J Biol Chem, Vol. 274, Issue 37, 26217-26224, September 10, 1999

Overexpression of Catalase in Cytosolic or Mitochondrial Compartment Protects HepG2 Cells against Oxidative Injury^*

Jingxiang Bai, Ana M. Rodriguez^§, J. Andres Melendez^§, and Arthur I. Cederbaum^¶

From the  Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029 and the ^§ Department of Biochemistry and Molecular Biology, Albany Medical College, Albany, New York 12208

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

HepG2 cells were transfected with vectors containing human catalase cDNA and catalase cDNA with a mitochondrial leader sequence to allow comparison of the effectiveness of catalase overexpressed in the cytosolic or mitochondrial compartments to protect against oxidant-induced injury. Overexpression of catalase in cytosol and in mitochondria was confirmed by Western blot, and activity measurement and stable cell lines were established. The intracellular level of H₂O₂ induced by exogenously added H₂O₂ or antimycin A was lower in C33 cell lines overexpressing catalase in the cytosol and mC5 cell lines overexpressing catalase in the mitochondria as compared with Hp cell lines transfected with empty vector. Cell death caused by H₂O₂, antimycin A, and menadione was considerably suppressed in both the mC5 and C33 cell lines. C33 and mC5 cells were also more resistant to apoptosis induced by H₂O₂ and to the loss of mitochondrial membrane potential induced by H₂O₂ and antimycin A. In view of the comparable protection by catalase overexpressed in the cytosol versus the mitochondria, catalase produced in both cellular compartments might act as a sink to decompose H₂O₂ and move diffusable H₂O₂ down its concentration gradient. The present study suggests that catalase in cytosol and catalase in mitochondria are capable of protecting HepG2 cells against cytotoxicity or apoptosis induced by oxidative stress.

	INTRODUCTION

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Hydrogen peroxide (H₂O₂), one of the major reactive oxygen species (ROS),¹ is produced at a relatively high rate as a product of aerobic metabolism. Under normal conditions, 1-2% of the oxygen reduced by mitochondria may be converted to O₂ at the NADH dehydrogenase and ubisemiquinone intermediate steps of the mitochondrial respiratory chain (1, 2). Superoxide can be readily converted by mitochondrial superoxide dismutase (MnSOD) into H₂O₂ (3). The primary cellular enzymatic defense systems against hydrogen peroxide are the glutathione redox cycle and catalase. GSH is a cofactor for glutathione peroxidase, which converts H₂O₂ to H₂O at the expense of oxidizing GSH to its disulfide form (GSSG). Glutathione reductase regenerates GSH from GSSG, using reducing equivalents from NADPH (4). Catalase also protects cells from the accumulation of H₂O₂ by converting it to H₂O and O₂ (5). The glutathione redox cycle system exists in both the cytosol and mitochondrial compartments of the cell. However, catalase is present only or primarily in the peroxisome fraction and is absent in mitochondria of mammalian cells, except rat heart mitochondria (6). Therefore, the only enzymatic defense system against hydrogen peroxide in mitochondria is the glutathione redox cycle system.

Pathological conditions, which increase the rate of H₂O₂ production or deplete components of the anti-oxidant system, e.g. GSH, will lead to the accumulation of H₂O₂ in the cytosol or mitochondria. In biological systems, H₂O₂ could readily diffuse across cellular membranes and lead to depletion of ATP, GSH, and NADPH. It could also induce a rise in free cytosolic Ca²⁺ and activate poly(ADP-ribose) polymerase activity and cause DNA damage (7). By scission of the peroxide bond through the Fenton reaction, hydrogen peroxide could generate hydroxyl radical, an extremely potent oxidant (5, 8). The hydroxyl radical is able to cause the degradation of most biological macromolecules, e.g. peroxidation of lipids, oxidation of sugars and of protein thiols, DNA base damage, and strand breakage of nucleic acids (9).

Low levels of H₂O₂ have been shown to trigger apoptosis, while high levels lead to necrosis (10, 11). Many reports indicate that hydrogen peroxide plays a very crucial role in cytotoxicity and apoptosis induced by stimuli such as ceramide (12), Antimycin A (AA) (13), arsenite (14), and tumor necrosis factor- (15). It might also contribute to human diseases such as Alzheimer's disease, diabetes, stroke, and AIDS dementia complex (16-18). Studies in our laboratory indicate that CYP 2E1-dependent cytotoxicity or apoptosis by ethanol to HepG2 cells was due to the generation of ROS such as H₂O₂ (19, 20).

