Mitochondrial Membrane Permeabilization and Superoxide Production during Apoptosis

A SINGLE-CELL ANALYSIS*

Heiko DüssmannDagger , Donat KögelDagger , Markus RehmDagger , and Jochen H. M. PrehnDagger §

From the Dagger  Interdisciplinary Center for Clinical Research (IZKF) and the § Department of Pharmacology and Toxicology, Westphalian Wilhelms-University, D-48149 Münster, Germany

Received for publication, October 22, 2002, and in revised form, December 19, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The temporal relationship between mitochondrial membrane permeabilization and reactive oxygen species production during apoptosis remains unknown. We analyzed the rate of superoxide production of human breast carcinoma cells expressing a cytochrome c-green fluorescent protein (cyt-c-GFP) fusion protein at the single-cell level during apoptosis. In cells treated with the proapoptotic agents staurosporine (3 µM) or tumor necrosis factor-alpha (100 ng/ml), the release of cyt-c-GFP was individually set for each cell, and the majority of the fusion protein was released in less than 10 min. Prior to the release of the fusion protein, cells demonstrated a constant rate of superoxide production determined with the probe hydroethidine. After the release was completed, the superoxide concentration increased rapidly to a level more than 3-fold above baseline. Treatment with the broad spectrum caspase inhibitor z-Val-Ala-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk; 200 µM) did not alter the kinetics of the cyt-c-GFP release but significantly reduced superoxide concentration after the release of cyt-c-GFP. Interestingly, treatment with z-VAD-fmk also reduced the increase in superoxide concentration in response to menadione in the absence of mitochondrial cyt-c-GFP release. Mitochondrial depolarization with the protonophore carbonyl cyanide p-trifluoromethoxy-phenylhydrazone per se did not trigger cyt-c-GFP release or an increase in superoxide production. Our data suggest that mitochondria increase their superoxide production during apoptosis directly after the quantitative release of soluble intermembrane proteins and demonstrate novel antioxidative effects of the commonly used caspase inhibitor z-VAD-fmk.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The release of soluble proteins from the mitochondrial intermembrane and intracristal space is a central, coordinating step in many apoptosis pathways (1, 2). The release of cytochrome c (cyt-c)1 and Smac/DIABLO triggers or potentiates the activation of caspases, a family of cytosolic cysteine proteases (3-5). Activation of executioner caspases 3, 6, and 7 is responsible for the majority of the biochemical and morphological changes during apoptosis. Mitochondria also release other factors that trigger caspase-independent apoptotic cell death, in particular the apoptosis-inducing factor AIF (6).

However, mitochondrial membrane permeabilization is also able to activate additional cell death pathways. A decrease in mitochondrial ATP generation, mitochondrial membrane potential (Delta Psi m) depolarization, activation of the permeability transition pore complex, and increased reactive oxygen species production are features of necrotic but also apoptotic cell death (7-13). Loss of cyt-c directly causes a respiratory inhibition and Delta Psi m depolarization (13). The increase in superoxide production during apoptosis has been shown to require mitochondrial respiratory chain activity and may also be a direct consequence of the loss of cyt-c (10, 12, 14). In neurons and other cell types susceptible to pro-oxidants, superoxide formation may play a prominent role in cell death execution during apoptosis (8, 15, 16). In some settings, inhibition of superoxide formation may exert stronger protective effects than inhibition of executioner caspases (12, 17). Despite the importance of mitochondrial superoxide production for cell death execution during apoptosis, the relationship between mitochondrial membrane permeabilization and superoxide production have not yet been determined at the single-cell level. In the present study, we investigated superoxide production during apoptosis by confocal time-lapse imaging of human breast carcinoma cells expressing a cyt-c-green-fluorescent-protein (GFP) fusion protein (11, 18).

