FASEB J. Avanti Polar Lipids
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(The FASEB Journal. 1998;12:1651-1663.)
© 1998 FASEB

RESEARCH COMMUNICATION

Glutathione is implied in the control of 7-ketocholesterol-induced apoptosis, which is associated with radical oxygen species production

Gerard Lizard1,a,2, Serge Gueldry1,a, Olivier Sordetb, Serge Moniera, Anne Athiasa, Carole Migueta, Ginette Bessedea, Stephanie Lemairea, Eric Solaryb and Philippe Gamberta

a INSERM U498 (Métabolisme des lipoprotéines humaines et interactions vasculaires), CHU/Hôpital du Bocage, 2 Bd Maréchal de Lattre de Tassigny, 21034 Dijon Cedex, France
b INSERM CJF 94/08, Faculté de Médecine, 21033 Dijon Cedex, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In a number of experimental systems, inhibition of apoptosis by antioxidants has led to the production of radical oxygen species (ROS) in certain apoptotic forms of cell death. Since antioxidant therapies can reduce vascular dysfunctions in hypercholesterolemic patients who frequently have increased plasma levels of oxysterols constituting potent inducers of apoptosis, we speculate that oxysterol-induced apoptosis could involve oxidative stress. Here, we tested the protective effects of the aminothiols glutathione (GSH) and N-acetylcysteine (NAC), which are two potent antioxidants, on apoptosis induced by 7-ketocholesterol in U937 cells, and we present evidence indicating that oxidative processes are involved in 7-ketocholesterol-induced cell death. Thus, GSH and NAC prevented phenomenona linked to apoptosis such as reduction of cell growth, increase cellular permeability to propidium iodide, and occurrence of nuclear condensation and/or fragmentation, and they delayed internucleosomal DNA fragmentation. In addition, cell treatment with GSH impaired cytochrome c release into the cytosol and degradation of caspase-8 occurring during cell death. During 7-ketocholesterol-induced apoptosis, we also observed a rapid decrease in cellular GSH content, oxidation of polyunsaturated fatty acids, and a production of ROS by flow cytometry with the use of the dye 2', 7'-dichlorofluorescin-diacetate; both phenomena were inhibited by GSH. Prevention of cell death by GSH and NAC does not seem to be a general rule since these antioxidants impaired etoposide (but not cycloheximide) -induced apoptosis. Taken together, our data demonstrate that GSH is implied in the control of 7-ketocholesterol-induced apoptosis associated with the production of ROS.—Lizard, G., Gueldry, S., Sordet, O., Monier, S., Athias, A., Miguet, C., Bessede, G., Lemaire, S., Solary, E., Gambert, P. Glutathione is implied in the control of 7-ketocholesterol-induced apoptosis, which is associated with radical oxygen species production. FASEB J. 12, 1651–1663 (1998)


Key Words: N-acetylcysteine • oxidation • oxysterol • low density lipoproteins • TUNEL


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OXIDIZED LOW DENSITY LIPOPROTEINS (LDL)3, which are increased in the plasma from hypercholesterolemic patients, (1), are a complex mixture of various components comprised of lipid hydroperoxides, aldehydes, and oxysterols (2). These latter compounds, mainly 7ß-hydroperoxycholesterol, 7ß-hydroxycholesterol, 7-ketocholesterol, and 5{alpha}, 6{alpha}-epoxycholesterol, can mimic the cytotoxic effects of oxidized LDL on various cell types (3, 4) including those of the vascular wall, i.e., endothelial cells (5, 6), smooth muscle cells (7, 8), and fibroblasts (9), and it has been shown that 7ß-hydroxycholesterol and 7-ketocholesterol are potent inducers of apoptosis in bovine and human endothelial cells (10–12). In addition, 7ß-hydroxycholesterol and 7-ketocholesterol favored interleukin 1ß secretion during the apoptotic process they induce in human promonocytic leukemia cells U937 (13). Therefore, these oxyterols, which are oxidized at position 7 and have also been identified in atheromatous plaques from hypercholesterolemic subjects (14–16) at different stages of development (17), could play a critical role in atherosclerosis.

As antioxidant therapies reverse endothelial dysfunction in patients with coronary artery disease (18, 19) and vitamin C reduces microcirculating changes in cholesterol-fed rabbits with high circulating oxysterol levels in the blood (20), we speculate that oxidative processes could occur during various cellular damages triggered by oxysterols, particularly during oxysterol-induced apoptosis. This hypothesis is reinforced by investigations that have shown the involvement of reactive oxygen species (ROS) in apoptosis induced by different agents (21). Indeed, lipid peroxidation (22, 23), production of ROS (21, 23, 24), and down-regulation of antioxidant defenses characterized by a reduced glutathione (GSH) level (25, 26), a progressive decline in the transcript levels for catalase, superoxide dismutase, DT diaphorase, and thioredoxin (27, 28), have been observed in some apoptotic processes. Moreover, ROS can also play an important role in apoptosis by regulating the activity of certain enzymes involved in the cell death pathway (29, 30). Thus, the ced 3 human analog CPP32 corresponding to caspase-3 (31), which belongs to the family of enzymes named cysteinyl aspartate-specific proteinases (caspases), is highly specific to the proteolytic cleavage of the poly (ADP-ribose) polymerase involved in internucleosomal DNA degradation (32), and its enzymatic activity depends on the nitrosilation state of its p17 subunit (33). Taken together, these different considerations have led us to determine whether oxysterol-induced apoptosis was associated with cellular features indicating the occurrence of oxidative processes during cell death.