Mitochondria are a main target for damage by ROS. Hydrogen peroxide is know to induce a mitochondrial permeability transition and disrupt the mitochondrial membrane potential (). Such conditions can cause the release of cytochrome c from the mitochondria to the cytosol, thereby triggering cells to undergo apoptosis by activating caspase 3 (21). There are no reports on the possible protective actions caused by expression of functionally active catalase in the mitochondrial compartment against toxicity by ROS. In this study, stable HepG2 cell lines that constitutively express catalase in the mitochondrial compartment were developed. The main goal of the present study was to compare the effect of overexpression of human catalase in the cell cytosol to that in the mitochondria on the ability to protect cells from cytotoxicity or apoptosis induced by hydrogen peroxide and AA.

	MATERIALS AND METHODS

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Reagents-- Rhodamine 123 (Rh123), propidium iodide (PI), and 2',7'-dichlorofluorescein diacetate (DCF-DA) were purchased from Molecular Probes (Eugene, OR). Polyclonal antibody raised in rabbit against human catalase was obtained from Calbiochem. Zeocin for cloning selection was from Invitrogen. Hydrogen peroxide, AA, horseradish peroxidase conjugated to goat anti-rabbit IgG, MEM, fetal bovine serum, and trypan blue were purchased from Sigma.

Plasmid Construction-- The pCAT10 plasmid containing the human catalase cDNA was obtained from American Type Culture Collection. PZeoSV2(+) mammalian expression vector was obtained from Invitrogen Corporation (San Diego, CA). A 900-base pair fragment containing the MnSOD mitochondrial signal peptide was PCR-amplified with HindIII-SalI ends from pRCMV/MnSOD (22). The resulting product was inserted into the pCR2.1 TA cloning vector (Invitrogen Corp.). A 1-kilobase pair fragment containing the mitochondrial signal peptide and the CMV promoter region of pCR/CMV plasmid was then removed from the resulting plasmid by digestion with HindIII, which cleaves at the newly introduced HindIII site and the naturally occurring HindIII 3' site in the MnSOD cDNA. This fragment was ligated into the pZeoSV2(+), precut with HindIII. The new PZeoSV-MSP was digested with BsaI, removing the CMV promoter region, and religated. The final pZeoSV-MSP was then digested with HindIII and NotI. The human fibroblast catalase cDNA from pCAT10 was equipped with a HindIII site at the 5' end and a NotI site at the 3' end with oligonucleotide linkers using PCR. The resulted 1.6-kilobase pair PCR product was ligated in frame into the pZeoSV-MSP to obtain the final recombinant plasmid pZeoSV/MSP-CAT. The mammalian expression vector pZeoSV-CAT was created by inserting the 1.6-kilobase pair catalase fragment into pZeoSV2(+) precut with HindIII and NotI. Each DNA insert was sequenced bidirectionally by using the Taq DyeDeoxy terminator cycle sequencing kit and an ABI model 310 DNA sequencer (Applied Biosystems).

Cell Culture and Transfection-- HepG2 cells (from ATCC) were cultured in MEM containing 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine in a humidified atmosphere, in 5% CO₂, at 37 °C. Before transfection, 5× 10⁵ HepG2 cells were seeded onto 10-cm culture dishes and grown to 60% confluence. The expression plasmid vectors pZeoSV2(+), pZeoSV2(+) containing human catalase cDNA (pZeoSV-CAT), as well as pZeoSV2(+) containing human catalase cDNA with a MnSOD mitochondrial leader sequence (pZeoSV/MSP-CAT) were transfected into HepG2 cells using the DOTAP transfection reagent (Roche Molecular Biochemicals) according to the instructions provided by the manufacturer. Forty-eight hours after transfection, cells were trypsinized and seeded at a low cell density onto 10-cm culture dishes in 10% fetal bovine serum plus MEM containing 300 µg/ml Zeocin. HepG2 cells not subject to transfection were cultured with the same concentration of Zeocin medium as a control for selection. Two weeks after transfection, the surviving clones were isolated, transferred to 24-well plates, and grown to large scale. Catalase expression in each clone was measured by Western blot and assays of activity. Stable cell lines with overexpression of catalase in the cytosol and in the mitochondria, as well as cells transfected with the empty vector, were selected and maintained in MEM containing 300 µg/ml Zeocin.

Preparation of Mitochondria-- Mitochondria were prepared by discontinuous Percoll gradient centrifugation using a modification of the method of Sims (23). Five 10-cm culture dishes of cells grown to 80% confluence were washed twice with PBS, scraped from the dishes, and suspended in 1 ml of cold isolation buffer (0.32 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4). Cells were homogenized using a glass homogenizer. The homogenate was centrifuged at 1330 × g for 5 min at 4 °C. The supernatants were centrifuged at 21,200 × g for 10 min at 4 °C. The pellet was suspended in a 15% Percoll solution, and aliquots were layered onto discontinuous Percoll gradients (23% over 40%) and then centrifuged for 15 min at 30,700 × g at 4 °C. The dense band of material at the interface between the 23% and 40% Percoll layers was collected, diluted 1:4 in fresh isolation buffer, and then centrifuged for 10 min at 16,700 × g at 4 °C. The pellet was resuspended in 2 ml of isolation buffer and centrifuged again for 10 min at 6900 × g. The mitochondrial pellet was suspended in 100 µl of PBS and used for Western blot analysis or catalase activity assay.

