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(The FASEB Journal. 2001;15:525-534.)
© 2001 FASEB

Prolonged hypoxia during cell development protects mature manganese superoxide dismutase-deficient astrocytes from damage by oxidative stress

JEAN-CHRISTOPHE COPIN*,{dagger}, YVAN GASCHE*, YIBING LI{dagger} and PAK H. CHAN*,{dagger}1

* Department of Neurosurgery, Department of Neurology and Neurological Sciences, and Program in Neurosciences, Stanford University School of Medicine, Stanford, California 94305-5487 USA; and
{dagger} Department of Neurological Surgery, University of California, School of Medicine, San Francisco, California 94143, USA

1Correspondence: Neurosurgical Labs, Stanford University, 1201 Welch Rd., MSLS P304, Stanford, CA 94305-5487 USA. E-mail: phchan{at}leland.stanford.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mouse astrocytes deficient in the mitochondrial form of superoxide dismutase do not grow in culture under 20% atmospheric O2 levels. By flow cytometry, immunocytochemistry, and enzymatic analysis we have shown that the oxygen block of cell division is due to a decrease in the number of cells entering the S phase of the cell cycle and is concomitant with higher DNA oxidation and impairment of mitochondrial functions. Seeding the cells under 5% O2 until the cultures become confluent can circumvent this problem. An initial hypoxic environment increases the resistance of manganese superoxide dismutase-deficient astrocytes to superoxide radicals artificially produced by paraquat treatment, preserves respiratory activity, and allows normoxic division during a subsequent passage. DNA oxidation is then not higher than in wild-type control cells. However, the adaptation of the cells is not due to compensation by other enzymes of the antioxidant defense system and is specific to cells totally lacking manganese superoxide dismutase. Alteration of the phenotype by prior hypoxia exposure in the SOD2-deficient mutant provide a unique model to study adaptative mechanisms of cellular resistance to oxygen toxicity.—Copin, J.-C., Gasche, Y., Li, Y., Chan, P. H. Prolonged hypoxia during cell development protects mature manganese superoxide dismutase-deficient astrocytes from damage by oxidative stress.


Key Words: paraquat • 8-oxo-7.8-dihydro-2'-deoxyguanosine • mitochondria • adaptation • preconditioning


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
AEROBIC ORGANISMS DEPEND on oxygen availability to generate their energy. Most of the oxygen molecules entering cells are tetravalently reduced to water during oxidative phosphorylation of adenosine diphosphate to adenosine triphosphate. However, 2 to 4% of the oxygen consumed by the cells is univalently reduced to superoxide radicals as a result of the leakage of electron transfer (1) . Superoxide radicals can be further reduced to hydrogen peroxide and hydroxyl radicals through reactions catalyzed by divalent metals or can react with nitric oxide to form peroxynitrites, which in turn generate hydroxyl radicals (2 , 3) . Superoxide radicals, hydrogen peroxide, hydroxyl radicals, and peroxynitrites, referred to as reactive oxygen species (ROS), are toxic when generated in excess in cells. They can affect various molecular components of the cells, damage DNA, inactivate enzymes, reduce the efficiency of the transcriptional machinery and peroxidize polyunsaturated lipids (4 5 6 7) . On the other hand, a low generation of ROS is responsible for the regulation of transcription factors such as AP-1 and NF-{kappa}B, which in turn regulate many effector genes (8) . ROS may also influence the genetic control of cell proliferation, and have been described as novel intra- and intercellular messengers (9) .

The reduction/oxidation state of cells is tightly regulated by an antioxidant defense system, which is constituted of diverse enzymes and free radical scavengers (10) . The first line of defense involves superoxide dismutases (SODs), which dismute superoxide into hydrogen peroxide. Copper/zinc SOD (CuZnSOD) is located in the cytoplasmic and nuclear compartments whereas manganese SOD (MnSOD) is found inside the mitochondria. Hydrogen peroxide is converted to water by catalase or glutathione peroxidase. The latter oxidizes glutathione, which is recycled by a glutathione reductase that uses reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cosubstrate. Free radical scavengers can be hydrophilic, such as ascorbate, urate and glutathione, or lipophilic such as tocopherols, flavonoids, and ubiquinol. Moreover, a cellular machinery, which maintains a reducing environment, is associated with the antioxidant defense system (e.g., glucose-6-phosphate dehydrogenase, which regenerates NADPH).