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Recombinant human tumor necrosis factor-alpha (TNF-alpha ), cycloheximide (CHX), carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), menadione, and embryo-tested paraffin oil were purchased from Sigma. Staurosporine (STS) and manganese tetrakis (4-benzoyl acid) porphyrin (MnTBAP) were from Alexis (Grünberg, Germany). The broad-spectrum caspase inhibitor z-Val-Ala-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk) was purchased from Bachem (Heidelberg, Germany). Hydroethidine (HEt) and tetramethylrhodamine methyl ester (TMRM) were from Molecular Probes (Leiden, The Netherlands).

Cell Culture and Transfection-- Generation and characterization of human breast carcinoma MCF-7 cells stably expressing a cytochrome c-green fluorescent protein (cyt-c-GFP) fusion protein have been described (18). We have previously shown that cyt-c-GFP is imported into mitochondria and co-released with endogenous cyt-c after selective outer membrane permeabilization with digitonin. Subcellular fractionation experiments confirmed the concomitant release of endogenous cyt-c and cyt-c-GFP from mitochondria during apoptosis (18).

Time-lapse Confocal Fluorescence Microscopy-- Cyt-c-GFP and ethidium fluorescence were monitored using an inverted Olympus IX70 confocal microscope equipped with a 488-nm argon laser (Fluoview; Olympus, Hamburg, Germany). HEt is a nonfluorescent compound that is oxidized by superoxide to its fluorescent product Et (19), allowing a semiquantitative determination of superoxide production (12, 20). HEt was used at an extracellular concentration of 1 µM. Measurement of Delta Psi m was performed after equilibration of MCF-7/cyt-c-GFP cells with 30 nM of the cationic, voltage-sensitive dye TMRM (21).

Fluorescence transmitted the first dichroic mirror above 505 nm, was divided with a second dichroic mirror at 550 nm, and detected after transmission of a 510-540-nm bandpass filter (GFP) or a 590-nm high pass emission filter (Et and TMRM). There was no Et or TMRM fluorescence detectable in the GFP channel. The cross-talk between the average pixel intensity of GFP in the Et or TMRM channel was also negligible. Fluorescence was detected from a 0.7-µm-thick confocal section (full width half-maximum). Horizontal resolution was 0.2 µm per pixel. Image data were obtained using Fluoview 2.0 software (Olympus) and Kalman-filtered from two scans (2.2 s/512 × 512 pixel) for each image.

For time-lapse imaging, culture dishes were mounted onto the microscope stage that was equipped with a temperature-controlled inlay (HT200, Minitüb, Tiefenbach, Germany). In control experiments constant fluorescence values were monitored for 24 h in case of cyt-c-GFP. Et fluorescence increase with constant slope was detected for 4 h under control conditions.

For induction of apoptosis, cells were incubated with 3 µM STS or 100 ng/ml TNF-alpha plus 1 µg/ml CHX (TNF-alpha /CHX). The medium was enriched with 10 mM HEPES (pH 7.4) and thoroughly mixed to ensure a proper distribution of the drugs. To prevent evaporation the media was covered with embryo-tested paraffin oil.

Image Processing-- The quantitative analysis of the fluorescence images was performed using UTHSCSA ImageTool program (University of Texas Health Science Center at San Antonio, TX). After background subtraction the average fluorescence intensity per pixel was calculated. The release kinetics of cyt-c-GFP of individual cells are shown as S.D. of the average pixel intensities (22). A compartmentalized cyt-c-GFP fluorescence contributes to a high S.D. of the pixel intensities, and a homogenous distribution after release of cyt-c-GFP is represented by a drop in S.D. Et fluorescence kinetics of individual cells were fitted with linear regression, and the ratios of the slopes after and before the release of cyt-c-GFP or after and before addition of menadione were calculated. Control ratios were calculated from Et fluorescence slopes during incubation with 1 µM HEt from 120 to 180 min divided by slopes from 60 to 120 min in cells treated with vehicle.

Cell Lysate Assay-- Under conditions of mitochondrial depolarization, mitochondrially generated Et may be released into the cytoplasm, resulting in an artificial fluorescence enhancement (23). We therefore additionally quantified Et production of whole cell cultures using a previously described cell lysate assay (12).

