggest that the functions of many growth-modulating proteins are dependent on their redox states (26). Redox regulation of such proteins could be achieved either through direct oxidative interactions with ROS or indirectly through changes in cellular levels of GSH and GSSG and subsequent thiol-disulfide exchange reactions. The present experiments were designed to test the hypothesis that enhanced mitochondrial GR activities in H441 cells would attenuate the cytostatic effects of exposure to 95% O2 (hyperoxia). We also investigated the effects of the adenoviral transfection-mediated enhancement of mitochondrial GR activities on cellular, mitochondrial, and extramitochondrial GSH and GSSG levels during exposure to hyperoxia. Cells transfected with LGR showed greater mitochondrial GR activities, were more effective in maintaining GSH levels, and sustained more normal growth patterns during exposure to hyperoxia than did cells transfected with an irrelevant gene or nontransfected control cells. These data suggest that augmentation of mitochondrial GR activities in lung-derived cells by adenovirus-mediated transfer of LGR can afford greater resistance to hyperoxia-induced growth inhibition.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Materials
H441 cells, a transformed human lung cell line with characteristics of Clara cells, were obtained from American Type Culture Collection (Manassas, VA). Media (RPMI 1640) used in all experiments were purchased from GIBCO BRL (Gaithersburg, MD). Reagents for protein assays were obtained from Bio-Rad Laboratories (Richmond, CA). All other reagents were obtained from Sigma Chemical Company (St. Louis, MO).
Vector Construction
Details on the cloning of human GR cDNA and addition of the
mitochondrial targeting signal (MTS) from the human manganese superoxide dismutase (MnSOD) gene have been described
previously (29). Using a GR cDNA with a functional MTS, we
constructed an adenoviral vector from the replication-defective
human adenovirus type 5 (Ad5). Briefly, human GR cDNA containing a functional MTS from the human MnSOD gene was ligated downstream from a elongation factor-1 alpha promoter
(LGR for GR construct containing an MTS), after which the promoter-cDNA sequence was inserted into the E1 region of the
shuttle plasmid p
E1sp1B (Microbix Biosystems, Inc., Toronto,
ON, Canada). The shuttle plasmid DNA and the circular Ad5 genome plasmid pBHGE3 (Microbix Biosystems, Inc.) were
cotransfected into 293 cells to generate recombinant adenovirus
AdLGR. Viral propagation, purification, and titration were carried out according to standard methods described elsewhere (30).
To provide experimental controls for the effects of viral transfection, we ligated the human MnSOD cDNA in the antisense orientation (DOS) into identically constructed adenoviral vectors.
Cell Transfection and Exposure to Oxygen
H441 cells (average of 0.5 × 106 cells/plate) were plated in 100-mm tissue culture dishes and incubated in media with 5% fetal bovine serum at 37°C. After 24 h, the cells were counted, the media changed, and the cell plates exposed to adenoviral vectors containing either LGR or DOS at a calculated multiplicity of infection (MOI). Cells (average of 0.9 × 106 cells/plate) were exposed to 21% O2 with 5% CO2 (normoxia) or to 95% O2 with 5% CO2 (hyperoxia) 24 h after transfection. After 24 or 48 h, the cells were collected, the cell monolayer in each plate was washed (three plates per group) with ice-cold phosphate-buffered saline (PBS) (GIBCO BRL), and the cellular material was collected by scraping in 700 µl of 0.2 M KPO4, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0, and 0.2% Triton X-100. After collection, the cell samples were incubated on ice for 1 h and centrifuged for 5 min at 10,000 × g. Lactate dehydrogenase (LDH) and GR activities, and GSSG, GSH, and protein concentrations were measured in the resulting supernatants. In subsets of transfected cells, subcellular fractions were prepared by differential centrifugation using an adaptation of the method described by Evans (31). In other studies, as indicated, the cells were collected after 48 h of exposure to normoxia or hyperoxia, the cell monolayer in each plate was washed (four plates per group) with ice-cold PBS and the cellular material was collected by scraping. After collection we employed the digitonin methods described by Andersson and Jones (32) for rapid separation of mitochondrial and postmitochondrial fractions, to provide subcellular preparations for measurements of GSH and GSSG concentrations.
Assessment of Cell Proliferation
Two different growth assays were used to determine cell growth. For the first assay, 100-mm tissue culture dishes containing either transfected (DOS, LGR) or untransfected cells (CON) were collected after exposure to 24 or 48 h of normoxia or hyperoxia. The plates were washed with ice-cold PBS and 0.5 ml of trypsin was added to each plate. After the cells were dislodged from the surface of the culture dishes, 0.5 ml of media was added to each plate to stop the trypsinization process. The cell suspension was then removed from each plate and the number of cells per plate was determined using a hemocytometer.