Variations in ambient oxygen concentration strongly modulate the developmental fate of embryonic tissues as well as the rate of cell growth in vitro, in both vertebrate and invertebrate species. Saccharomyces cerevisiae deficient in MnSOD is hypersensitive to oxygen, and increasing concentrations of oxygen lead to a progressive inhibition of growth (11) . In contrast, MnSOD-deficient yeast with complete absence of electron transport grows normally in hyperoxia, suggesting a role for superoxide radicals in growth arrest (12) . Exposure of bovine endothelial and smooth muscle cells to hyperoxia delays the entry of the cells into the S phase of the cell cycle and blocks the cells in the S and G2 phases (13) . Embryos generated by in vitro fertilization and exposed to 20% O2 instead of the relatively low O2 tension present in the oviducal and uterine environments become retarded or arrested in culture in vitro (14) . The in vitro block phenomenon of embryo development is related to ROS generation. It can be bypassed by performing cultures under 5% O2 conditions and may be partially explained by oxidation of the thiol group of proteins (15 , 16) .

Genetically modified animals with alterations in the antioxidant defense system are valuable tools for studying the effect of oxygen on cellular functions in vivo and in vitro. Two MnSOD-deficient mouse models have been produced by homologous recombination techniques (17 , 18) . Only heterozygous MnSOD knockout animals (Sod2-/+) are viable, whereas homozygous animals (Sod2-/-) die shortly after birth. Studies performed on heterozygous animals demonstrate that an increase in mitochondrial ROS can result in biochemical alterations with features reminiscent of genetic mitochondrial disorders (19) . Altered mitochondrial functions in Sod2-/+ mice contrast with the absence of change in oxidation of cytosolic proteins or nuclear DNA (20) . Permanent focal cerebral ischemia performed on Sod2-/+ animals leads to a bigger infarct size and higher neurological deficits in vivo, whereas glutamate toxicity is exacerbated in Sod2-/+ cortical neurons in vitro (21 , 22) . However, some results suggest that in mice only 50% of MnSOD activity may be sufficient for normal resistance to 100% oxygen toxicity (23) .

Sensitivity of MnSOD homozygous knockout cells to oxygen toxicity depends on the type of cells that are cultured. Neuronal cultures of Sod2-/- mice do not survive under normoxia (22) . Cultivation of Sod2-/- fetal fibroblasts is possible and their growth is ~60% that of wild-type cells (24) . In this study, we show that Sod2-/- growing astrocytes are highly sensitive to 20% oxygen in culture. Cultures of cerebral cells deficient in MnSOD are valuable models to study the critical role played by MnSOD under normal physiological conditions, as well as to better understand the consequences of mitochondrial oxidative imbalance in pathophysiological pathways such as excitotoxicity, hypoxia/reperfusion or apoptosis. Thus, we cultured Sod2-/- astrocytes under 5% O2. Our results indicate that MnSOD is necessary for the normoxic development of primary astrocytes in vitro by preventing mitochondrial injury, but is less necessary when the cells reach confluency. Furthermore, during secondary culture, Sod2-/- astrocytes do not have to be kept under 5% O2 for successful dividing. They also become less sensitive to paraquat treatment. The results are explained in light of a possible adaptation of the cells and prompt further studies, which may give new insight into the role of oxygen in gene regulation and the effect of low oxygenation on phenomenon such as preconditioning.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
MnSOD-deficient mice
The animals used in this study were mutated with a replacement type targeting vector that inactivated the MnSOD gene (18) and were extensively bred on a CD-1 background. Homozygous MnSOD knockout animals were produced by crossing together heterozygous mice. Immediately after birth, the animals were typed by polymerase chain reaction amplification of mutant and normal fragments of the MnSOD gene from tail DNA obtained by proteinase K digestion as described previously (22) .

Primary culture of astrocytes
Astrocytes were cultured as described by Juurlink and Hertz (25) with minor modifications. Briefly, cortical tissues of newborn mice were dissociated by mechanical action and passed sequentially through 80-µm and then 10-µm filtration meshes. Cell suspensions in minimum essential medium (MEM, Gibco BRL, Grand Island, N.Y.) supplemented with 20% fetal bovine serum (Gemini Bio-Products Inc., Calabasas, Calif.) were plated into 24 multi-well plates, 35 mm or 60 mm dishes (Falcon; Becton Dickinson, Franklin Lakes, N.J.), at a dilution of one brain per 48 cm2 and incubated at 37°C under 5% CO2-air atmosphere in a water-jacketed incubator (Forma Scientific Inc., Marietta, Ohio) or under 5% O2-5% CO2-90% N2 in a gas-tight humidified chamber (modular incubator chamber; Billups-Rothenberg, Del Mar, Calif.). After 4 days the culture medium was replaced with fresh MEM containing 10% fetal bovine serum, and the astrocytes were fed twice a week.