To this end, human promyelocytic leukemia cells U937 were treated with 7-ketocholesterol alone [which is a potent inducer of apoptosis in these cells (13, 34)] or in combination with the antioxidants GSH or N-acetylcysteine (NAC), a precursor of glutathione (35). U937 cells were used because they are sensitive to oxysterols in the same range of concentrations as those observed in endothelial (10, 11) and smooth muscle cells (8); they are frequently used as macrophage reference models in studies to investigate the cytotoxicity of oxysterols in humans (13, 34). It is well recognized that macrophages play important roles in the atherosclerotic process since increased concentrations of oxysterols in macrophage foam cells (resulting from the phagocytosis of oxidized LDL by macrophages) may lead to cell death and plaque rupture (36). GSH and NAC were chosen because decreased GSH levels were observed during apoptosis that resulted either from the formation of oxidized glutathione (GSSG) (37) or GSH exclusion (37, 38). It has been shown at the cellular level that GSH is implied in the regulation of events leading to apoptosis. Thus, GSH controls the transmembrane mitochondrial potential ({Delta}{Psi}) (26) regulating the release of inner mitochondrial molecules, such as apoptosis-inducing factor (39) and/or cytochrome c (40–42), which is able to activate the caspases leading to internucleosomal DNA fragmentation; it has also been reported that GSH can regulate the activity of the neutral magnesium-dependent sphingomyelinase (43) that catalyzes the hydrolysis of sphingomyelin in ceramide, an early messenger implied in the cell death signal involved in apoptosis (44).

In the present study, the effects of GSH and NAC on 7-ketocholesterol-induced apoptosis were characterized and quantified after various periods of time (6, 12, 18, and 24 h) by different methods: cell counting, flow cytometric analysis of cell permeability with propidium iodide (45), in situ visualization of DNA fragmentation with the TdT-mediated dUTP-biotin nick end labeling method (TUNEL method) (46), determination of the proportion of apoptotic cells displaying condensed and/or fragmented nuclei after DNA staining with Hoechst 33342 (47), and analysis of the DNA fragmentation pattern by electrophoresis on 1.8% agarose gel (10, 11). At 24 h of treatment, the effects of GSH on 7-ketocholesterol-induced apoptosis were also characterized by Western blot analysis of some biochemical markers involved in the transmission of the cell death signal: cytochrome c which is released from mitochondria to the cytosol (48); procaspase-8/Flice/MACH/Mch5, an early caspase involved in signal transduction resulting from various ligand/receptor interactions (49, 50); procaspase-3/CPP32/Yama/apopain and procaspase-7, which are late effector caspases (31). During cell death, GSH content was followed with the use of the dye monochlorobimane (51, 52), and ROS production was investigated by oxidation of the dye 2', 7'-dichlorofluorescin-diacetate (DCFH-DA) determined by flow cytometry (53) and by quantification of polyunsaturated fatty acids determined by gas chromatography coupled with mass spectrometry (54). Addition of GSH and NAC in the cells treated with 7-ketocholesterol was able to impair the apoptotic program and some associated events. This led us to conclude that 7-ketocholesterol-induced apoptosis depends on the cellular GSH level and is associated with ROS production. However, prevention of apoptosis by GSH and NAC does not seem to be a general rule since these antioxidants impaired etoposide (but not cycloheximide) -induced apoptosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
U937 cells were grown in suspension in culture medium consisting of RPMI 1640 medium (Gibco, Eragny, France), 2 mmol/l L-glutamine (Gibco), and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin) (Gibco) supplemented with 10% (v/v) heat-inactivated fetal calf serum (Boehringer-Mannheim, Meylan, France). The cells were seeded at 5 x 105 per ml of culture medium, passaged twice a week, and incubated at 37°C under a 5% CO2/95% air atmosphere.

Cell treatments
The purity of 7-ketocholesterol(provided by Sigma, St. Louis, Mo.) was determined by CPG/SM to be 100%. For all experiments, a 7-ketocholesterol stock solution was prepared at 800 µg/ml as previously described (6, 10, 11, 13). Briefly, to realize this initial solution, 800 µg of 7-ketocholesterol was dissolved in 50 µl of absolute ethanol and 950 µl of culture medium was added. To obtain a 40 µg/ml final concentration, 50 µl of this initial solution was introduced per milliliter of culture medium at the beginning of the culture; the cells were further incubated with 7-ketocholesterol during various periods of time: 6, 12, 18, and 24 h. Under these experimental conditions, the final concentration of ethanol in the culture medium was 0.25% (v/v); at this concentration, ethanol did not affect cell growth characteristics (6, 10, 11, 13). Etoposide (VP-16) and cycloheximide (CHX) were obtained from Sigma. Stock solutions of VP-16 (20 mmol/l) and CHX (10 mg/ml) were prepared by diluting these reagents in dimethylsulfoxide (DMSO) (Sigma) and in culture medium, respectively. Further dilutions were made in culture medium in order to obtain VP-16 and CHX at 50 µmol/l and 50 µg/ml final concentrations, respectively. With VP-16, the final concentration of DMSO in the culture medium was 0.25% (v/v). The antioxidants GSH and NAC (Sigma) were diluted in the culture medium to obtain a 100 mmol/l initial concentration and used at 10 mmol/l final concentration, which is in the same range as GSH measured in mammalian cells (55, 56). When the cells were treated simultaneously with 7-ketocholesterol and GSH (or NAC), the antioxidants were introduced in the culture medium 30 min before 7-ketocholesterol, VP-16, or CHX.

Cell counting
Cell counting was performed with a hematocytometer under an inverted phase contrast microscope Laborlux IX 70 (Olympus, Tokyo, Japan) on cells seeded in 6-well plates (Falcon/Becton-Dickinson, Plymouth, U.K.) at a concentration of 1.5 x 105 cells in 3 ml of culture medium per well.

Determination of cell permeability with propidium iodide
Cell permeability was determined after staining with the phenanthrene dye propidium iodide ({lambda} Ex max: 540 nm, {lambda} Em max: 625 nm) (Sigma), which enters only dead cells (45, 47). A stock solution of propidium iodide was prepared in phosphate-buffered saline (PBS) at a concentration of 10 µg/ml and kept in the dark at room temperature. Propidium iodide was used at a final concentration of 4 µg/ml on the cell suspension adjusted to 106 cells/ml. Fluorescence was immediately quantified by flow cytometry in 10,000 cells on a logarithmic scale of fluorescence of four decades of log on a FACScan flow cytometer (Becton Dickinson, Mountain View, Calif.) at excitation and emission wavelengths of 488 nm and 585/42 nm, respectively.