The effects of exposure to 95% O2 on cell proliferation were also
determined by a colorimetric assay based on the ability of viable cells
to reduce the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan (33). Cells treated in a
similar fashion (CON, DOS, or LGR) were placed in 96-well plates and incubated in hyperoxia. Each well was paired with cells treated identically and placed in normoxia (control). At 24 or 48 h of exposure, MTT reduction activities were determined and activities in cells
exposed to hyperoxia were expressed as a percentage of the reduction activities in corresponding cells incubated in normoxia, calculated as: (absorbance of cells exposed to normoxia) 
(absorbance of
cells exposed to hyperoxia)/(absorbance of cells exposed to normoxia) × 100.
GR Assay
H441 cells were transfected with an adenovirus containing LGR or with an adenovirus containing DOS. Control cells were not exposed to virus. Cells were harvested and subcellular fractions were prepared by differential centrifugation using an adaptation of the method described by Evans (31) 48 h after transfections. Supernatant (57 µl) was mixed with 193 µl of assay mixture (0.1 M Tris-HCl, pH 8.0, 7 mM GSSG, and 0.225 mM NADPH) in one well of a 96-well microtiter plate, and the rate of consumption of NADPH was measured as the change in optical density per min at 340 nm. In this reaction, 1 U of GR is defined as the oxidation of 1 µmol of NADPH per min at 25°C (34).
LDH Assay
A total of 15 µl of cellular supernatants, appropriately diluted with 1× PBS in order to measure the activity within the standard curve, or 50 µl of media was mixed with 200 µl of a reaction mixture (216 µM nicotinamide adenine dinucleotide [NADH] in 60 mM KPO4, 0.72 mM pyruvate, pH 7.5). LDH activities were determined by the rates of NADH oxidation measured as the changes in optical density at 340 nm (34).
GSH and GSSG Assays
GSH and GSSG concentrations were determined by an adaptation of the method described by Tietze (35). For determination of GSH + 2GSSG concentrations, 30 µl of cellular supernatants, appropriately diluted with 1× PBS, or 200 µl of media were mixed with 50 µl of reaction mixture 1 (18.75 µl of 0.1 M phosphate buffer, 5 mM EDTA, pH 7.5, 25 µl of 10 mM 5,5'-dithio-bis[2-nitrobenzoic acid], 6.25 µl of GR [5 U/ml]) and 50 µl of reaction mixture 2 (37.5 µl of 0.1 M phosphate buffer, 5 mM EDTA, pH 7.5, 12.5 µl of 0.225 mM NADPH). The GSH + 2GSSG concentrations were determined from measurements of rates of change in absorbance at 410 nm and calculated from experimentally derived standard curves. Similarly, for GSSG determinations, 30 µl of Sep-Pak (Waters Corp., Milford, MA) eluent of cellular supernatants were mixed with 50 µl of reaction mixture 1 and 50 µl of reaction mixture 2, and GSSG concentrations were determined from measurements of rates of change in absorbance at 410 nm and calculated from experimentally derived standard curves. The GSH concentrations were calculated from the differences in the values, and GSH and GSSG concentrations are expressed per milligram of protein.
Protein Assay
The protein concentrations of the supernatants were measured using a modification of the Bradford assay (36). A total of 25 µl of cellular supernatant, diluted with distilled water as needed to measure the activity within the standard curve, was mixed with 200 µl of dye (Bio-Rad reagent diluted 1:5) and the absorbances at 600 nm were measured with a microtiter plate reader (Dyna-tech Laboratories, Chantilly, VA). The protein concentrations were calculated using experimentally derived standard curves constructed with bovine serum albumin.
Statistics
All data are expressed as means ± standard error of the mean
(SEM). Data were analyzed with two- or three-way analysis of variance (ANOVA), as indicated. When a three way ANOVA
indicated statistically significant differences or significant interactions, subsequent two-way ANOVAs were carried out as indicated. When two-way ANOVAs indicated statistically significant
differences or an interaction, appropriate post hoc testing was
performed with one-way ANOVA or modified t tests. All analyses were performed using SPSS for windows (version 7.0; SPSS,
Chicago, IL). Statistical significance was attributed to P 
0.05.