Western Blot-- To assay for total cellular catalase protein levels, cells were grown to 80% confluence in 10-cm dishes, washed twice with PBS, harvested by scraping, and subsequently sonicated at duty cycle 50% and output control 4 for 20 s. To assay for mitochondrial catalase protein levels, the intact mitochondria prepared as described above were sonicated (Heat Systems-Ultrasonics, Inc.) at duty cycle 30 and output control 3 for 10 s. The sonicated suspensions were centrifuged at 8000 × g for 10 min at 4 °C. The supernatant was transferred to a new tube, and the protein concentration was measured (DC protein assay reagent, Bio-Rad). Ten µg of denatured protein were resolved on 10% SDS-PAGE and electroblotted onto nitrocellulose membranes (Bio-Rad). The membrane was incubated with rabbit anti-human catalase polyclonal antibody as the primary antibody (1:1000), followed by incubation with horseradish peroxidase conjugated to goat anti-rabbit IgG (Sigma) as the second antibody (1:5000). Detection by the chemiluminescence reaction was carried out for 1 min using the ECL kit (Amersham Pharmacia Biotech), followed by exposure to Kodak X-Omat x-ray film (Eastman Kodak Co.).

Catalase Activity Assay-- Fresh sonicated extracts from cells and mitochondria were used. Catalase activity was determined at 25 °C according to Claiborne and Fridovich (24). The decomposition of hydrogen peroxide by catalase was followed by ultraviolet spectroscopy at 240 nm. The reaction was performed using a solution of 20 mM hydrogen peroxide in 50 mM KH₂PO₄ containing 10 µg of total cellular or mitochondrial protein in a final volume of 1 ml. Specific activity of catalase was calculated from the equation: specific activity (units/mg of protein/min) = A_{240 nm} (1 min) × 1000/43.6 × mg protein.

Intracellular H₂O₂ Measurement-- Fluorescence spectrophotometry and confocal microscopy were used to measure intracellular H₂O_2, with 2',7'-DCF-DA as the probe. DCF-DA readily diffuses through the cell membrane and is enzymatically hydrolyzed by intracellular esterases to the nonfluorescent DCFH, which can then be rapidly oxidized to highly fluorescent DCF in the presence of ROS. Cells incubated in medium alone or cells treated with H₂O₂ or AA were incubated with 5 µM DCF-DA in MEM for 30 min at 37 °C in the dark. The cells were washed in PBS, trypsinized, resuspended in 3 ml of PBS, and the intensity of fluorescence was immediately read in a fluorescence spectrophotometer (Perkin-Elmer 650-10S, Hitachi, Ltd.) at 503 nm for excitation and at 529 nm for emission. For confocal microscopy, slides with control, non-treated cells or cells treated with H₂O₂ and DCF-DA were washed in PBS and covered with glass covers, followed by observation under a confocal microscope.

Cell Viability-- Cell viability was measured either by trypan blue exclusion or by vital dye reduction. For menadione toxicity experiments, the cell titer 96 nonradioactive cell proliferation assay kit (Promega) as described by Chen et al. (25) was used. 1 × 10⁵ cells were seeded onto 24-well plates and incubated with medium containing different concentrations of menadione for 18 h. After addition of the "stop" solution, the absorbance of the reaction solution at 570 nm was recorded. The absorbance at 630 nm was used as reference. The net A₅₇₀  A₆₃₀ was taken as the index of cell viability, The net absorbance from the wells of cells cultured with control medium was taken as the 100% viability value. The percentage of viability of the treated cells was calculated by the formula (A₅₇₀  A₆₃₀)sample/(A₅₇₀  A₆₃₀)control × 100.

For experiments involving toxicity of H₂O₂ and AA, cell viability was detected by the trypan blue exclusion assay. 1× 10⁵ cells were seeded onto 24-well plates and first incubated with medium containing 0.3 mM BSO for 16 h, followed by incubation with medium containing 100 µM H₂O₂ or 15 µM AA. At different time points, cells were trypsinized and diluted followed by staining with 0.2% trypan blue. The number of cells excluding trypan blue or staining with trypan blue were counted under the light microscope. The percentage of cells excluding trypan blue was taken as an index of cell viability. Cell morphology was also visualized under the light microscope, and pictures were taken.