Identification and quantification of apoptotic cells after nuclei staining with Hoechst 33342
Nuclear morphology of control and treated cells was studied by fluorescence microscopy after staining with Hoechst 33342 (Sigma); apoptotic cells were essentially characterized by nuclear condensation of chromatin and/or nuclear fragmentation (47). Hoechst 33342 was prepared extemporaneously in distilled water at 1 mg/ml and added to the culture medium at a final concentration of 10 µg/ml. After 1 h of incubation at 37°C, cells were washed twice in PBS and resuspended at a concentration of 106 cells/ml in PBS containing 1% (w/v) paraformaldehyde. Cell deposits of about 40,000 cells were applied to glass slides by cytocentrifugation for 5 min at 15,000 rpm with a cytospin 2 (Shandon, Cheshire, U.K.), mounted in Fluoprep (Biomérieux, Marcy l'Etoile, France), coverslipped, and stored in the dark at 4°C. The morphological aspect of cell nuclei was observed with an inverted microscope Laborlux IX70 (Olympus) by using an UV light excitation; for each sample, 300 cells were examined.

Characterization of apoptotic cells by in situ detection of DNA fragmentation
In situ visualization of DNA fragmentation at the single cell level was performed by the TUNEL method developed by Gavrieli et al. (46) by using the MEBSTAIN Apoptosis kit (Immunotech, Marseille, France) according to the manufacturer's procedure. Briefly, U937 cells were collected by centrifugation, the cell pellet was washed twice in phosphate-buffered saline (PBS, pH 7.2), and the cells were applied to glass slides (40,000 cells/slide) by centrifugation for 5 min at 15,000 rpm with a Cytospin 2 (Shandon, Cheshire, U.K.). After fixation at 4°C for 15 min with a solution of 4% (w/v) paraformaldehyde (Sigma) prepared in PBS, the cells were permeabilized at room temperature for 15 min with a solution of PBS containing 0.5% (v/v) Tween 20 (Sigma)-0.2% (w/v) bovine serum albumin (BSA) (Sigma), washed three times with deionizing water, and incubated with the TdT solution for 1 h at 37°C in a humidified atmosphere. The nuclear signal given by the TdT-mediated dUTP-biotin nick end labeling method was then detected by incubation for 30 min at room temperature with peroxidase-conjugated streptavidin (Dako, Copenhagen, Denmark) diluted 1/300 in PBS containing 0.2% (w/v) BSA, and revelation of peroxidase activity was performed with the Dako Liquid DAB Substrate Chromogen System (Dako). U937 cells were further counterstained with methylene blue (RAL/Rhône Poulenc, Villers St. Paul, France); the slides were mounted in Fluoprep (Biomérieux, Marcy l'Etoile, France), coverslipped, and stored in the dark at 4°C until observation with an inverted microscope Laborlux IX70 (Olympus).

DNA fragmentation assay on agarose gel
DNA fragmentation assays were performed by electrophoresis on 1.8% agarose gel. To this end, cellular DNA was extracted as previously described (57) by using a DNA extraction kit (Stratagene, La Jolla, Calif.). Briefly, after overnight cell lysis at 37°C in a lysis buffer containing 10 mmol/l EDTA, 400 mmol/l NaCl, 1 mg/ml proteinase K, 35 mmol/l sodium dodecyl sulfate (SDS), and 10 mmol/l Tris-HCl (pH 8.2), each tube was centrifuged and the supernatant containing the DNA was precipitated by two volumes of 100% ethanol and left overnight at -20°C. After centrifugation, DNA was resuspended in 100 µl TE buffer (10 mmol/l tris-HCl, 0.2 mmol/l Na2 EDTA, pH 7.5) before quantitation by spectrofluorometry. The size of DNA standards used to evaluate DNA fragmentation ranged from 100 to 2072 bp (Gibco). Electrophoresis was carried out for 15 h at 20 volts in 1.8% agarose gel prepared in TBE buffer [80 mmol/l Tris-Borate (pH 8.0), 2 mmol/l EDTA] and containing 0.1 µg/ml ethidium bromide. After electrophoresis, gels were examined under ultraviolet light and photographed or computerized with an image analysis system (Biocom, Les Ulis, France).

Subcellular fractionation and Western blot analysis
Cytochrome c release into the cytosol and degradation of procaspase-3, -7 and -8 were investigated by Western blot analysis on U937 cells incubated for 24 h in culture medium in the absence or presence of GSH (10 mmol/l), 7-ketocholesterol (40 µg/ml), or 7-ketocholesterol plus GSH, as well as in U937 cells treated for 4 h with VP-16 (50 µmol/l) used as positive control. For analysis of cytochrome c release, cells were harvested at the end of the treatment and washed twice with ice-cold PBS. Cells were resuspended in buffer A (20 mmol/l HEPES-KOH, pH 7.5, 10 mmol/l KCl, 1.5 mmol/l MgCl2, 1 mmol/l sodium EDTA, 1 mmol/l sodium EGTA, 1 mmol/l dithiothreitol) containing 250 mmol/l sucrose and a mixture of protease inhibitors (1 mmol/l phenylmethylsulfonylfluoride, 1% aprotinin, 1 mmol/l leupeptin, 1 µg/ml pepstatin A, and 1 µg/ml chymostatin). The cells were homogenized by successive passages through a 26 G fine needle. Unbroken cells, large plasma membrane pieces, and nuclei were removed by centrifuging the homogenates at 1000 x g at 4°C for 10 min. The resulting supernatant was subjected to 10,000 x g centrifugation at 4°C for 20 min. The supernatant was recentrifuged at 100,000 x g (4°C, 1 h) to generate cytosol. For analysis of procaspase-3, -7, and -8, cells were collected by centrifugation at the end of the incubation time, washed twice in PBS, and lysed in Ripa buffer (150 nmol/l NaCl, 50 mmol/l Tris-HCl, pH 8.0, 0.1% NaSDS, 0.5% Na-desoxycholate) in the presence of protease inhibitors (0.1 mmol/l phenylmethylsulfonylfluoride, 2.5 µg/ml pepstatin, 10 g/ml aprotinin, 2.5 µg/ml trypsin inhibitor, 5 µg/ml leupeptin) for 30 min, then centrifuged (20 min, 15,000 x g).