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Results |
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Introduction
Materials and Methods
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Discussion
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In H441 cells transfected with adenovirus containing LGR at 10 MOI, the dose that we previously found to be optimal for maximal increases in GR activities with minimal toxicity (22), total cellular GR activities were 4-fold higher, cytosolic GR activities were approximately 1-fold higher, and mitochondrial GR activities were approximately 18-fold higher than in nontransfected control cells or in cells transfected with adenovirus containing DOS (Figure 1). Although we have previously reported greater increases in GR activities in the cytosolic compartments of H441 cells transiently transfected with adenovirus containing LGR at 10 MOI, using gentler subcellular fractionation techniques we have found that the increases in GR activities achieved by the construct and methods described in the present studies are even more selective for expression in the mitochondria.
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Microscopic examination of the cells after 48 h revealed no obvious visual differences in cellular morphologies between untransfected control cells and cells transfected with LGR or DOS, either maintained in normoxia or hyperoxia (data not shown). Hyperoxia inhibited growth and proliferation of CON and DOS-transfected cells but did not similarly affect cells transfected with the LGR construct (Figure 2). Higher cell counts in cells transfected with LGR were evident at 24 h and persisted through 48 h of exposure to hyperoxia. Exposure to hyperoxia also diminished cellular capacities to reduce MTT (Figure 3). The LGR-transfected cells showed no differences from control cells or DOS-transfected cells in the effects of 24 h of hyperoxia on MTT reduction, all cells showing declines in MTT reduction of 20%. However, after 48 h of hyperoxia the LGR-transfected cells showed no further decreases in MTT reduction activities, whereas the abilities of both the control cells and the DOS-transfected cells to reduce MTT declined further. No differences in MTT reduction were observed between transfected (DOS, LGR) and nontransfected (CON) cells maintained in normoxia (data not shown). The release of cellular LDH into the incubation media ranged between 3 and 4.5% and was not different in any group with any treatment (data not shown), indicating that the mechanisms responsible for sustaining cell numbers during hyperoxia in cells transfected with LGR are not readily explained by increased cell death in the control or DOS-transfected cells.
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Cellular GSH levels were 33% higher in the DOS-transfected cells grown in normoxia than in the CON-AIR cells, but the cells transfected with LGR and cultured in normoxia were not different from the CON cells (Figure 4A). GSH levels in nontransfected control cells exposed to hyperoxia were diminished by 46%, and concentrations in DOS-transfected cells exposed to hyperoxia were 36% lower than in the same cells cultured in normoxia. In contrast, cellular GSH levels in LGR-transfected cells were 25% greater in cells exposed to hyperoxia than in LGR cells maintained in normoxia (Figure 4A). Cellular GSSG levels were similar in all cells maintained in normoxia, with levels uniformly less than 1% of the GSH levels (GSH/GSSG ratios greater than 100), and the cellular GSSG concentrations were increased in cells exposed to hyperoxia (Figure 4B). Interestingly, cells transfected with LGR and exposed to hyperoxia, which had maintained cellular GSH levels in hyperoxia, exhibited the highest cellular GSSG levels, whereas the lowest levels of cellular GSSG in hyperoxia were observed in the control cells. Intermediate levels of GSSG were observed in the DOS-transfected cells. In view of this finding, we measured the cellular release of GSSG into the incubation media in all cells over the time course of the study. Slight increases in the medium GSSG concentrations were observed in all cells exposed to hyperoxia; however, no differences were observed between control cells or cells transfected with LGR or DOS (data not shown).
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We also employed the digitonin fractionation procedure described by Andersson and Jones (32) for measurements of GSH and GSSG contents in the mitochondrial and postmitochondrial fractions isolated by these methods. We recognized the potential for cellular disruption and fractionation procedures to allow or cause changes in thiol disulfide status, but the predominant effect on thiol/disulfide status of transfection with the LGR construct would be expected to be expressed in the quantitatively minor mitochondrial compartment. In cells maintained in normoxia, mitochondrial (digitonin) GSH levels estimated by these methods were not different in nontransfected cells and in cells transfected with the DOS construct, but mitochondrial (digitonin) GSH levels in the LGR cells were more than double the levels in the other two cell lines (Figure 5A). Exposure to hyperoxia diminished GSH levels in digitonin-mitochondrial fractions of CON cells by 73% and in DOS-transfected cells by 76% from the levels observed in cells cultured in normoxia, whereas GSH levels in mitochondrial fractions of LGR-transfected cells were only 27% lower in cells exposed to hyperoxia than in LGR cells maintained in normoxia (Figure 5A). Mitochondrial GSSG levels were similar in all cells maintained in normoxia and were not affected by exposure to hyperoxia (Figure 5B). Mitochondrial GSH/GSSG ratios calculated from these measurements were lower in nontransfected control cells and in DOS-transfected cells exposed to hyperoxia than in the respective cells cultured in normoxia, whereas GSH/GSSG ratios in the mitochondrial fractions of LGR-transfected cells were maintained through 48 h of hyperoxia (Figure 5C). GSH and GSSG concentrations measured in postmitochondrial fractions from the digitonin procedures were similar in all cells maintained in normoxia. The GSH levels and GSH/GSSG ratios were lower in cells exposed to hyperoxia for 48 h than in the cells in normoxia, but no differences among the cells were associated with transgene transfection (data not shown). After exposure to hyperoxia, lower GSH levels and GSH/GSSG ratios were observed in the posmitochondrial fractions of cells transfected with virus (DOS and LGR) than in the untransfected CON cells, but in all samples the GSH/GSSG ratios were far more oxidized than we observed in the same cells not subjected to these suspension, permeabilization, and fractionation procedures before acidification or derivatization.