Apoptosis Assay-- The DNA fragmentation pattern (DNA ladder) was carried out by agarose gel electrophoresis. Cells (1× 10⁶) were scraped and centrifuged at 1200 rpm for 10 min. The cell pellet was resuspended in 1 ml of lysis buffer consisting of 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 10 mM EDTA, 100 µg/ml proteinase K, and 0.5% SDS and incubated for 2 h at 50 °C. DNA was extracted with 1 ml of phenol, pH 8.0, followed by extraction with 1 ml of phenol/chloroform (1:1) and chloroform. The aqueous phase was precipitated with 2.5 volumes of ice-cold ethanol and 0.1 volume of 3 M sodium acetate, pH 5.2, at 20 °C overnight. The precipitates were collected by centrifugation at 13000 × g for 10 min. The pellets were air-dried, resuspended with 50 µl of TE buffer supplemented with 100 µg/ml RNase A and incubated at 37 °C for 30 min. DNA was loaded onto a 1.5% agarose gel containing ethidium bromide, electrophoresed in TAE buffer for 2 h at 50 V, and photographed under UV illumination.

DNA analysis by flow cytometry was used to quantify the percentage of apoptotic cells. 5 × 10⁵ cells were seeded onto six-well plates and incubated with medium containing 0.3 mM BSO for 16 h, followed by treatment with 100 µM H₂O₂. At different time points, cells were harvested by trypsinization, washed with PBS, followed by centrifugation at 2000 rpm for 10 min. The pellet of cells was resuspended in 80% ethanol and stored at 4 °C for 24 h. Cells were washed twice with PBS. The pellet was resuspended in PBS containing 100 µg/ml RNase A, incubated at 37 °C for 30 min, stained with PI (50 µg/ml), and analyzed by flow cytometry for DNA analysis.

Flow Cytometry Analysis of the Mitochondrial Membrane Potential-- Changes in the integrity of the plasma membrane and in the mitochondrial membrane potential were examined by monitoring the cells after double staining with PI and Rh123. Cells (5 × 10⁵) were seeded onto six-well plates and incubated with medium containing 0.3 mM BSO for 16 h followed by treatment with 100 µM H₂O₂ or 15 µM AA for different times. The cells were then incubated with medium containing 5 µg/ml Rh123 for 1 h. Cells were harvested by trypsinization, and resuspended in 1 ml of MEM containing 5 µg of PI. The intensity of fluorescence from PI and Rh123 was analyzed by flow cytometry.

	RESULTS

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Overexpression of Catalase in Cytosol and in Mitochondria-- Surviving clones of HepG2 cells transfected with the empty plasmid, or plasmid containing human catalase cDNA, or plasmid containing catalase cDNA with a 80-base pair MnSOD mitochondrial leader sequence were assayed for catalase expression by Western blot and catalase catalytic activity. Two high expression cytosol catalase clones (C1 and C33) and two high expression mitochondrial catalase clones (mC5 and mC26) were selected for detailed evaluation. Fig. 1 shows the expression of catalase in the total cell extract (panel a) and in the mitochondrial extract (panel b) from these cells and cells transfected with empty vector (Hp) as well as the parental HepG2 cells as determined by Western blot. Results from densitometric analyses of the intensity of the various bands indicated that the expression of catalase in total cell extracts of cell lines C1, C33, mC5, and mC26 was about 2-fold higher than that in the Hp cells and the parental HepG2 cells. A high amount of catalase was found in the mitochondrial extracts from mC5 and mC26 cells, but very low levels of catalase were present in the mitochondrial extracts from C1, C33, Hp, and parental HepG2 cells. The catalase content in mitochondrial extracts from mC5 and mC26 cells was about 20-fold higher than that in HepG2 cells and at least 5-10-fold higher than the other transfected cell lines. The same results were obtained for the catalase activity assay (Fig. 2). The catalase activity in total extracts of cell lines C1, C33, mC5, and mC26 was 2-3-fold higher than that of the control cell line Hp and parental HepG2 cells. With respect to catalase activity in the mitochondria, high activity was found only in the mitochondrial extracts of cell lines mC5 and mC26. For the experiments shown below, results were compared between C33, mC5, and Hp cell lines, in order to assess the effectiveness of mitochondrially expressed catalase with that of cytosolically expressed catalase in protecting against oxidative stress and cellular toxicity.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Expression of catalase in different transfected clones and parent HepG2 cells. Ten micrograms of protein from cellular extraction (a) or from mitochondrial extraction (b) were loaded into each lane for 10% SDS-PAGE, followed by Western blot analysis with polyclonal rabbit anti-human catalase antibody as described under "Materials and Methods." Lane 1, HepG2 cells (H); lane 2, cells transfected with empty vector (Hp); lanes 3 and 4, two different clones transfected with catalase (C1, C33); lanes 5 and 6, two different clones transfected with catalase with a mitochondria leader sequence (mC5, mC26).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Catalase activity in different transfected clones and parental HepG2 cells. Ten micrograms of protein from cellular extraction (a) or from mitochondrial extraction (b) were used to measure the catalytic activity of catalase. Specific catalase units (units/mg protein/min) were calculated as described under "Materials and Methods." Bar 1, HepG2 cells; bar 2, cells transfected with empty vector (Hp); bars 3 and 4, two different clones transfected with catalase (C1, C33); bars 5 and 6, two different clones transfected with catalase with a mitochondria leader sequence (mC5, mC26). Data are mean ± S.E. of triplicate experiments.