Enzymatic activities
For enzymatic assays, astrocytes were cultured in 60 mm dishes. SOD, catalase, glutathione peroxidase, and intracellular lactate dehydrogenase (LDH) activity was measured in the supernatant obtained after sonication of the cells and centrifugation at 20,800 g for 15 min at 5°C. Total SOD and MnSOD activity, which was determined after 30 min inactivation of CuZnSOD with 2 mM KCN, was measured by the method of Crapo et al. (26) . One unit of SOD was defined as the quantity of enzyme necessary to inhibit by 50% the rate of reduction of ferricytochrome c. Catalase activity was measured by the method of Lück (27) . One unit of catalase was defined as the quantity of enzyme necessary to consume half of the hydrogen peroxide in the reactive buffer in 1 min. Glutathione peroxidase activity was measured by the method of Paglia and Valentine (28) . LDH activity present inside the cells or released into the culture medium was measured spectrophotometrically at 340 nm by following the rate of reduction of NAD in the presence of lactate with a kit from Sigma (LD-L; Sigma, St. Louis, Mo.).

Superoxide radical generation
Intracellular production of superoxide radicals was achieved by treatment of the cells for 15 h with doses of paraquat (Sigma) ranging from 2.5 to 100 mM. Paraquat was directly dissolved in MEM, which was used as a replacement medium for the cultures at the beginning of the experiment. Cellular toxicity was measured by LDH release into the medium and expressed as a percent of maximum release that was obtained for a dose of 100 mM of paraquat.

Mitochondrial activity
The rate of respiration was measured with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) as an alternative electron acceptor, according to the method described by Manthorpe et al. (29) . Results were normalized with the protein content.

Protein and DNA contents
For developmental study, astrocytes were cultured in 24 multi-well plates. After washing, the cells were resuspended in 300 µl of water per well and sonicated. Protein concentration was determined spectrophotometrically by the bicinchoninic method (BCA Protein Assay Reagent kit; Pierce, Rockford, Ill.) according to the procedure provided by the manufacturer. DNA concentration, as an index of the cell number, was determined fluorometrically by using TO-PRO-1 iodide (Molecular Probes, Eugene, Oreg.). Results were compared to a standard curve made with calf thymus DNA (Sigma).

Immunocytochemistry
For immunostainings, astrocytes were cultured on glass coverslips. Glial fibrillary acidic protein was detected after formaldehyde fixation, with a rabbit anti-cow GFAP (Dako, Carpinteria, Calif.; Ref. Z-0334, dilution 1:500, 1 h incubation at room temperature), followed by a fluorescein isothiocyanate (FITC) -conjugated swine anti-rabbit immunoglobulin G (Dako; Ref. F-0205, dilution 1:30, 1 h incubation at room temperature in the dark). 8-Oxo-7.8-dihydro-2'-deoxyguanosine (8-oxo-dG) was detected after formaldehyde fixation with mouse anti-8-oxo-dG (Trevigen, Gaithersburg, Md.; Ref. 4355-MC-100, dilution 1:100, overnight incubation at 5°C), followed by FITC-conjugated goat anti-mouse immunoglobulin G (Dako; Ref. F-0479, dilution 1:20, 1 h incubation at room temperature in the dark). To differentiate between 8-hydroxyguanine formation on DNA or RNA, the cells were treated for 1 h before immunostaining with 5 µg/µl RNase A or 1 U/µl DNase I (Roche Diagnostics Corp., Indianapolis, Ind.). Coloration of the nuclei was done by 15 min of incubation with 4 µg/ml of the DNA-specific fluorochrome Hoechst 33258.

Flow cytometry
For flow cytometry experiments, astrocytes were cultured in 35 mm dishes. To prevent the problem of contact inhibition during cell division studies, the cells were passaged after 5 days in primary culture, at a ratio of 1 to 3, and treated for 6 h with 10 µM of bromodeoxyuridine (BrdU; Sigma) 1 to 3 days after passage. BrdU incorporation into the DNA was detected with FITC-conjugated mouse anti-human BrdU (PharMingen, San Diego, Calif.; Ref. 36634K, dilution 1:5, 1 h incubation at room temperature in the dark). Analysis of cell cycle distribution was performed using 5 µg/ml propidium iodide. DNA oxidation was analyzed by anti-8-oxo-dG immunostaining under the same conditions of concentration and incubation time as those used in immunocytochemistry. All samples were run on a FACScan flow cytometer equipped with a single 488 nm argon laser (Becton Dickinson) and 10,000 events per sample where collected.