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The present studies demonstrate that adenovirus-mediated gene transfer of LGR can increase the mitochondrial GR activities in H441 cells and confer partial protection from the growth inhibitory effects of exposure to hyperoxia. Using the methods employed in our studies, efficiencies of transfection of marker genes approaching 100% have been reported (37, 38). Although significant increases in cytosolic GR activities are achieved by transfection with LGR at a viral dose of 10 MOI, the present studies indicate that the mitochondria-specific increases in GR activities are elevated to a much greater extent than are the cytosolic levels. The greater selectivity for the enhancement of GR activities in the mitochondria observed in the present studies than what we reported previously (22) are attributed to the gentler cellular homogenization and subcellular fractionation techniques used in the present studies (31). Immunohistochemical analyses of CHO cells stably transfected with LGR provide additional evidence that the mitochondrial targeting sequence is highly effective in directing the GR protein derived from the LGR construct to the mitochondria (39). Whether the increases in GR activities observed in the cytosolic fractions from cells transfected with LGR represent real increases in this cellular compartment or imperfect separation of the cellular compartments is not completely resolved.
The lower cell counts observed after exposure to hyperoxia of nontransfected cells and cells transfected with DOS than of cells transfected with LGR (Figure 2) could result from decreased cell division or increased cell death. Hyperoxia did not increase LDH activities in the media, suggesting that the difference in cell counts observed in the studies are not attributable to greater rates of cell death. Increased rates of apoptotic cell death might avoid detection through measurements of medium LDH activities if apoptotic bodies were phagocytosed by adherent cells with very high efficiencies. In addition, we did not observe any histologic evidence of morphologic changes in the cells or of cells detaching from the culture plate. The more straightforward inhibition of cell proliferation thus appears to provide a more plausible explanation of the effects of hyperoxia on attenuation of increases in cell numbers.
Resistance to hyperoxia-induced growth inhibition was clearly evident after 24 h of exposure to hyperoxia in cells with enhanced mitochondrial GR activities. Although the initial rates of increase in cell counts had somewhat slowed after 48 h of exposure to hyperoxia, the doubling time of the LGR-transfected cells was similar to the times observed in cells maintained in normoxia. In contrast, differences between hyperoxia-exposed groups in the ability to reduce MTT only became evident at 48 h. This discrepancy in the timing between the observed effects of LGR transfections on cell counts (24 and 48 h) and MTT reduction (48 h) may indicate a lower sensitivity of the MTT assay for detecting differences in cell numbers, although other possibilities, such as the induction of other common antioxidant enzymes, could explain these observations. Hyperoxia may have diminished cellular abilities to reduce MTT, but such an interpretation would require that this effect be exerted preferentially in LGR cells because DOS-transfected cells showed decreases both in cell numbers and in MTT reduction activities. The observed rates of increases in cell numbers were similar in all cells maintained in room air. In addition, cell counts in nontransfected cells and DOS-transfected cells maintained in hyperoxia were indistinguishable from each other. Cells transfected with LGR accumulated higher cell counts in hyperoxia than did the DOS cells exposed to hyperoxia, indicating that the attenuation of hyperoxia-induced cytostasis was due to transgene-specific effects rather than to effects caused by exposure to adenovirus. Inhibition of growth of pulmonary endothelial type cells during exposure to hyperoxia has been observed in other studies (24, 25). The present studies demonstrate that resistance to hyperoxia-induced growth inhibition in lung-derived cells can be conferred by enhancing mitochondrial GR activities.