Intracellular H₂O₂-- Fig. 3A shows the result of intracellular H₂O₂ levels in cell lines C33, mC5, Hp, and parental HepG2 cells as determined by fluorescence spectrophotometry using the oxidant-sensitive dye 2',7'-DCF-DA. Higher H₂O₂ levels were detected in Hp cells and HepG2 cells than that found in C33 and mC5 cells after treatment with either 500 µM H₂O₂ or 15 µM AA for 2 h followed by incubation with 5 µM 2',7'-DCF-DA for 30 min. Cytosolic and mitochondrial expressed catalase were equally effective in lowering the fluorescence associated with oxidation of DCFH after treatment with either H₂O₂ or AA. We also analyzed the intracellular level of H₂O₂ by confocal microscopy after treating cells with 500 µM H₂O₂ for 2 h, followed by incubation with 2',7'-DCF-DA (Fig. 3B). The intensity of fluorescence in Hp cells was much stronger than that observed for the C33 and mC5 cells.

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Determination of intracellular H2O2. A, cells were treated with buffer (first set of bars) or 15 µM AA (second set) or 500 µM H2O2 (third set) for 2 h, followed by incubation with 5 µM 2',7'-DCF-DA for 30 min. Cells were washed in PBS, trypsinized, resuspended in PBS, and the intensity of fluorescence was immediately read in a fluorescence spectrophotometer at wavelengths of 503 nm for excitation and at 529 nm for emission. The results were expressed as relative units with the fluorescence intensity of control Hp cells assigned a value of 1. Data are mean ± S.E. of triplicate experiments. B, cells were grown on slides. After treatment with 500 µM H2O2 for 2 h followed by 5 µM 2',7' DCF-DA for 30 min, cells were washed in PBS, observed under the confocal microscope, and pictures taken. a, Hp cells; b, C33 cells; c, mC5 cells.

Suppression of Menadione-, H₂O₂-, and Antimycin A-induced Cytotoxicity-- The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay was used to determine the cytotoxicity induced by the redox cycling agent, menadione. HepG2, Hp, C33, and mC5 cells were treated with different concentrations (0-20 µM) of menadione for 18 h, and the viability of the cells was determined. The percentage of surviving cells decreased in a dose-dependent manner, and the decrease was similar for the parental HepG2 cells and the Hp cell line (Fig. 4). mC5 cells were more resistant to the cytotoxic effects of menadione than Hp and wild HepG2 cells, and C33 cells were even more resistant. The resistance against menadione toxicity by the mC5 cells was maintained up to menadione concentrations of 15 µM but then decreased at the menadione concentration of 20 µM; C33 cells still showed strong resistance even at 20 µM menadione.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Cytotoxicity of menadione to different cell lines. HepG2 (), Hp (), C33 (), and mC5 () cells were exposed to different concentrations of menadione for 18 h. Cell viability was detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium method. The percentage of viability of the treated cells was calculated by the formula (A570  A630) sample/(A570  A630)control × 100.