Statistics-- Data are given as means ± S.E. For statistical comparison, analysis of variance and subsequent Tukey test were employed (SPSS 10.0, SPSS Inc., Chicago, IL). p values smaller than 0.05 were considered to be statistically significant.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Rapid Increase in Superoxide Production after Mitochondrial Membrane Permeabilization-- MCF-7 cells stably expressing a cyt-c-GFP fusion protein were exposed to the protein kinase inhibitor STS (activation of the mitochondrial pathway) or to TNF-alpha /CHX (activation of death receptors). After 120 min, the culture medium was supplemented with 1 µM HEt. The kinetics of cyt-c-GFP release and HEt oxidation were monitored by confocal fluorescence microscopy. Control cells treated with vehicle demonstrated a constant rate of HEt oxidation in the absence of cyt-c-GFP release (Figs. 1A and 2B). In cultures exposed to STS, individual cells released the cyt-c-GFP fusion protein at different time points after onset of the treatment (Fig. 1, B and C). The release was characterized by a sudden redistribution from a punctate, filamentous to a diffuse, homogeneously distributed GFP signal (Fig. 1B) (18, 22). The cyt-c-GFP redistribution was indicated by a rapid (<10 min) decrease in the S.D. of the average GFP pixel intensity (Fig. 1C). Confocal imaging of the kinetics of cyt-c-GFP release and the rate of superoxide production demonstrated that the rate of HEt oxidation increased significantly after the release of the cyt-c-GFP fusion protein. Similar results were obtained in cells exposed to TNF-alpha /CHX (Fig. 1D).


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Fig. 1.   The release of cyt-c-GFP during apoptosis increases HEt oxidation. A, MCF-7/cyt-c-GFP cell incubated with vehicle for 120 min. After addition of 1 µM HEt, GFP and Et fluorescence were monitored confocally at the times indicated. Scale bar, 5 µm. B, MCF-7/cyt-c-GFP cell incubated with the apoptosis-inducing kinase inhibitor STS (3 µM) for 120 min. After addition of 1 µM HEt, GFP and Et fluorescence were monitored confocally. Note the rapid redistribution of the cyt-c-GFP signal and the rapid increase in HEt oxidation after the release. Scale bar, 5 µm. C, traces of two cells treated with 3 µM STS. HEt was added 120 min into the STS exposure, and cells were monitored subsequently. The release of cyt-c-GFP was detected by a change in the S.D. of the GFP fluorescence pixel intensity. Note the rapid increase in HEt oxidation after the cyt-c-GFP redistribution. Squares and diamonds indicate corresponding cyt-c-GFP and Et changes of the two cells, respectively. Similar traces were recorded from 22 cells in three independent experiments. D, traces of two cells treated similarly with 100 ng/ml TNF-alpha and 1 µg/ml CHX and analyzed as described above. Similar traces were recorded from 10 cells in two independent experiments.

The release of cyt-c triggers Delta Psi m depolarization (13), a process that has also been observed during the release of cyt-c-GFP (11). Delta Psi m depolarization can lead to an artificial Et fluorescence enhancement when large amounts of mitochondrially generated Et are released into the cytosol ("unquenching" effect) (23). To avoid this artifact, our study was performed using a low HEt concentration (1 µM). Under these conditions, rapid and complete mitochondrial depolarization with the protonophore FCCP (10 µM) did not result in an increased Et fluorescence signal, even after prolonged pre-exposure to HEt (Fig. 2, A and B). Parallel measurements of TMRM uptake demonstrated that FCCP depolarized Delta Psi m within minutes, but it did not lead to the release of cyt-c-GFP (Fig. 2, C and D).