The most likely mechanism for attenuation of hyperoxia-induced growth inhibition in cells transfected with LGR is that intramitochondrial GSH levels and GSH/GSSG ratios during exposure to hyperoxia are maintained more effectively in cells with enhanced mitochondrial GR activities (Figure 5). Hyperoxia exposure may alter the regulation of cell division by redox modification of proteins essential for progress through the cell cycle (6, 27, 28). Redox regulation of such proteins could be achieved either through direct oxidative interactions with ROS or indirectly through changes in cellular levels of GSH and GSSG and subsequent thiol/ disulfide exchange reactions. In particular, mitochondria have a limited ability to import GSH or export GSSG (40, 41). These limitations place an additional burden on mitochondrial GR to maintain intramitochondrial GSH concentrations and GSH/GSSG ratios. Hyperoxia increases the production of ROS in the mitochondria, and consumption of mitochondrial GSH with formation of GSSG often follows. If this additional oxidative load cannot be accommodated by mitochondrial GR, changes in redox tone with subsequent protein modifications may ensue. In the present studies, cells with enhanced mitochondrial GR activities had higher cellular GSH levels, maintained higher mitochondrial GSH/ GSSG ratios, and resisted oxygen-induced growth inhibition, suggesting that GSH homeostasis determined critical aspects of cell division under these experimental conditions.
Our data indicate that hyperoxia increases cellular GSSG levels. While this is an appropriate response to the oxidative challenge, it is interesting that cells exposed to virus (LGR or DOS) and hyperoxia had significantly higher GSSG levels than did similarly treated nontransfected control cells. Although the combination of virus- dependent effects and hyperoxia on cellular oxidative metabolism probably accounts for the association between viral transfection, hyperoxia, and increased cellular GSSG levels, what is curious and mechanistically more difficult to explain is why hyperoxia-exposed cells transfected with LGR, in addition to maintaining cellular GSH levels after exposure to hyperoxia, also had higher cellular GSSG levels than did similarly exposed cells transfected with DOS. One explanation may be related to the cellular compartmentalization of GSH (42). The mitochondria constitute about 10% of the cell mass and a comparable fraction of the total cellular GSH, whereas the cytoplasmic GSH pool is larger, containing about 90% of the total cellular GSH. Therefore, any changes in total cellular GSSG levels will be dominated by effects in the cytoplasmic pool. Although this could partially explain our findings, the physiologic consequences of enhancing mitochondrial GR activities, particularly regarding compartmental responses to oxidative stresses, have yet to be determined.
Adenovirus-mediated gene transfer of LGR to H441 cells can increase mitochondrial GR activities and enhance cellular resistance to hyperoxia-induced growth inhibition. Although many details and questions regarding the mechanisms of cellular resistance to hyperoxia- induced growth inhibition in cells with enhanced mitochondrial GR activities are not evident from the present data, cells transfected with the LGR construct maintained normal cellular GSH concentrations during exposure to hyperoxia. Studies of compartmental GSH and GSSG levels using other methods indicated that substantial thiol oxidation occurs during processing of cultured cells (43), but the data suggest that the GSH levels and GSH/GSSG ratios are higher in the LGR cells than in CON or DOS-transfected cells, both in normoxia and in hyperoxia. Adenovirus-mediated LGR transfection provides a cellular basis for studying the role of mitochondrial GSH homeostasis in hyperoxia-induced growth inhibition and suggests a cellular mechanism for preventing inhibition of cell growth in patients exposed to high concentrations of oxygen. The development of strategies to prevent hyperoxia-induced growth inhibition in pulmonary cells is an important goal for research that could prove to be particularly useful for prematurely born infants, in whom exposure to hyperoxia can alter pulmonary maturation and alveolarization irreversibly.
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Address correspondence to: Stephen E. Welty, Dept. of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. E-mail: swelty{at}neo.bcm.tmc.edu
(Received in original form June 21, 1999 and in revised form December 9, 1999).
Abbreviations: analysis of variance, ANOVA; Chinese hamster ovary, CHO; complementary DNA, cDNA; control cells, CON; superoxide dismutase in reverse orientation, DOS; ethylenediaminetetraacetic acid, EDTA; glutathione, GSH; glutathione disulfide, GSSG; glutathione reductase, GR; lactate dehydrogenase, LDH; GR cDNA with a mitochondrial leader sequence, LGR; manganese superoxide dismutase, MnSOD; multiplicity of infection, MOI; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT; phosphate-buffered saline, PBS; reactive oxygen species, ROS; standard error of the mean, SEM.Acknowledgments: This study was supported in part by an NIH grant GM44263.
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