Statistical analysis
All data obtained from 3 to 5 independent experiments are expressed as means ± SE or medians, and for each experiment multiple measurements were made on 3 to 6 different dishes or wells. Statistical analyses were performed by either the Mann Whitney nonparametric test, the paired t test, or one-way analysis of variance, followed by the Scheffe’s post hoc test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Oxygen blocks MnSOD-deficient astrocyte development by reducing DNA synthesis activity during cell division
MnSOD-deficient astrocytes in primary culture do not grow if maintained in normoxia after initial plating. As an index of cell number, DNA content drops to almost zero in SOD2-/- cultures maintained in normoxia for 8 days (Fig. 1a ). In contrast, SOD2-/- cells cultured under 5% O2 develop similarly to wild-type cells. In normoxia, SOD2-/- cells remain attached to the plates and increase in size as demonstrated by a higher protein content 16 days in vitro (DIV) compared to 8 DIV (Fig. 1b ). However, protein content in SOD2-/- astrocytes cultured in normoxia is statistically lower than in the SOD2-/- astrocytes cultured in hypoxia and in wild-type cells. Since MnSOD heterozygous knockout astrocytes grow to the same extent as wild-type astrocytes in either normoxia or hypoxia, the developmental block by oxygen is specific to cells that totally lack MnSOD.



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  		Figure 1. Hypoxia is required for SOD2-/- astrocytes to grow in primary culture. a, b) DNA and protein content changes in SOD2-/-, SOD2-/+, and wild-type astrocytes. Astrocyte cultures were maintained for 12 days under 5% O2 and returned to normoxia (black squares, wild-type; black triangles, SOD2-/+; black circles, SOD2-/-) or immediately incubated under 20% O2 (white squares, wild-type; white triangles, SOD2-/+; white circles, SOD2-/-). DNA and protein content were measured periodically during the entire month of the culture. *P < 0.01 vs. wild-type maintained in normoxia. +P < 0.01 vs. SOD2-/- that remained 12 days in hypoxia. c) Protein content in cultures maintained for increasing period of time under 5% O2. Cultures of SOD2-/- (black circles) and wild-type (white squares) astrocytes were maintained for 0 to 30 days under 5% O2 and then returned to 20% O2. Protein content was measured after 30 DIV. *P < 0.01 vs. wild-type. +P < 0.01 vs. SOD2-/- maintained for 12 days in hypoxia. d) DNA content in cultures placed under 5% O2 9 days after initial plating in normoxia. Nine days after initial plating and incubation under 20% O2, cultures of SOD2-/- (black circles) and wild-type (white circles) astrocytes were placed under 5% O2. DNA contents were measured periodically during the entire month of the culture. Median values are represented by black bars for SOD2-/- and white bars for wild-type astrocytes. *P < 0.005 vs. each group at the initial time in hypoxia (day 9). +P < 0.005 vs. wild-type. e) LDH activity in astrocytes maintained for increasing period of time under 5% O2. The conditions of the culture were the same as described for panel c (black bars, SOD2-/-; white bars, wild-type). LDH activity was measured after 30 DIV. *P < 0.0001 vs. LDH activity in wild-type cells cultured only in normoxia (bar at 505.1 mU/mg). f) Respiratory rate in SOD2-/- and wild-type astrocytes. Respiratory rates were determined in 21-day-old cultures of SOD2-/- and wild-type astrocytes maintained (black bars) or not (white bars) for 12 days in hypoxia before return to normoxia using MTT as an alternative electron acceptor. *P < 0.0001 vs. wild-type cultured only in normoxia. +P < 0.0001 vs. SOD2-/- maintained for 12 days in hypoxia.

SOD2-/- astrocytes do not need to be constantly maintained in hypoxia. The first 12 to 15 days after plating are important, and the longer the cells are kept in hypoxia during this period of time the better they develop during the month after initial plating (Fig. 1c ). The shorter amount of time in hypoxia needed to obtain SOD2-/- cultures similar to wild-type cultures corresponds to the time necessary for the cells to achieve confluency. Maintaining SOD2-/- astrocytes in hypoxia for a period shorter than 12 days leads to cultures with fewer cells than normal (Fig. 2 ). Without an initial hypoxic environment or a hypoxic environment reduced to only 3 days, SOD2-/- astrocytes develop poorly and use all of the space spared by no division, which features hypertrophic cells (Fig. 2a , Fig. 2b ). On the other hand, there was no morphological difference at the age of 30 DIV between SOD2-/- astrocytes that were maintained in hypoxia for 12 or 30 days (Fig. 2c , 2d ).



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  		Figure 2. Normoxic exposure leads to low density of SOD2-/- astrocytes in primary culture featuring hypertrophic cells. Cultures of SOD2-/- astrocytes were maintained for 0 (a), 3 (b), 12 (c), and 30 (d) days in hypoxia and returned to 20% O2. Photomicrographs represent cells immunostained after 30 DIV with an FITC-conjugated antibody recognizing the anti-GFAP antibody and counterstained with Hoechst 33258 revealing the nuclei. Scale bar: 80 µm (applies to all panels).