The protein concentration was measured by using bicinchoninic acid reagent (Pierce, Rockford, Ill.) according to the method of Smith et al. (58). Proteins (50 µg) were incubated in loading buffer (125 mmol/l Tris-HCl, pH 6.8, 10% ß-mercapto-ethanol, 4.6% SDS, 20% glycerol and 0.003% bromophenol blue), separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and electroblotted to polyvinylidine difluoride membrane (BioRad, Ivry sur Seine, France). After blocking nonspecific binding sites overnight by 5% nonfat milk in TPBS (PBS, Tween 20 0.1%), the membranes were incubated for 2 h at room temperature with primary antibody: procaspase-3 (Transduction Laboratories, Lexington, Ky.), procaspase-7, and procaspase-8 (Pharmingen, San Diego, Calif.). After two washes in TPBS, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse (Jackson ImmunResearch Laboratories, West Grove, Pa.) for 30 min at room temperature and washed twice in TBPS. Immunoblot was revealed using enhanced chemoluminescence detection kit (Amersham, Les Ulis, France) by autoradiography. Each experiment was repeated four to six times with identical results.

Quantification of cellular glutathione content by flow cytometry
The level of cellular GSH per cell was determined by flow cytometry after staining with monochlorobimane (Molecular Probes, Eugene, Oreg.) as previously described (52). Monochlorobimane [(syn-(ClCH2, CH3)-1,5-diazabicyclo-[3.3.0]-octa-3,6-dione-2,8-dione); {lambda} Ex max: 380 nm, {lambda} Em max: 461 nm], which binds to GSH under the action of glutathione S-transferase (51, 52), was prepared as a 4 mmol/l solution in 100% ethanol and stored in the dark at 4°C. It was added at 200 µmol/l in cell suspensions adjusted at 106 cells per ml. After 30 min of incubation at 37°C, cells were washed twice in PBS, resuspended at a concentration of 106 cells/ml in PBS, and analyzed on a BRYTE HS flow cytometer (BioRad, Hercules, Calif.). Monochlorobimane was excited at 360/370 nm with a 75W mercury/xenon lamp and the fluorescence was collected with a 450/20 nm band pass filter. Analyses were performed on 5000 cells and fluorescence intensities were measured on a logarithmic scale of fluorescence of four decades of log. The data were collected, stored, and analyzed with the WinBryte software (BioRad).

Characterization and quantification of 7-ketocholesterol by capillary gas chromatography and mass spectrometry
U937 cells incubated for 6, 12, 18, and 24 h in their culture medium in the absence or presence of GSH (10 mmol/l), 7-ketocholesterol (40 µg/ml), or 7-ketocholesterol plus GSH were collected by centrifugation, washed twice in PBS, and enumerated with a hematocytometer. Total lipids from cells were further extracted by the methods of Folch et al. (54). The extract was saponified at 60°C for 60 min with potassium hydroxide (13.2 g/l), followed by esterification at 60°C for 60 min with boron trifluoride (BF3) -methanol to give fatty acid methyl esters, and cholesterol oxides were analyzed by capillary gas chromatography (59, 60) on a Hewlett-Packard 12.5 m-long fused silica, cross-linked methylsilicone column with the use of a Hewlett-Packard 5890 gas chromatograph attached to a 5971A mass detector (Hewlett-Packard, Palo Alto, Calif.). Heptadecanoic acid (17:0) was added as an internal standard to each sample before extraction and 7-ketocholesterol content was determined from the ratio of the peak area of the sample to the peak area of the internal standard. Data are presented as the quantity of 7-ketocholesterol in µg per 106 cells.

Characterization of polyunsaturated fatty acids oxidation by capillary gas chromatography and mass spectrometry
U937 cells were incubated for 6, 12, 18, and 24 h in their culture medium in the absence or presence of GSH (10 mmol/l), 7-ketocholesterol (40 µg/ml), or 7-ketocholesterol plus GSH. Total lipids were extracted by the methods of Folch et al. (54). The extract was further saponified at 60°C for 60 min with potassium hydroxide (13.2 g/l), followed by esterification at 60°C for 60 min with BF3-methanol to give fatty acid methyl esters. Samples were analyzed by capillary gas chromatography on a Hewlett-Packard 5890 chromatograph attached to a 5971A mass detector (Hewlett-Packard). They were injected onto a Hewlett-Packard 12.5 m-long methylsilicone fused silica column by using a 7673A auto-injector (Hewlett-Packard). The temperature of the injector and mass detector was 250°C and 280°C, respectively. Temperature was set at 140°C for 3 min and then programmed to reach 220°C at the rate of 1°C/min and 280°C at the rate of 10°C/min; helium pressure was 35 kPa. Heptadecanoic acid (17:0) was added as an internal standard to each sample before extraction and fatty acid content was determined from the ratio of the peak area of the sample to the peak area of the internal standard. To demonstrate the involvement of oxidation processes occurring during cell death, data are presented by the ratio [unsaturated fatty acids]/[saturated fatty acids].

Flow cytometric measurement of reactive oxygen species production with DCFH-DA
To measure the production of ROS, cells (5x105 cells/ml of culture medium) were incubated for 1 h at 37°C in the dark with 10 µmol/l of DCFH-DA (Molecular Probes), as described previously (53), and a positive control was performed with H2O2 used at 1 volume final concentration. The 2',7'-dichlorofluorescein (DCF) fluorescence resulting from the oxidation of DCFH-DA was measured in 10,000 cells on a logarithmic scale of fluorescence of four decades of log by using a FACScan flow cytometer (Becton-Dickinson) at excitation and emission wavelengths of 488 nm and 524/44 nm, respectively.

Statistical methods
Statistical analyses were performed with SYSTAT software (Evanston, Ill.) using a two-way analysis of variance .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Protective effects of glutathione and of N-acetylcysteine on 7-ketocholesterol induced apoptosis
Characterization of cell death induced by 7-ketocholesterol (40 µg/ml) on U937 cells was performed at 6, 12, 18, and 24 h of treatment. As shown in Fig. 1A, treatment of U937 cells with 7-ketocholesterol significantly inhibited cell growth at 18 and 24 h, but no effect was observed before. Because of the complex relationships between inhibition of cell growth and modification of the cellular membrane integrity, flow cytometric analyses of cell permeability were performed with propidium iodide, and a significant increase in the number of propidium iodide permeable cells was observed at 24 h ( Fig. 1B). 7-Ketocholesterol-induced cell death was also characterized by in situ detection of DNA fragmentation with the TUNEL method as well as by nuclei staining with Hoechst 33342 ( Fig. 2) in order to distinguish between apoptosis and necrosis. By using the TUNEL method, fragmented DNA was found in cells exhibiting typical morphological features of apoptosis such as condensed and/or fragmented nuclei ( Fig. 2B), which were also identified after staining with Hoechst 33342 ( Fig. 2D). This latter method, which was used to quantify apoptosis, showed a significant increase of the proportion of apoptotic cells at 12, 18, and 24 h of treatment ( Fig. 1C). Under treatment with 7-ketocholesterol, the occurrence of apoptotic cells and the reduction of cell growth preceded the increase permeability of U937 cells to propidium iodide.



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  		Figure 1. Protective effects of glutathione and N-acetylcysteine on 7-ketocholesterol-induced apoptosis. Cell growth, permeability to propidium iodide, and induction of apoptotic cells in untreated U937 cells (control) and in U937 cells treated for 6, 12, 18, and 24 h with either 7-ketocholesterol (40 µg/ml) (7-keto), glutathione (10 mmol/l) (GSH), N-acetylcysteine (10 mmol/l) (NAC), or 7-ketocholesterol associated with glutathione (7-keto+GSH) or N-acetylcysteine (7-keto+NAC). At the end of the incubation period, the following parameters were measured: total number of cells per ml (A), proportions of propidium iodide permeable cells (B), and proportions of apoptotic cells (C). Data are mean ±SEM of four independent experiments. Significance of the differences between control and 7-ketocholesterol-treated cells (*P<0.05); significance of the differences between 7-ketocholesterol-treated cells and (7-ketocholesterol + glutathione or N-acetylcysteine)-treated cells (**P<0.05).



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  		Figure 2. Morphological characterization of 7-ketocholesterol-treated cells. U937 cells were incubated for 6, 12, 18, and 24 h in culture medium with or without 7-ketocholesterol (40 µg/ml). Subsequently, apoptosis was characterized either by in situ detection of DNA fragmentation with the TUNEL method or after nuclei staining with Hoechst 33342. With the TUNEL method, the fragmented DNA localized in the nuclei of apoptotic cells is shown in brown (B) and is not detected in untreated cells (A). After Hoechst staining, cells with condensed and/or fragmented nuclei are detected in 7-ketocholesterol-treated cells (D) but not in untreated cells (C) (x1000). The data shown with the TUNEL method and after staining with Hoechst 33342 correspond to a 18 h incubation time.

When U937 cells were simultaneously incubated with 7-ketocholesterol and GSH or NAC [which at the concentration used (10 mmol/l) had no effect on cell growth, cell permeability and apoptosis ( Fig. 1)], we observed that GSH and NAC impaired 7-ketocholesterol-induced cytotoxicity ( Fig. 1). Thus, GSH and NAC counteracted cell growth inhibition induced by 7-ketocholesterol at 18 and 24 h ( Fig. 1A), and also strongly reduced the proportion of propidium iodide-permeable cells at 24 h ( Fig. 1B) as well as the proportions of apoptotic cells at 12, 18, and 24 h ( Fig. 1C).

Effect of glutathione and N-acetylcysteine on 7-ketocholesterol-induced DNA fragmentation
Under treatment with 7-ketocholesterol (40 µg/ml), the DNA fragmentation pattern was characterized by electrophoresis on 1.8% agarose gel. In these conditions, no DNA fragmentation was detected in untreated cells ( Fig. 3, lane 1) or GSH- and NAC-treated cells ( Fig. 3, lanes 2–3), whereas a typical internucleosomal DNA fragmentation characteristic of 7-ketocholesterol-induced apoptosis was detectable at 18 and 24 h of treatment ( Fig. 3, lanes 5–6), but not at 12 h ( Fig. 3, lane 4). This internucleosomal DNA fragmentation was strongly inhibited by GSH ( Fig. 3, lane 8) or NAC ( Fig. 3, lane 11) at 18 h, but not at 24 h ( Fig. 3, lanes 9 and 12). As shown with 7-ketocholesterol, no internucleosomal DNA fragmentation was detected at 12 h in cells incubated simultaneously with 7-ketocholesterol and GSH ( Fig. 3, lane 7) or NAC ( Fig. 3, lane 10). As similar results were obtained with GSH and NAC, only GSH was used for additional experiments performed in association with 7-ketocholesterol.



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  		Figure 3. Effect of the antioxidants glutathione and N-acetylcysteine on the internucleosomal DNA fragmentation induced by 7-ketocholesterol. Analysis of DNA fragmentation by electrophoresis on 1.8% agarose gel was performed at 24 h of culture on untreated U937 cells (1), U937 cells incubated with glutathione (10 mmol/l) (GSH) (2), or N-acetylcysteine (10 mmol/l) (NAC) (3), and on U937 cells treated with 7-ketocholesterol (40 µg/ml) for 12, 18, and 24 h in the absence of antioxidant (lanes 4, 5, and 6) or in the presence of GSH (lanes 7–9) or of NAC (lanes 10–12)

Effect of glutathione on the uptake of 7-ketocholesterol
To define whether GSH did not interact with the uptake of 7-ketocholesterol, the cellular concentration of 7-ketocholesterol was quantified by capillary gas chromatography in U937 cells incubated for 6, 12, 18, and 24 h in their culture medium in the absence or presence of GSH (10 mmol/l), 7-ketocholesterol (40 µg/ml), or 7-ketocholesterol plus GSH ( Table 1). In these conditions, whatever the time of culture, the uptake of 7-ketocholesterol was not affected by the presence of GSH, and was similar in 7-ketocholesterol-treated cells and 7-ketocholesterol plus GSH-treated cells.


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  		Table 1. Effect of glutathione on 7-ketocholesterol uptake after 6, 12, 18, and 24 h of culture1tfa

Modulation by glutathione of cytochrome c release and of procaspases activation
In 7-ketocholesterol-treated U937 cells (40 µg/ml) and after 24 h incubation, cytochrome c release into the cytosol and activation of procaspase-3, -7 and -8 were investigated by Western blot analysis in the absence or presence of GSH (10 mmol/l). A positive control was performed on the cells treated for 4 h with VP-16 (50 µmol/l) ( Fig. 4). In untreated and GSH-treated cells, similar results were obtained. As observed in VP-16-treated cells, 7-ketocholesterol-induced apoptosis was associated with an important release of cytochrome c into the cytosol and a noticeable degradation of procaspase-8. However, in contrast to VP-16-treated cells, 7-ketocholesterol-induced apoptosis was associated only with a slight degradation of procaspase-3 and -7. The minor degradation of caspase-3 in 7-ketocholesterol-induced apoptosis was confirmed by the low intensity of p17 subunit observed by Western blot analysis, whereas in VP-16-treated cells a net cleavage of procaspase-3 into subunits p17 and p19 was observed ( Fig. 4).



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  		Figure 4. Modulation by glutathione of cytochrome c release and of procaspase activation occurring during 7-ketocholesterol-induced apoptosis. Cytochrome c release into the cytosol and activation of the procaspase-3, -7 and -8 were investigated at 24 h by Western blot analysis in control cells (control), glutathione-treated cells (10 mmol/l) (GSH), 7-ketocholesterol (40 µg/ml) -treated cells (7-keto), and (7-ketocholesterol plus glutathione)-treated cells (7-keto+GSH). A positive control was performed on the cells treated for 4 h with etoposide (50 µmol/l).

During 7-ketocholesterol-induced apoptosis, GSH impaired cytochrome c release into the cytosol and procaspase-8 degradation, but had no effect on the minor degradations observed in procaspase-3 and -7.

Decrease of glutathione content under treatment with 7-ketocholesterol
Since GSH protected from 7-ketocholesterol induced apoptosis and its side effects, we asked whether 7-ketocholesterol could act by decreasing the GSH content per cell and whether GSH could counteract this phenomenon. To evaluate this hypothesis, GSH content was measured by flow cytometry with monochlorobimane at 30 min, 6 h, 12 h, 18 h, and 24 h in untreated and GSH-treated cells, and at 6 h, 12 h, 18 h and 24 h in 7-ketocholesterol and (7-ketocholesterol plus GSH)-treated cells. In these conditions, the GSH content per cell in 7-ketocholesterol-treated cells as well as the proportion of GSH positive cells measured by the mean fluorescence intensity began to decrease significantly by 6 h of treatment ( Fig. 5). Thus, the decrease in GSH content constitutes an early event of 7-ketocholesterol-induced apoptosis. Whatever the time of culture, cells treated with GSH did not have a higher GSH content than untreated cells. However, the decrease in GSH content and the proportions of GSH-positive cells was significantly less important in 7-ketocholesterol plus GSH-treated cells than in 7-ketocholesterol-treated cells ( Fig. 5).



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  		Figure 5. Impairment by glutathione of the decrease of cellular glutathione content induced by 7-ketocholesterol. Glutathione (GSH) content was measured by flow cytometry with monochlorobimane at 30 min, 6 h, 12 h, 18 h, and 24 h in untreated and GSH-treated cells, and at 6, 12, 18, and 24 h in 7-ketocholesterol and (7-ketocholesterol plus GSH)-treated cells. GSH (10 mmol/l) was added in the culture 30 min before 7-ketocholesterol (40 µg/ml). At the end of the indicated incubation times, the cellular GSH content was quantified by flow cytometry after staining with monochlorobimane (A) and the proportion of monochlorobimane-positive cells was determined (B). Data are mean ±SEM of four independent experiments. Significance of the differences between control and 7-ketocholesterol-treated cells (*P<0.05); significance of the differences between 7-ketocholesterol-treated cells and (7-ketocholesterol + glutathione)-treated cells (**P<0.05).

Occurrence of fatty acids oxidation during 7-ketocholesterol-induced apoptosis: partial inhibition by glutathione
Oxidation of unsaturated fatty acids leading to an increase of saturated fatty acids can occur under the action of various ROS. To show the involvement of ROS during 7-ketocholesterol-induced apoptosis, the ratio [unsaturated fatty acids]/[saturated fatty acids] was determined by capillary gas chromatography. In these conditions, the production of ROS indirectly revealed by the decrease in the ratio became detectable at 18 h ( Fig. 6). As the highest decrease in the ratio was observed at 24 h, the production of ROS was investigated at that time by flow cytometry with the use of DCFH-DA. An oxidation of the intracellular DCFH to fluorescent DCF was observed in 7-ketocholesterol-treated cells as indicated by the mean fluorescence value, which was higher in 7-ketocholesterol-treated cells (mean fluorescence value = 69, 81) ( Fig. 7C) than in untreated cells (mean fluorescence value = 37, 47) ( Fig. 7A). This data confirmed the production of ROS during 7-ketocholesterol-induced apoptosis. At 18 and 24 h, GSH inhibited the oxidation of polyunsaturated fatty acids as shown by the highest values of the ratios [unsaturated fatty acids]/[saturated fatty acids] ( Fig. 6). At 24 h, ROS production was also prevented by GSH: the fluorescence in (7-ketocholesterol + GSH)-treated cells (mean fluorescence = 46.42) ( Fig. 7D) was lower than in 7-ketocholesterol-treated cells (mean fluorescence value = 69, 81) ( Fig. 7C); at that time the mean fluorescence values in untreated cells (mean fluorescence value = 37,47) ( Fig. 7A) and GSH-treated cells (mean fluorescence value = 37,48) ( Fig. 7B) were similar.



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  		Figure 6. Oxidation of unsaturated fatty acids during 7-ketocholesterol-induced apoptosis and the protective effect of glutathione. Analysis of the oxidation of unsaturated fatty acids by gas chromatography coupled with mass spectrometry was performed on untreated U937 cells (control), and on U937 cells incubated with either glutathione (10 mmol/l) (GSH), 7-ketocholesterol (40 µg/ml) (7-keto), or 7-ketocholesterol and glutathione (7-keto + GSH) at 6, 12, 18, and 24 h of treatment. The oxidation process was measured by the ratio [unsaturated fatty acids]/[saturated fatty acids]. Data are mean ±SEM of four independent experiments. Significance of the differences between control and 7-ketocholesterol-treated cells (*P<0.05); significance of the differences between 7-ketocholesterol treated cells and (7-ketocholesterol + glutathione)-treated cells (**P<0.05).



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  		Figure 7. Impairment of ROS production by glutathione during 7-ketocholesterol-induced apoptosis. Analysis of ROS production in untreated U937 cells (control) (A), glutathione-treated cells (10 mmol/l) (GSH) (B), 7-ketocholesterol-treated cells (40 µg/ml) (7-keto) (C), and 7-ketocholesterol (40 µg/ml) plus glutathione (10 mmol/l) -treated cells (7-keto+GSH) (D). At the end of the incubation time, ROS production was quantified by flow cytometry after staining with DCFH-DA, and a positive control (E) was performed with H2O2 used at 1 volume final concentration. The DCF fluorescence resulting from the oxidation of DCFH-DA was measured on 10,000 cells on a logarithmic scale of fluorescence of four decades of log by using a FACScan flow cytometer; data shown are representative of three independent experiments. Shaded histograms: ROS production revealed by the fluorescence of DCF resulting from the oxidation of DCFH-DA; unshaded histograms: spontaneous fluorescence of the cells.

Effect of glutathione and N-acetylcysteine on etoposide and cycloheximide-induced apoptosis
Finally, we asked whether GSH and NAC were able to inhibit apoptosis induced by molecules other than 7-ketocholesterol. To this end, U937 cells were treated by VP-16, an inhibitor of topoisomerase II, or by CHX, an inhibitor of protein synthesis, known inducers of apoptosis (61, 62). When U937 cells were incubated simultaneously with VP-16 or CHX associated with GSH or NAC [which at the concentration used (10 mmol/l) had no effect on cell growth and apoptosis ( Table 2) ], we observed that GSH and NAC impaired VP-16-induced cytotoxicity but not CHX-induced cytotoxicity ( Table 2). Thus, when U937 cells were treated with VP-16, GSH and NAC counteracted cell growth inhibition and strongly reduced the proportion of apoptotic cells quantified after nuclei staining with Hoechst 33342.


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  		Table 2. Effect of the antioxidants glutathione (GSH) and N-acetylcysteine (NAC) on etoposide (VP-16) and cycloheximide (CHX)-induced apoptosis2tfa


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The involvement of ROS species in apoptosis induced by different agents has been suggested by some experiments: induction of apoptosis by hydrogen peroxide (63), oxygen radicals (64), or nitric oxide donors (65); potentiation of apoptosis by molecules decreasing intracellular stores of GSH (66); down-regulation of antioxidant defenses (27); lipid peroxidation (23, 67); inhibition of apoptosis by antioxidants and by superoxide dismutase or catalase (23). In addition, the Bcl-2 oncogene, which prevents apoptosis in various systems (68) and partially inhibits 7ß-hydroxycholesterol and 7-ketocholesterol-induced apoptosis, has also been shown to block oxidative damage (22). However, so far the production of ROS during apoptosis does not seem to be a general rule (69–71). Therefore, we asked whether the apoptotic process triggered by 7-ketocholesterol was associated with an oxidative stress and have demonstrated that GSH is implied in the control of 7-ketocholesterol-induced apoptosis, which is associated with the production of ROS.

The role of GSH in 7-ketocholesterol induced apoptosis was investigated since the GSH redox cycle isan enzyme-coupled system, constituting one of the most important cellular defense systems against oxidative stress (35, 72). In addition, GSH is involved in the control of apoptosis induced by different agents, as indicated by various investigations. GSH depletion is an early event in thymocyte apoptosis (73); the reduced GSH prevents nitric oxide-induced apoptosis in vascular smooth muscle cells (74). NAC, which increases the cellular GSH content, protects from apoptosis triggered by tumor necrosis factor {alpha} (75) or by transforming growth factor ß1 (76). Slater and colleagues (25) have demonstrated that thymocytes apoptosis is associated with a 40% decrease in intracellular GSH content; furthermore, Kane and colleagues (77) have shown that expression of the proto-oncogene Bcl-2, a known inhibitor of apoptosis, is associated with an increase of intracellular GSH.

As we report here that GSH or NAC impaired various phenomena linked to apoptosis (reduction of cell growth, enhanced permeability to propidium iodide, occurrence of nuclear condensation and/or fragmentation, release of cytochrome c into the cytosol, degradation of procaspase-8) and delayed internucleosomal DNA fragmentation, our findings demonstrate for the first time that GSH is involved in the control of 7-ketocholesterol-induced apoptosis. This involvement of GSH in the control of 7-ketocholesterol-induced apoptosis is clearly supported by the ability of GSH to inhibit cytochrome c release into the cytosol and to counteract procaspase-8 degradation, early signals of activation in different forms of apoptosis (49); however, no protective effects of GSH were found on the late effectors caspase-3 (CPP32) and -7. Because of the slight degradation of these late effector caspases observed under treatment with 7-ketocholesterol, we must consider different hypotheses.

In a first hypothesis, we can suppose that caspase-3 and -7 play a minor role in 7-ketocholesterol-induced apoptosis and that GSH has no effect on these caspases. Such a mechanism can occur since CPP32 (caspase-3) -independent pathways leading to apoptosis have been described (78); although involvement of CPP32 has previously been suggested in 7-ketocholesterol-induced apoptosis (8, 79), our data do not contradict this result. Indeed, in this previous study, the involvement of CPP32 in 7-ketocholesterol-induced apoptosis was based only on the ability of the tetrapeptide DEVD (an inhibitor of cysteine protease considered specific to CPP32) to decrease DNA fragmentation. However, at that time it was not clearly established that DEVD was also able to inhibit caspase-6, -8, and -7 (80). Therefore, as shown in the present study that caspase-8 is involved in the metabolic pathway leading to 7-ketocholesterol-induced apoptosis and considering that caspase-3 plays a minor role, the impairment of apoptosis by the tetrapeptide DEVD could in fact be due to caspase-8 inhibition.

In a second hypothesis, since 7-ketocholesterol-induced apoptosis is a low process (opposite of those observed under treatment with VP-16) and is accompanied by a rapid secondary necrosis characterized by an increased permeability of the cells to propidium iodide, we can speculate that the activation of caspase-3 and -7 could be difficult to detect because these caspases would be transiently present in dead cells due to the proteolytic activities that are probably important in the late phases of apoptosis. Therefore, even if caspase-3 and -7 are involved, a protective effect of GSH would be difficult to establish. According to our data, which demonstrate that GSH strongly reduces cytochrome c release into the cytosol as well as caspase-8 degradation but only delays DNA fragmentation, it seems likely that GSH inhibits early signals of 7-ketocholesterol-induced apoptosis but not the late signals implied in DNA degradation. In the present study, we also described that 7-ketocholesterol induced a rapid decrease of the GSH content, occurring as soon as 6 h of treatment; this event constitutes, to our knowledge, the earliest cellular change associated with apoptosis triggered by this oxysterol. As GSH content has been measured with the use of monochlorobimane, which binds to thiol groups by an enzymatic reaction catalyzed by glutathione S-transferase (51, 52), we can consider that the decrease in GSH level results either from a reduction of glutathione S-transferase activity or from a down-regulation of this enzyme at the transcriptional or transductional level (27). We can also suppose that the overall GSH depletion is due to active GSH extrusion from apoptotic cells (37, 38) or that the cellular oxidized form of glutathione (GSSG) increased as a consequence of ROS production occurring during apoptosis. Indeed, ROS production has soon been revealed during spontaneous apoptosis described throughout the degeneration of cultured Down's syndrome neurons (23), as well as in apoptosis induced by biological, chemical, or physical agents (21, 67).

Another major new finding of our study is the identification of ROS production during 7-ketocholesterol-induced apoptosis, as shown by the time-dependent decrease of the ratio [unsaturated fatty acids]/[saturated fatty acids] measured by gas chromatography coupled with mass spectroscopy and by oxidation of the dye DCFH-DA quantified by flow cytometry. In addition, inhibition of the oxidation of fatty acids and of the dye DCFH-DA by GSH are also in favor of ROS production during 7-ketocholesterol-induced apoptosis. Moreover, as ROS react with cellular macromolecules, either damaging them directly or setting in motion a chain reaction wherein the free radical is passed from one macromolecule to another, resulting in extensive damages to cellular structures such as membranes (81), ROS production could induce some modifications at various cellular levels. In the present work, one of the consequences of fatty acids oxidation could be the membrane damages revealed in 7-ketocholesterol-treated cells by enhanced permeability to propidium iodide. Previously, we also showed a decrease in the mitochondrial potential ({Delta}{Psi}) of apoptotic cells under treatment with 7-ketocholesterol (6); this observation agrees with ROS production and with the cytosolic release of cytochrome c presently observed. Indeed, when {Delta}{Psi} decreases, the breakdown of the outer membrane of mitochondria occurs and the cytochrome c is released in the cytosol (24, 26, 39). During this process, ROS could react with the thiol groups of GSH localized at the mitochondrial membrane level (82) and contribute to lower GSH level. Furthermore, a low level of GSH could favor the decrease of {Delta}{Psi}, which would subsequently activate the opening of permeability transition pores to finally induce the release of apoptosis-inducing factor (26) and/or of cytochrome c (83). It is tempting to speculate that this mechanism probably occurs. Indeed, the impairment of 7-ketocholesterol-induced apoptosis by GSH is associated with a lower cytosolic release of cytochrome c (reported in this study) and with an inhibition of the decrease of {Delta}{Psi} measured with rhodamine 123 (unpublished results).

Finally, to determine whether impairment of apoptosis by GSH was a general phenomenon, we studied the effect of GSH on U937 cells treated with either VP-16 or CHX, two potent inducers of apoptosis. The ability of GSH to inhibit VP-16-induced apoptosis but not CHX-induced apoptosis is in favor of a protective effect of GSH, depending of the inducer of cell death considered.

In conclusion, our data demonstrate that GSH is involved in the control of 7-ketocholesterol-induced apoptosis associated with the production of ROS and underline the ability of antioxidants to counteract the cytotoxic effects of oxysterols that are at increased levels in the plasma of hypercholesterolemic patients at high cardiovascular risk (1, 84). Our data also open new areas of investigation to explain the mode of action of oxysterols that could contribute in vivo to the development of the atherosclerotic lesion by inducing endothelium damage (10, 11) and by initiating inflammatory processes resulting from the release of interleukin 1ß (13) and ROS in the vicinity of apoptotic cells.


   ACKNOWLEDGMENTS
 
This work was supported by the Université de Bourgogne, the Conseil Régional de Bourgogne, the Institut National de la Santé et de la Recherche Médicale (INSERM), the Comité Français de Coordination des Recherches sur l'Athérosclérose et le Cholestérol (ARCOL), and the Fondation pour la Recherche Médicale. We express our gratitude to Dr. Corinne Laplace-Builhe (BIO-RAD S.A., Ivry-sur-Seine, France) for flow cytometric quantification of glutathione content with monochlorobimane.


   FOOTNOTES
 
1 The first two authors contributed equally to this work.

2 Correspondence: CHU/Hôpital du Bocage, INSERM U 498, Laboratoire de Biochimie Médicale, BP 1542, 21034 Dijon Cedex, France. E-mail: Gerard.Lizard{at}u.bourgogne.fr

3 Abbreviations: BF3, boron trifluoride; BSA, bovine serum albumin; CHX, cycloheximide; DCF, 2',7'-dichlorofluorescein; DCFH-DA, 2', 7'-dichlorofluorescin-diacetate; GSH, glutathione; DMSO, dimethylsulfoxide; LDL, low density lipoproteins; NAC, N-acetylcysteine; PBS, phosphate-buffered saline; ROS, reactive oxygen species; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TPBS, PBS, Tween 20 0.1%; TUNEL, TdT-mediated dUTP-biotin nick end labeling; VP-16, etoposide.

Received for publication February 26, 1998. Accepted for publication July 24, 1998.


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