A trypan blue exclusion assay was used to detect the cytotoxicity induced by H₂O₂ and AA. In order to show the maximum protective effects of catalase and to lower the influence of the GSH system, BSO, an inhibitor of the synthesis of glutathione, was used to lower the level of GSH in the cells. Hp, C33, and mC5 cells were pretreated with 0.3 mM BSO for 16 h, followed by incubation with 100 µM H₂O₂ or 15 µM AA for different times. The number of surviving Hp cells rapidly decreased after 8 h of incubation with 100 µM H₂O₂, and almost none of the Hp cells were viable after 48 h (Fig. 5a). Both C33 and mC5 cells showed a comparable resistance to the cytotoxic effects of H₂O₂ as compared with the Hp cells; 60-70% of cells were still viable even after 48 h of treatment with H₂O₂ (Fig. 5a). Hp cells were also quite sensitive to toxicity by 15 µM AA, as only 50% of cells were viable after 4 h of treatment while few cells were alive after 12 h (Fig. 5b). Both C33 and mC5 cells were significantly resistant to the toxicity of AA, with about 85% and 50% of cells viable after 4 and 12 h treatment, respectively (Fig. 5b). There is no significant difference between C33 and mC5 cells in protecting against the toxicity induced by 100 µM H₂O₂ or 15 µM AA. Essentially similar results were obtained after morphological visualization of the cells under the light microscope (data not shown). Most of the Hp cells lost normal morphology when treated with H₂O₂ for 24 h or AA for 12 h, whereas the C33 cells and the mC5 cells retained their shape and structure.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Cytotoxicity of H2O2 and AA to different cell lines. Hp (), C33(), and mC5() cells were seeded onto 24-well plates and incubated with medium containing 0.3 mM BSO for 16 h, followed by incubation with medium containing 100 µM H2O2 (a) and 15 µM AA (b). At different time points, cells were trypsinized, diluted, and stained with 0.2% trypan blue. The number of cells excluding or staining for trypan blue were counted under the light microscope. Cell viability was expressed as the percentage of the cells excluding trypan blue out of the total number of cells. Data are the mean ± S.E. of triplicate experiments.

DNA Fragmentation Induced by H₂O₂-- DNA ladders are believed to be a biochemical marker for apoptosis. The cells were pretreated with 0.3 mM BSO for 16 h, followed by incubation with 100 µM H₂O₂ for 48 h. The DNA was isolated and electrophoresed on a 1.5% agarose gel. Hp and parental HepG2 cells showed a clear DNA ladder in response to the H₂O₂ treatment, whereas no DNA ladder was seen in either the C33 or the mC5 cells (Fig. 6).

View larger version (78K): [in this window] [in a new window]   Fig. 6.   DNA fragmentation induced by H2O2 in different transfected cell lines. Cells were seeded onto six-well plates and pretreated with 0.3 mM BSO for 16 h, followed by treatment with 100 µM H2O2 for 48 h. DNA was extracted and electrophoresed on a 1.5% agarose gel as described under "Materials and Methods." Lane 1, 100-base pair standard ladder; lanes 2, 3, 4, and 5 were HepG2, Hp, C33, and mC5 cells pretreated with BSO for 16 h, followed by treatment with 100 µM H2O2 for 48 h.

DNA analysis by flow cytometry after staining with PI was used to measure the percentage of apoptotic cells. Hp, C33, and mC5 cells were pretreated with 0.3 mM BSO for 16 h, followed by incubation with 100 µM H₂O₂ for 0, 4, 8, 12, and 24 h. DNA analysis was carried out as described under "Materials and Methods." There were less than 5% apoptotic cells after the BSO treatment for all three cell lines not incubated with the H₂O₂ (0 h samples) (Fig. 7, A, panels a-c, and B, bar graph 2). In the Hp cells, the percentage of apoptotic cells increased to 17%, 40%, and 48% after 8, 12, and 24 h of treatment with H₂O₂, respectively (Fig. 7B, bar graphs 4-6; A, panels d and g show the 8- and 24-h data). The C33 and mC5 cells were resistant to the H₂O₂-induced apoptosis, e.g. after the 24-h treatment, the percentage of apoptotic cells was 48% in Hp cells and only 17% and 6% in C33 and mC5, cells respectively (Fig. 7, A, panels g-i; B, bar graph 6).

View larger version (26K): [in this window] [in a new window]   Fig. 7.   DNA analysis by flow cytometry. Hp, C33, or mC5 cells were seeded onto six-well plates and incubated with medium containing 0.3 mM BSO for 16 h, followed by incubation with medium containing 100 µM H2O2. At different time points, cells were harvested by trypsinization and washed with PBS, followed by centrifugation. The cell pellet was resuspended in 80% ethanol and stored at 4 °C for 24 h. Cells were washed twice with PBS, resuspended in PBS containing 100 µg/ml RNase, and incubated at 37 °C for 30 min. The cells were stained with PI, and analyzed by flow cytometry. A, flow cytometry DNA histographs of Hp (a, d, g), C33 (b, e, h), and mC5 (c, f, i) cells treated with 100 µM H2O2 for 0 h (a, b, c), 8 h (d, e, f), or 24 h (g, h, i). The percentage of cells in the zone 1 hypodiploid area is depicted on the graphs. B, bar graph showing the percentage of apoptotic cells measured by flow cytometry. Bar 1, cells incubated with control medium; bar 2, BSO-treated cells; bar 3, BSO treatment followed by 100 µM H2O2 for 4 h; bar 4, BSO + 100 µM H2O2 for 8 h; bar 5, BSO + 100 µM H2O2 for 12 h; bar 6, BSO + 100 µM H2O2 for 24 h. Data are expressed as mean ± S.E.