SOD2-/- astrocytes that did not grow in normoxia could be forced to divide by placing them in hypoxia as late as 9 days after initial plating (Fig. 1d ). This indicates that normoxic exposure does not irreversibly block cell development. However, cell division remained slow and only a very small number of plates showed an efficient salvage. Overall, there is a statistical difference between SOD2-/- astrocytes forced to divide after initial normoxic insult and wild-type astrocytes.

To demonstrate that the cells were halted in their division, SOD2-/- astrocytes were incubated in normoxia and hypoxia in the presence of BrdU for 6 h before analysis by flow cytometry. To work with enough cells, SOD2-/- astrocytes were initially maintained for 5 days in hypoxia, which did not suppress oxygen toxicity as demonstrated before (see Fig. 1c ), and then placed in normoxia. Twenty-four hours after return to normoxia, the cells showed a slight but statistical reduction of BrdU incorporation when compared to sister cultures maintained in hypoxia (Fig. 3b compared to Fig. 3a ; Table 1 ). After 48 h in normoxia, the cells showed a dramatic reduction of BrdU incorporation (Fig. 3d compared to Fig. 3c ; Table 1 ). After 72 h in normoxia, more than half the SOD2-/- astrocytes that would enter the S phase of the cell cycle, if maintained in hypoxia, were repressed by oxygen toxicity (Table 1) . BrdU incorporation in wild-type astrocytes was similar under 5% and 20% O2, although a significant drop in BrdU incorporation was noted the second and third day after passage (day 1: 8.1% ± 0.3; day 2: 5.1% ± 0.3; day 3: 4.4% ± 0.2). This decrease, which may be explained by a more rapid inhibition of contact in wild-type astrocytes, prevented a direct comparison between these cells and SOD2-/- astrocytes.



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  		Figure 3. Normoxic exposure dramatically decreases BrdU incorporation into the DNA of SOD2-/- astrocytes in primary culture. Five days after initial plating and incubation in 5% oxygen, SOD2-/- astrocytes were returned to normoxic conditions and treated for 6 h with 10 µM of BrdU the first or second day in normoxia. BrdU incorporation was analyzed by flow cytometry and compared with incorporation in cells that remained in hypoxic conditions (see Table 1 for quantitative results). BrdU positive cells are set in the upper right quadrant. a, b) Day 1 in hypoxia and normoxia, respectively. c and d) Day 2 in hypoxia and normoxia, respectively.


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  		Table 1. Normoxic exposure decreases the number of SOD2-/- astrocytes entering cell divisiona

Hypoxia preconditions MnSOD-deficient astrocytes and suppresses oxygen toxicity when the cells reenter a new cycle of cell division in normoxia
Twelve to 15 days after plating, MnSOD-deficient astrocytes can be removed from their hypoxic chamber and will still survive under 20% O2 (trypan blue exclusion experiments; data not shown). Since superoxide radicals are constantly generated by cells and since MnSOD is a primary line of defense against superoxide radicals, a possibility exists that the reason for MnSOD-deficient cells to survive in normoxia is a diminution of mitochondrial activity and a stimulation of the glycolytic pathway. A period of hypoxia longer than 8 days stimulates LDH activity in SOD2-/- astrocytes (Fig. 1e ). This is a direct effect of hypoxia that is also observed in wild-type astrocytes maintained for 30 days in low oxygen concentration, and cannot be the only reason that MnSOD-deficient cells do not suffer from oxidative injury after the return to normoxia, since 8 days in hypoxia are not enough to release the blockade of cell division due to normoxia (see Fig. 1c ). Furthermore, no mitochondrial activity disruption was observed by MTT staining in SOD2-/- astrocytes that were maintained for 12 days in hypoxia before the return to normoxia (Fig. 1f ). On the other hand, a dramatic reduction of respiratory activity occurred in MnSOD-deficient astrocytes that were not preserved by an initial period of growth in hypoxia.

A modification of the antioxidant system in SOD2-/- astrocytes initially plated under hypoxic conditions for 2 wk cannot explain the survival of the cells under normoxia. There was no difference in CuZnSOD, catalase and glutathione peroxidase activity between wild-type cells and MnSOD-deficient cells (Table 2 ). Only MnSOD activity was undetectable in SOD2-/- astrocytes, as expected.


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  		Table 2. MnSOD deficiency does not alter other antioxidant activitiesa

Moreover MnSOD-deficient astrocytes developed identically under 5% and 20% O2 (Fig. 4 ). The rate of division of SOD2-/- astrocytes in secondary culture was also similar to the rate of division of wild-type astrocytes. This observation is opposite to the high degree of sensitivity of SOD2-/- astrocytes to normoxic conditions during initial plating and suggests that a consequence of hypoxia is to suppress oxygen toxicity when the cells reenter a new cycle of cell division after resting at confluency.



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  		Figure 4. SOD2-/- and wild-type astrocytes grow similarly in secondary culture. Astrocyte cultures were maintained for 12 days under 5% O2 and then returned to normoxia. At the age of 21 DIV, the cells were passaged at a ratio of 1 to 3 and cultured either in hypoxia (black squares, wild-type; black circles, SOD2-/-) or normoxia (white squares, wild-type; white circles, SOD2-/-). DNA content was measured periodically for 25 days in secondary culture. *P<0.001, values statistically different in the SOD2-/- group cultured in normoxia, when compared within the group to the values obtained the first day after passage. There was no statistical difference between groups, when observed at the same age.

To further investigate the apparent adaptation of MnSOD-deficient astrocytes to oxidative stress, we treated SOD2-/- and wild-type astrocytes with paraquat, a superoxide radical generator. Astrocytes showed a dose- and time-dependent sensitivity to paraquat. Treatment with 5 mM paraquat for 15 h significantly damaged 21-day-old primary cultures of wild-type astrocytes initially maintained for 12 days in hypoxia (Fig. 5c compared to Fig. 5a ), but did not affect primary cultures of SOD2-/- astrocytes (Fig. 5d compared to 5b ). Higher doses damaged SOD2-/- cultures, but for doses lower than 10 mM SOD2-/- astrocytes were statistically less sensitive than wild-type astrocytes (Fig. 6a ).



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  		Figure 5. Paraquat at 5 mM injures wild-type but not SOD2-/- astrocytes, either at confluence or during division in secondary culture. Astrocyte cultures were maintained for 12 days under 5% O2 and returned to normoxia. At the age of 21 DIV, the cells were treated with 5 mM of paraquat for 15 h or passaged at a ratio of 1 to 3 and treated 1 day later under the same conditions. Photomicrographs in phase contrast represent primary cultures of wild-type (a) and SOD2-/- (b) control, wild-type (c) and SOD2-/- (d) treated cells, and secondary cultures of wild-type (e) and SOD2-/- (f) control and wild-type (g) and SOD2-/- (h) treated cells. In primary cultures, scale bar is 200 µm; and in secondary cultures scale bar is 100 µm.



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  		Figure 6. Hypoxia during cell development protects mature SOD2-/- astrocytes from paraquat toxicity. Cultures of wild-type (white bars) and SOD2-/- astrocytes (black bars) were maintained for 5 days under 5% O2 and treated for 15 h with doses of paraquat ranging from 0 to 100 mM (dividing cells, a), maintained for 12 days under 5% O2 and then returned to normoxia to be treated under the same conditions, at the age of 21 DIV (confluent cells, b), or passaged to a ratio of 1 to 3 and treated 1 day later (dividing cells in secondary culture, c). Cellular toxicity was measured by LDH release into the medium and expressed as a percent of maximum release that was obtained for a dose of 100 mM of paraquat. Values statistically different from values in the nontreated control groups (+P<0.002), and during comparison between the wild-type and SOD2-/- groups treated with the same doses of paraquat (*P<0.002) are indicated.

The lower sensitivity of SOD2-/- astrocytes to paraquat treatment was specific, in primary culture, to cells that reached confluency after initial hypoxic growth. This was not seen in primary cultures in division, which showed a sensitivity similar to wild-type cultures for doses as small as 2.5 mM (Fig. 6b ). In contrast, in secondary dividing cultures, SOD2-/- astrocytes remained less sensitive than wild-type astrocytes to paraquat treatment. Paraquat at 2.5 mM produced a significant release of LDH in the medium of wild-type cells but did not lead to any detectable increase in LDH release in SOD2-/- cells (Fig. 6c ). A dose of 5 mM dramatically destroyed wild-type cultures but still had no statistical effect on LDH release (Fig. 6c ) or on the morphology of MnSOD-deficient cultures (Fig. 5e-h ). Stronger paraquat treatments similarly injured SOD2-/- and wild-type cells (Fig. 6c ).

DNA oxidation is inversely correlated to the ability of MnSOD-deficient astrocytes to divide
Since MnSOD-deficient astrocytes become insensitive to oxygen toxicity after a prolonged period in hypoxia and since they are then able to divide in normoxia, it was interesting to analyze the relationship between oxidative stress and cell division. As a marker of oxidative stress, we measured by flow cytometry and immunocytochemistry the presence of the oxidative DNA adduct 8-oxo-dG, since guanine is the most susceptible DNA target to a wide variety of oxidation reactions mediated by ROS (30) .

Five days after plating, primary cultures of wild-type and SOD2-/- astrocytes were removed from the hypoxic chamber and incubated in normoxia for 24 h before analysis. A subpopulation of MnSOD-deficient cells was highly positively stained for 8-oxo-dG compared to wild-type cells (Fig. 7a , c ). This population was diploid as indicated by the propidium iodide fluorescence intensity, which was twice the intensity of the main population. By gating the cells in the G2/S phase, it was shown that MnSOD deficiency led to a higher oxidation of the DNA in ~15% of the dividing cells (Fig. 7d ). This value is statistically different from wild-type cells (Fig. 7b ). In contrast, SOD2-/- astrocytes in secondary culture were stained for 8-oxo-dG to the same extent as wild-type cells (Fig. 7e f g h ), which is consistent with their relative insensitivity to paraquat treatment.



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  		Figure 7. DNA oxidation is higher in dividing SOD2-/- astrocytes in primary than in secondary culture. Cultures of wild-type and SOD2-/- astrocytes were either maintained for 5 days under 5% O2 and returned to normoxia (primary culture) or maintained for 12 days under 5% O2, returned to normoxia until the age of 21 DIV and then passaged at a ratio of 1 to 3 to induce a new cell division (secondary culture). Twenty-four hours after return to normoxia (in the case of primary cultures) or 5 days after passage (in the case of secondary cultures) the cells were harvested and processed for 8-oxo-dG detection. Wild-type astrocytes in primary culture (a, b) and in secondary culture (e, f). SOD2-/- astrocytes in primary culture (c, d) and in secondary culture (g, h). Contour plots show the total cell population. Histograms show the repartition of 8-oxo-dG immunoreactivity inside the groups of cells in the G2/S phase of cell division that are gated on the upper panels (rectangles). The number of cells with high immunoreactivity relative to the total number of cells in the G2/S phase is indicated as percent. *P < 0.002 vs. wild-type groups.

Immunocytochemistry showed that 8-oxo-dG staining was limited to the cytosolic regions of the cells (Fig. 8a ). Granular staining, mainly perinuclear, suggested a mitochondrial localization of 8-oxo-dG. Furthermore, the signal could not be abolished by RNase treatment (Fig. 8b ), whereas it disappeared by DNase treatment (Fig. 8c ). Microscopic observation of 8-oxo-dG immunoreactivity remained difficult due to the autofluorescence that masks the specific signal (Fig. 8d ).



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  		Figure 8. 8-Oxo-dG immunoreactivity is localized in mitochondria. After initial cell isolation and seeding on glass coverslips, cultures of SOD2-/- astrocytes were maintained for 5 days under 5% O2 and returned to normoxia. Twenty-four hours later the cells were fixed and processed for 8-oxo-dG immunodetection, without further treatment (a) or after RNase A (b) or DNase I (c) treatments. Photographs represent cells immunostained with an FITC-conjugated antibody recognizing the anti-8-oxo-dG antibody. d) Cells incubated only in presence of the secondary antibody (negative control). Scale bar: 25 µm (applies to all panels).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The main result of this study is that astrocytes lacking the mitochondrial form of superoxide dismutase adapted to oxygen toxicity by a temporary stage under low O2 (5%) during initial plating. This adaptation resulted in a higher degree of resistance to free radical generation, as demonstrated by a lower sensitivity to paraquat treatment, and may explain the acquired ability of SOD2-/- astrocytes to divide under 20% O2.

ROS have been implicated in the regulation of several physiological processes, including cell proliferation, differentiation, apoptosis, and senescence (9 , 31 , 32) . Deregulation of these processes leads to anarchic growth, characteristic of cancer cells. Reduced MnSOD activity in certain tumor types may lead to a higher oxidative state that favors such deregulated proliferation (33) . Attempts to counteract this by treatment with SOD mimetics or by SOD gene transfer have inhibited tumor progression or reversed the tumor phenotype (34 , 35) . Our results show that the blockade of cell division in SOD2-/- astrocytes can be bypassed by a mechanism that is not a simple compensation due to overexpression of other antioxidant enzymes. However, the cells became less sensitive to free radical generation. Indeed, DNA oxidation, which was higher in normoxic dividing cells in primary cultures, was not more intense than in wild-type cells in secondary cultures. DNA oxidation was localized in the mitochondrial compartments, which is in accordance with a previous study performed on SOD2-/+ mice showing no change in oxidation of cytosolic proteins or nuclear DNA (20) . Since the respiratory rate totally collapsed in nonadapted SOD2-/- astrocytes and was well preserved in adapted cells, a relationship can be suggested between energetic status of the cells, protection against ROS, and cell division, the key component being the new ability of the cells to counteract oxidative stress and therefore to preserve mitochondrial functions. Recently a quantitative correlation between the level of mitochondrial impairment and cell respiration, cell growth, free radical production, lipid peroxidation, mitochondrial membrane potential, and cell death was established (36) .

The underlying mechanisms involved in SOD2-/- astrocyte adaptation to normoxic conditions of growth may be diverse. Transient adaptation to oxidative stress in several mammalian cell lines involves the de novo synthesis of at least 20 proteins, although Northern blot and enzyme activity analyses revealed no significant increase in transcription or translation of the classical antioxidant enzymes (37) . An extensive transcriptional remodeling occurs in S. cerevisiae, which lacks a functional CuZnSOD gene, under dioxygen stress. This remodeling results in a pattern of expression of gene products needed for defense and repair, and suppression of activities associated with normal cell proliferation (38) . Oxidative damage in eukaryotic cells can be prevented through alterations in manganese homeostasis or in mutation in the BSD1 (bypass SOD defects) gene (39) . Overexpression of Bcl-2 protects cultured lymphocytes from oxidative death caused by either hydrogen peroxide or the redox-cycling agent menadione (40) and prevents neuronal death as a result of glutathione depletion (41) . Various biomolecules may act as antioxidants either in the cytosol, such as pyruvate (42) , or at the cell membrane level, such as plasmalogens (43) . Therefore, our current results open the door to many more investigations to elucidate the mechanism of adaptation of SOD2-/- cells to oxidative stress. The progress of such empiric studies can be boosted by the use of the newly developed gene chips, which allow the screening of thousand of genes in one single trial (44 , 45) .

Independent of the understanding of the molecular mechanisms that give unexpected competence to proliferate in an oxidative environment to cells that lack the mitochondrial form of the superoxide dismutase and that may provide significant insight into important problems, such as the two-cell block of embryo development or the activity of aberrant genes that induce cells to enter uncontrollable cycles of division, the current results raise a fundamental question about the confidence, sometimes too quickly developed, in experimental data generated with mutant animals. Gene function is often studied by making irreversible changes to the genome. This approach has a major drawback in that the function of the gene in question must be deducted from the phenotype of animals that are deficient in the product of the disrupted gene throughout their development. Compensation for the loss of the gene of interest could yield an apparently unaltered phenotype (46) . Alternatively, changes in the regulation of other genes could yield a misleading phenotype. Consideration must also be given to the genetic background of the mutant animal. The animals used in our study were bred on a CD-1 background. The SOD2-/- pups died within the first 10 days of life without neurological deficit (18) . Lebovitz et al. (17) reported an independently derived MnSOD null mouse on a mixed C57BL/6 and 129Sv background with a different phenotype. The SOD2-/- pups were able to survive up to 18 days; animals living for more than 15 days displayed neurological abnormalities, including ataxia and seizures. By crossing MnSOD-deficient animals from different strains, the influence of the genetic background has been clearly demonstrated (47) . Some of the drawbacks seen in mutations expressed throughout ontogeny and in a systemic fashion could be circumvented if the expression of a particular gene could be restricted both temporarily and spatially through the use of an inducible genetic system (48) . This could be a good alternative to studying the effects of MnSOD deficiency in cell culture in relation to oxidative imbalance, since the only way so far to culture SOD2-/- astrocytes prevents confident interpretation of the results that can be generated because of the adaptation of the cells and the development of an unexpected higher resistance to oxidative stress.

Nevertheless, the model that we use needs to be further explored, since the higher resistance developed by MnSOD-deficient astrocytes undoubtedly is the consequence of an interaction between the lack of MnSOD and the lower oxygenation. It may be visualized as a biphasic oxidative stress inducer, the first phase being sublethal and the second more intense and damaging. The cells could not resist the second phase if they had not been, in the first place, preconditioned by a temporary stage in hypoxia. Understanding the mechanisms leading to those new cellular capabilities to resist to a higher level of injury will help in understanding similar phenomena seen in ischemic preconditioning. Indeed, it has become apparent that exposure of tissues to brief periods of ischemia protects them from harmful effects of subsequent, prolonged ischemia. This was first described in the heart, but has subsequently been demonstrated in many other organs as well as in patients with cardiovascular diseases (49 50 51) . In the heart, there are actually two types of protection afforded by preconditioning, an immediate and a more delayed preconditioning. Delayed preconditioning, or ischemic tolerance, is dependent on altered gene expression as well as the synthesis of new proteins, including antioxidant enzymes, heat shock proteins, and nitric oxide synthase (52) . Similarly, ROS may act as intracellular messengers in a chemically induced preconditioning of the brain to ischemic injury (53) . The mechanisms leading to altered gene expression and details concerning the transcription-dependent synthesis of proteins that are associated with preconditioning are largely unknown. Our in vitro model may become relevant in this important field of research.


   ACKNOWLEDGMENTS
 
This work was supported by National Institutes of Health grants NS14543, NS25372, NS36147, NS37530, NS38653, and NO1 NS82386. The authors thank Jane O. Kim for breeding the animals.

Received for publication May 25, 2000. Revision received July 17, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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