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Fig. 2.   Depolarization of Delta Psi m does not increase Et fluorescence. A, GFP and Et fluorescence images of a MCF-7/cyt-c-GFP cell exposed to 1 µM HEt for 240 min and then exposed to 10 µM FCCP. Images are shown before (-5 min) and after (+5 min) addition of FCCP. Scale bar, 5 µm. B, corresponding traces of a cell exposed to HEt for 240 min and then exposed to 10 µM FCCP. Note that the exposure to FCCP did not increase the slope of the Et fluorescence trace. Similar traces were recorded from 24 cells in three independent experiments. C, GFP and TMRM fluorescence images of a MCF-7/cyt-c-GFP cell incubated for 240 min with 30 nM TMRM and then exposed to 10 µM FCCP. Images are shown before (-5 min) and after (+5 min) addition of FCCP. Scale bar, 5 µm. D, corresponding traces of a cell incubated with 30 nM TMRM for 240 min and then exposed to 10 µM FCCP. Note that the exposure to FCCP caused a rapid decrease in TMRM fluorescence. Similar traces were recorded from 15 cells in two independent experiments. E, Et fluorescence trace of a cell recorded over 5 h. FCCP (10 µM) was added after 120 min (arrow). Note that an increase in superoxide production or a change in the S.D. of the GFP signal (indicative of cyt-c-GFP release) could not be detected. Similar traces were recorded from 26 cells in three independent experiments. F, confocal images of single MCF-7/cyt-c-GFP cells treated for 6 h with 10 µM FCCP or vehicle. Scale bar, 5 µm.

The above experiments also suggested that mitochondrial depolarization per se was not sufficient to acutely trigger an increase in superoxide production. Indeed, even prolonged treatment with FCCP (10 µM) neither induced cyt-c-GFP release from mitochondria nor triggered the generation of superoxide (Fig. 2, E and F).

The Broad-spectrum Caspase Inhibitor z-VAD-fmk Inhibits the Increase in Superoxide Production after Mitochondrial Membrane Permeabilization-- A treatment with the SOD mimetic MnTBAP (100 µM) potently blocked the increase in superoxide production after STS-induced cyt-c-GFP release (Fig. 3A). Interestingly, treatment with the broad-spectrum caspase inhibitor z-VAD-fmk did not inhibit cyt-c-GFP release or alter its kinetics (18) but also potently inhibited the increase in superoxide production after the cyt-c-GFP release (Fig. 3B). Quantification of the Et fluorescence slopes before and after the STS-induced cyt-c-GFP release demonstrated a significant, more than 3-fold slope increase. This increase was significantly reduced in STS- plus z-VAD-fmk-treated cells (Fig. 3C). Quantification of Et production of whole cell cultures treated for 6 h with 3 µM STS also showed a 21.5 ± 1.9% increase in Et fluorescence per µg of protein compared with vehicle-treated controls (p < 0.05; n = 12 and 10 cultures, respectively). Et fluorescence in whole cell lysates was significantly reduced in 3 µM STS- plus 200 µM z-VAD-fmk-treated cells to a level of 12.6 ± 1.5% above control (p < 0.05 compared with the STS treatment; n = 12 cultures). The level of Et production in these bulk analyses was significantly smaller than expected from the single-cell measurements, suggesting that not all cells had released cytochrome c or that superoxide production had ceased in severely damaged cells.


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Fig. 3.   The broad spectrum caspase inhibitor z-VAD-fmk blocks the increase in superoxide production after mitochondrial membrane permeabilization. A, trace of an individual MCF-7/cyt-c-GFP cell treated with 3 µM STS and 100 µM of the SOD mimetic MnTBAP. Cells were incubated with 1 µM HEt 120 min after treatment with the drugs. Note that the release of cyt-c-GFP was not followed by an increased HEt oxidation. Similar traces were recorded from 10 cells in two independent experiments. B, traces of two MCF-7/cyt-c-GFP cells treated with 3 µM STS and caspase inhibitor z-VAD-fmk (200 µM). Cells were incubated with 1 µM HEt 120 min after treatment with the drugs. Similar traces were recorded from 20 cells in three independent experiments. C, quantification of Et fluorescence slopes. The ratio of the Et fluorescence intensity slopes after and before the release of cyt-c-GFP was significantly higher in cells treated with 3 µM STS compared with cells treated with 3 µM STS plus 200 µM z-VAD-fmk. Control ratios were calculated from Et fluorescence slopes during incubation with 1 µM HEt from 120 to 180 min divided by slopes from 60 to 120 min in cells treated with vehicle. No release of cyt-c-GFP was observed under these conditions. Data are means ± S.E. from the n = 20-24 cells in the three separate experiments per treatment.

Menadione-induced Superoxide Production Is Sensitive to z-VAD-fmk-- Treatment with 100 µM menadione acutely increased the superoxide production of MCF-7/cyt-c-GFP cells in the absence of any detectable cyt-c-GFP release (Fig. 4A). Surprisingly, cells pretreated with 200 µM z-VAD-fmk and then treated with menadione did not show such increase (Fig. 4B), again in the absence of any detectable changes in cyt-c-GFP redistribution. Quantification of the Et fluorescence slopes in individual cells before and after the addition of menadione confirmed the inhibitory effect of z-VAD-fmk quantitatively (Fig. 4C).


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Fig. 4.   Superoxide production generated by menadione is inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. A, traces of two MCF-7/cyt-c-GFP cells incubated with 1 µM HEt during the entire experiment. Menadione (100 µM) was added after 60 min (arrow). B, traces of two MCF-7/cyt-c-GFP cells pretreated for 60 min with 200 µM z-VAD-fmk and subsequently incubated with 1 µM HEt. Menadione (100 µM) was added after 60 min (arrow). C, quantification of changes in Et fluorescence slopes. The ratio of the Et fluorescence intensity slopes after and before the addition of 100 µM menadione was significantly higher compared with vehicle-treated controls and to cells treated with 100 µM menadione plus 200 µM z-VAD-fmk. Data are means ± S.E. from the n = 15-22 cells in the three separate experiments per treatment. Control values were calculated as described in the legend to Fig. 3C.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activation of death receptors or activation of the mitochondrial apoptosis pathway is known to increase the production of reactive oxygen species, and this has been demonstrated both during TNF-alpha - and STS-induced apoptosis (8-10, 17). Our data now demonstrate at the single-cell level that superoxide production during STS- and TNF-alpha -induced apoptosis starts directly after the mitochondrial release of soluble intermembrane proteins. We did not detect a significant increase in superoxide production prior to the cyt-c-GFP release, and the SOD mimetic MnTBAP did not alter the kinetics of STS-induced cyt-c-GFP release. These results suggest that superoxide anions do not significantly modulate the process of outer mitochondrial membrane permeabilization during STS-induced apoptosis in MCF-7 cells. However, the increase in superoxide production may well play an important role in mitochondrial transition pore opening (24). Interestingly, treatment with the broad-spectrum caspase inhibitor z-VAD-fmk inhibited the increase in superoxide production after STS-induced cyt-c-GFP release, suggesting that this was a caspase-dependent process. Since the increased superoxide during apoptosis is generated by the mitochondrial respiratory chain (10, 12), it is possible that caspases directly target mitochondrial proteins. However, mitochondrial inner membrane or matrix proteins have not yet been identified among the more than 250 caspase substrates known to date.

Interestingly, z-VAD-fmk also inhibited superoxide production triggered by an acute administration of menadione. Menadione is a prototype of a redox cycling compound that is reduced by complex I of the respiratory chain and subsequently generates superoxide (25). Under our experimental conditions, release of cyt-c-GFP could not be detected in response to menadione, even in cells monitored up to 120 min after the addition of menadione. Cells also exhibited normal morphology,2 suggesting that apoptotic pathways were not activated. These results suggest that the widely used broad spectrum caspase inhibitor z-VAD-fmk also exerts direct or indirect antioxidative effects and that inhibition of caspase activation is not solely responsible for the effects of z-VAD-fmk on superoxide production during apoptosis.

At least two separate processes may account for potential caspase-independent superoxide production after mitochondrial membrane permeabilization: (i) increased superoxide production due to reverse electron flow after the loss of cyt-c and (ii) decreased antioxidant defense capacity in the mitochondrial intermembrane space. Cyt-c may function as a direct antioxidant in living cells (26). It is known that a large fraction of cyt-c does not participate in electron transport (27) and hence that cyt-c is present in excess in the mitochondrial intermembrane space. Electron microscopy studies have indicated that the release of cyt-c during apoptosis is a complete process, leading to a total depletion of cyt-c in affected mitochondria (28). There is also evidence that cyt-c is released specifically out of apoptotic cells prior to a loss of plasma membrane integrity (18, 29). It is therefore possible that the loss of cyt-c out of apoptotic mitochondria or cells increases their oxidative stress burden.

However, this view is challenged by a recent study performed in isolated mitochondria that suggests that increased superoxide production during apoptosis may be related to the cyt-c-linked electron transport activity rather than the direct radical scavenging activity of cyt-c (14). Mitochondria that are energized with malate and glutamate have been shown to generate superoxide via the flavin mononucleotide group of complex I in response to inhibition of mitochondrial electron flow (14, 30, 31). Superoxide generation due to reversed electron transport to complex I has been shown to be dependent on Delta Psi m (30). Indeed, the qualitatively similar increase in superoxide generation of isolated mitochondria depleted of cyt-c compared with mitochondria treated with the complex I inhibitor rotenone, as well as its dependence on the NADH/NAD+ ratio, suggests that complex I is a primary site of superoxide production during apoptosis (14). However, in depolarized mitochondria superoxide is primarily generated via the ubiquinone in the inner mitochondrial membrane (30, 32). Delta Psi m depolarizes after the release of cyt-c or cyt-c-GFP (11, 13, 33). It is therefore possible that ubiquinone is also a major source of superoxide after mitochondrial membrane permeabilization. Indeed, an electron paramagnetic resonance study performed in digitonin-permeabilized mitochondria has demonstrated that ubiquinone autoxidation at the outer site of the complex III ubiquinone pool increases superoxide formation toward the intermembrane space (34).

In conclusion, our study demonstrates that increased superoxide production and hence the activation of additional cell death pathways starts immediately after mitochondrial membrane permeabilization during STS- and TNF-alpha -induced apoptosis. The increase in superoxide production was sensitive to the broad spectrum caspase inhibitor z-VAD-fmk. However our data also indicate that this commonly used inhibitor may exert antioxidant activities unrelated to caspase inhibition.

    ACKNOWLEDGEMENTS

We thank Petra Mech and Christiane Schettler for technical assistance and Drs. A.-L. Nieminen and J. Ma for the cyt-c-GFP construct.

    FOOTNOTES

* These experiments were supported by the Deutsche Forschungsgemeinschaft and the Interdisciplinary Center for Clinical Research, University Münster Clinics.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address and to whom correspondence should be addressed: Experimental Neurosurgery, Klinikum der Johann Wolfgang Goethe-Universität, Haus 25 B, 4. OG, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. Tel.: 49-69-6301-6930; Fax: 49-69-6301-5575; E-mail: prehn@em.uni-frankfurt.de.

Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M210826200

2 H. Düssmann, D. Kögel, M. Rehm, and J. H. M. Prehn, unpublished data.

    ABBREVIATIONS

The abbreviations used are: cyt-c, cytochrome c; CHX, cycloheximide; cyt-c-GFP, cytochrome c green fluorescent protein fusion protein; Et, ethidium; FCCP, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone; HEt, hydroethidine; MnTBAP, manganese tetrakis(4-benzoyl acid)porphyrin; SOD, superoxide dismutase; STS, staurosporine; TMRM, tetramethylrhodamine methyl ester; TNF-alpha , recombinant human tumor necrosis factor-alpha ; z-VAD-fmk, z-Val-Ala-Asp(O-methyl)- fluoromethylketone.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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