Mitochondrial Membrane Potential () and the Integrity of the Plasma Membrane-- Rh123, a lipophilic cation, is selectively taken up by mitochondria, and uptake is directly proportional to mitochondrial (26, 27). PI is imported into cells and binds to cellular DNA when the integrity of the plasma membranes is lost. After incubation with BSO, those Hp, C33, and mC5 cells not treated with H₂O₂ or AA were predominantly located in the PI-negative and high (strong Rh123 fluorescence) field (PI()- high) reflective of viable, intact cells (Fig. 8, a-c, and insets a-c). A small percentage of cells were located in the (PI(+)- low) field, reflective of damaged cells (Fig. 8, a-c). Hp cells that were pretreated with 0.3 mM BSO for 16 h followed by incubation with 100 µM H₂O₂ for 24 h moved to the (PI()- low) and (PI(+)- low) field, and 67% of the cells displayed low Rh123 intensity (Fig. 8d, M1 population, compared with 27% of Hp control cells in panel a). However, only 42% and 30% of C33 and mC5 cells treated with H₂O₂ were present in the low field, respectively (Fig. 8, e and f). Hp cells incubated with 15 µM AA for 8 h also moved to the (PI(+)- low) field, and 72% of the cells displayed low Rh123 intensity (Fig. 8g, M1 population). However, only 29% and 34% of C33 and mC5 cells were present in the low field, respectively (Fig. 8, h and i). These results indicate that both cytosolic catalase and mitochondrial catalase protected the cells from loss of mitochondrial membrane potential and loss of membrane integrity produced by either H₂O₂ or AA.

View larger version (57K): [in this window] [in a new window]   Fig. 8.   Flow cytometry analysis of the mitochondrial membrane potential. Hp, C33, and mC5 cells were seeded onto six-well plates and incubated with medium containing 0.3 mM BSO for 16 h, followed by incubation with 100 µM H2O2 for 24 h or 15 µM AA for 8 h, The cells were then incubated with medium containing 5 µg/ml Rh123 for 1 h. Cells were harvested by trypsinization and resuspended in 1 ml of MEM medium containing 5 µg of PI. The intensity of fluorescence from PI and Rh123 was analyzed by flow cytometry. Panels a, b, and c refer to Hp, C33, and mC5 cells, respectively, incubated just with culture medium. Cells not treated with H2O2 or AA were predominantly located in the PI-negative and high (Rh123-positive) field (PI()- high) while a smaller percentage of cells were in the (PI(+)- low) field (insets of a-c). Panels d, e, and f refer to Hp, C33, and mC5 cells incubated with 100 µM H2O2 for 24 h. Panels g, h, and i refer to Hp, C33, and mC5 cells incubated with 15 µM AA for 8 h. M1 and M2 are two population of cells with low and high Rh123 fluorescence intensity, and the percentage of cells with low Rh123 fluorescence is shown in each panel. The figure is one representative experiment out of three.

	DISCUSSION

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Because mitochondria are an important target for interaction with H₂O₂ and since these organelles lack catalase, we developed a HepG2 cell line that expresses catalase in mitochondria by transferring a plasmid containing catalase cDNA with the peptide leader sequence of MnSOD into HepG2 cells. Western blot and catalase activity assay showed that higher amounts of catalase were present in mitochondria of these cells, compared with mitochondria from cells transfected with plasmid containing only catalase cDNA or cells transfected with empty vector or parental HepG2 cells. Thus, the MnSOD leader sequence could be used to successfully import catalase into mitochondria. Most of the increase in cellular catalase content and activity in the mC5 (and in mC26) clones is due to the expression of catalase in the mitochondrial fraction of these cells with just a small increase in cytosolic catalase.

Intracellular hydrogen peroxide generation induced by exogenous H₂O₂ or AA was suppressed by cytosolic catalase and by mitochondrial catalase. In general, the overexpression of cytosolic and mitochondrial catalase was equally effective in lowering DCF-DA fluorescence induced by exogenous H₂O₂ or by AA.

The overexpression of catalase in the cytosol and mitochondria protected the cells from cytotoxicity caused by menadione. Comparing C33 and mC5 cells, the cytosol catalase showed stronger protective effect than the mitochondria catalase at higher menadione concentrations. However, comparable protection against menadione toxicity was observed in the C1 and mC26 clones (data not shown). Cytosolic catalase and mitochondrial catalase equally protected cells from cytotoxicity induced by H₂O₂ and AA. Mitochondrial catalase was protective against exogenously added H₂O₂, which suggests that some of the added H₂O₂ diffuses into the mitochondria and perhaps damage to the mitochondria is an important factor contributing to H₂O₂ toxicity. Similarly, cytosolic catalase was protective against AA-induced H₂O₂ production and toxicity, which suggests that mitochondrially produced H₂O₂ diffuses into the cytosol where it may exert cytotoxic action. It would appear that catalase produced in any cellular compartment might act as a sink for H₂O₂ and promote H₂O₂ movement down its concentration gradient. There are reports that catalase added to the culture medium might protect cells against oxidant-induced injury; while this may reflect some uptake of catalase into the cells by endocytosis, it may also reflect the removal of diffusable H₂O₂ from the cell.

Under physiological conditions, H₂O₂ may play an important role in signal transduction pathways (28), and activation of the transcription factor NF-B (29). However, under pathogenic conditions, H₂O₂ can produce apoptosis or necrosis (30, 31). HepG2 cells pretreated with BSO followed by exposure to 100 µM H₂O₂ could undergo apoptosis as a DNA ladder was detected in parental HepG2 and Hp cells after 48 h of incubation. Consistent with the protection afforded by cytosolic or mitochondrial catalase, a DNA ladder was not detected in the C33 and mC5 cells under the same conditions. Flow cytometry DNA analysis showed that 48% of Hp cells were in the apoptotic zone after incubation with BSO and 100 µM H₂O₂ for 24 h. However, only 17% and 6% percent apoptotic cells were detected in C33 and mC5 cells, respectively, under the same conditions. Thus both mitochondrial catalase and cytosolic catalase protect HepG2 cells from apoptosis induced by H₂O₂. It is also likely that some of the H₂O₂ toxicity may be necrotic in nature; for example, a DNA ladder was observed at 48 h but not at 24 h after treatment with 100 µM H₂O₂, yet 75% of Hp cells lost their viability at 24 h (Fig. 5). It would appear that cytosolic and mitochondrial catalase can protect against the apoptotic and the necrotic effects of H₂O_2.

Mitochondria permeability transition (MPT) and mitochondrial membrane potential () are markers for mitochondrial damage and dysfunction (32-34). Mitochondrial dysfunction caused by ROS, especially H₂O₂, can lead to necrosis and apoptosis (22, 35-38). We determined the mitochondrial membrane potential in Hp, C33, and mC5 cells pretreated with 0.3 mM BSO followed by incubation with either 100 µM H₂O₂ or 15 µM AA. The decline in caused by these agents was much less in C33 and mC5 cells than the Hp cells. These results suggest that both cytosolic catalase and mitochondrial catalase protected cells from oxidant-induced loss of mitochondrial potential, which may play an important role in the overall protection against oxidant-induced cytotoxicity.

Both bcl-2 and bcl-xL prevent cell death by inhibiting the loss of mitochondrial membrane potential and by inhibiting the release of cytochrome C or other apoptotic-inducing factors from mitochondria to cytosol (39-42). bcl-2 prevents cells from undergoing necrosis caused by inhibitors of the respiratory chain (chemical hypoxia) (43). Heat shock protein 70 also can prevent changes in mitochondrial membrane potential induced by H₂O₂ (44). These experiments demonstrate that protection of mitochondria might be an important target for prevention against oxidative injury. Overexpression of phospholipid hydroperoxide glutathione peroxidase in mitochondria was much more effective than overexpression in the cytosol of RBL-2H3 cells in protecting from oxidative injury (45). We found that cytosolic and mitochondrial catalase were equally effective in preventing loss of and loss of cellular viability. Perhaps one advantage of catalase over the GSH-glutathione peroxidase system in these actions is the lack of requirement for recycling of GSH and consumption of NADPH and perhaps avoiding the accumulation of GSSG and mixed protein disulfides.

In summary, the present study suggests that mitochondria are an important target for oxidative damage and that both catalase in cytosol and catalase in mitochondria are capable of protecting HepG2 cells against cytotoxicity or apoptosis induced by oxidative stress.

	FOOTNOTES

^* This work was supported by Grant AA 03312 from the NIAAA and CA 77068 from NCI National Institutes of Health.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.

^¶ To whom all correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, Box 1020, One Gustave L. Levy Pl., New York, NY 10029. Tel.: 212-241-7285; Fax: 212-996-7214; E-mail: acederb@smtplink.mssm.edu.

	ABBREVIATIONS

The abbreviations used are: ROS, reactive oxygen species; MnSOD, manganese superoxide dismutase; BSO, L-buthionine sulfoximine; DCF-DA, 2',7'-dichlorofluorescein diacetate; DCF, 2',7'-dichlorofluorescein; DCFG, 2',7'-dichlorofluorescein; AA, antimycin A; PI, propidium iodide; Rh123, rhodamine 123; , mitochondrial membrane potential; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; CMV, cytomegalovirus; CAT, chloramphenicol acetyltransferase; MEM, minimal essential medium.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES