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INTRODUCTION |
Reactive oxygen species
(ROS)1 such as hydrogen
peroxide may contribute to several diseases (1-5) and play a role in
the cytotoxicity of agents such as ceramide, arsenite, and tumor
necrosis factor-
(6-8). In the presence of transition metals such
as ferric and copper, hydrogen peroxide and superoxide generate
hydroxyl radical by the Fenton reaction or the metal-catalyzed
Haber-Weiss reaction (9, 10). Hydroxyl radical is able to cause the
degradation of most biological macromolecules, e.g.
peroxidation of lipids, oxidation of sugars and protein thiols, DNA
base damage, and strand breakage of nucleic acids (11).
Cytochrome P450 2E1 (CYP2E1), the ethanol-inducible form, is of
interest because of its ability to metabolize and activate many
toxicologically important substrates to more toxic products (12,
13). There is considerable interest in the mechanism by which ethanol
is hepatotoxic (14, 15). In the intragastric model of ethanol feeding,
prominent induction of CYP2E1 occurs, and significant alcohol liver
injury occurs only in the presence of polyunsaturated fatty acids
(16-18). In these models, large increases in lipid peroxidation have
been observed, and the ethanol-induced liver pathology has been shown
to correlate with CYP2E1 levels and elevated lipid peroxidation. In our
laboratory, stable HepG2 cell lines that express human CYP2E1 were
established; ethanol, arachidonic acid (AA), or iron was toxic to cells
expressing CYP2E1, but not to control cells lacking CYP2E1 (19-21).
Induction of a state of oxidative stress appears to play a central role
in this CYP2E1-dependent cytotoxicity.
Mitochondria are an important organelle in the cell and are not
only a major source of generation of ROS from the mitochondrial respiratory chain (22), but also a critical target of ROS that are
generated either in the mitochondria or derived from outside of
the mitochondria. ROS, including superoxide radical or hydrogen peroxide and especially hydroxyl radical, attack a variety of molecules
present within the mitochondria that subsequently cause loss of
mitochondrial membrane potential, decrease in ATP generation, and
release of cytochrome c, events that lead cells to undergo necrosis or apoptosis (23-25).
Catalase is a very efficient enzyme to protect cells from the
accumulation of H2O2, catalyzing the conversion
of H2O2 to H2O and O2;
catalase is effective at high levels of H2O2
(9). Catalase is mainly located in the cytoplasm (erythrocyte) and
peroxisomes (most cells, especially liver). With the exception of rat
heart (26), catalase is not normally present in the mitochondria. Another major system that catalyzes the reduction of
H2O2 is glutathione peroxidase, which is
localized in both the cytosolic and mitochondrial compartments. This
system also requires NADPH plus glutathione reductase to catalyze the
reduction of GSSG back to reduced GSH.
In a previous study, we established HepG2 cells with overexpressed
catalase in the cytosolic and mitochondrial compartments by
transfection with plasmids containing catalase cDNA and catalase cDNA linked to the leader sequence of manganese-superoxide
dismutase (27). The HepG2 cell lines that constitutively expressed
catalase in the mitochondrial as well as cytosolic compartments were
protected from cytotoxicity or apoptosis induced by hydrogen
peroxide and antimycin A (27). In the present study,
adenovirus-mediated gene transfer was used to compare the effect of
overexpression of human catalase in the cell cytosol with that in the
mitochondria on the ability to protect HepG2 cells from
CYP2E1-dependent cytotoxicity induced by arachidonic acid
and iron. It was hoped that studies with these adenoviral constructs
and cell culture would be useful for eventual in vivo
studies employing catalase localized in different cellular compartments.
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MATERIALS AND METHODS |
Reagents--
Rhodamine 123, propidium iodide (PI), and
MitoTracker® Red were purchased from Molecular Probes,
Inc. (Eugene, OR). Polyclonal antibody raised in rabbit against human
catalase was obtained from Calbiochem. Arachidonic acid, horseradish
peroxidase-conjugated goat anti-rabbit IgG, fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG, minimal essential
medium, and fetal bovine serum were purchased from Sigma.
Recombinant Adenovirus Production--
Ad5 adenoviral vector
with compensating deletions in the early region 1 (E1), as developed by
Graham and co-workers (28, 29), was purchased from Microbix Biosystems
Inc. Human catalase cDNA and catalase cDNA with a
manganese-superoxide dismutase mitochondria leader sequence were
obtained by digestion of plasmids pZeoSV-CAT and pZeoSV/MSP-CAT,
respectively (kindly provided by Dr. J. Andres Melendez, Albany Medical
College, Albany, NY). The plasmid shuttle vectors pAd5-CMV-Cat and
pAd5-CMV-mCat were constructed by inserting catalase cDNA or
catalase cDNA with a manganese-superoxide dismutase mitochondria
leader sequence, respectively, into the Ad5 shuttle vector pCA13. These
adenoviral shuttle plasmids together with the Ad5 genomic DNA JM17 were
transfected into human embryonic kidney 293 cells, which provide the
E1A gene product necessary for viral replication during transfer. After
transfection, plates were overlaid with agar, and initial plaques were
harvested, amplified, and screened for enzymatic activity. Adenovirus
containing no cDNA (AdNull) was used as a control.
Virus possessing cytosolic catalase (AdCat) and mitochondrial catalase
(AdmCat) as well as AdNull were plaque-purified two times and amplified
in 293 cells. Purified high titer stocks of recombinant adenovirus were
generated by two sequential rounds of CsCl density purification. The
preparations were dialyzed and stored in dialysis buffer (10 mM Tris-Cl (pH 7.8), 15 mM NaCl, 10 mM MgCl2 and 10% glycerol) and stored at
80 °C. The titer of each viral stock was determined by plaque
assay on 293 cells; titers were consistently ~1× 1011
plaque-forming units/ml. The concentration of recombinant adenovirus was quantified also by absorbance, and the ratio of particles to
plaque-forming units consistently ranged between 20 and 30.
Cell Culture and Infection--
E47 cells, a HepG2 cell line
overexpressing CYP2E1, and C34 cells, a HepG2 cell line transfected
with empty plasmid (pCI-neo) (30), were cultured in minimal essential
medium 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 infection, E47
cells were seeded onto dishes or plates; grown to 60% confluence; and
infected with AdCat, AdmCat, and AdNull at m.o.i. = 100. Forty-eight
hours after infection, cells were collected and assayed for catalase
expression or treated with pro-oxidants.
Western Blotting--
Cell lysates were prepared by sonicating
cells, followed by centrifugation. The protein concentration of the
supernatant was measured (DC protein assay reagent, Bio-Rad),
and 10 µg of denatured protein was resolved on 10%
SDS-polyacrylamide gel and electroblotted onto nitrocellulose membrane
(Bio-Rad). The membrane was incubated with rabbit anti-human catalase
polyclonal antibody (1:1000 dilution), followed by incubation with
horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000
dilution). Detection by the chemiluminescence reaction was carried out
for 1 min using the ECL kit (Amersham Pharmacia Biotech,
Buckinghamshire, United Kingdom), followed by exposure to Kodak X-Omat
x-ray film.
Catalase Activity Assay--
To validate expression of
functional catalase, the catalase activity of fresh sonicated cell
extracts was determined at 25 °C according to Claiborne and
Fridovich (31). 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 K2HPO4 containing 10 µg of
total cellular protein in a final volume of 1 ml. The specific activity
of catalase was calculated from the following equation: specific
activity (units/mg of protein/min) =
A240 nm(1 min) × 1000/43.6 × mg
of protein.
Immunofluorescence Microscopy--
E47 cells were grown in
minimal essential medium containing 10% fetal bovine serum on glass
coverslips and infected with AdCat, AdmCat, or AdNull adenovirus.
Forty-eight hours after infection, cells were incubated for 30 min with
50 nM MitoTracker® Red. The incubation medium
was removed, and the cells were fixed at room temperature for 10 min
with freshly prepared 2.5% paraformaldehyde in PBS. Cells were washed
three times for 10 min in 0.3 M glycine prepared in PBS and
permeabilized for 5 min in 0.5% Triton X-100 at room temperature,
followed by three washes in PBS. Cells were incubated for 1 h with
a 1:500 dilution of a rabbit anti-human erythrocyte catalase polyclonal
antibody, followed by three washes in PBS, and incubated with a 1:160
dilution of a goat anti-rabbit antibody labeled with fluorescein
isothiocyanate. After three washes in PBS, cells were incubated with
0.1% Triton X-100 for 5 min, followed by an additional three washes in
PBS. The coverslips were mounted on slides and examined by confocal microscopy.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide Assay--
Cell viability was measured by vital dye
reduction using the cell titer 96 non-radioactive cell proliferation
assay kit (Promega). Cells (1 × 105) were seeded onto
24-well plates and infected with adenovirus, followed by treatment with
different concentrations of AA (0, 10, 20, and 30 µM)
plus 30 µM Fe-NTA. Similarly treated uninfected E47 cells
were used as a control. The medium was removed, and cell viability was
evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described previously (27).
LDH Release Assay--
LDH activity was measured as another
index of cytotoxicity. E47 cells were plated onto 6-well plates and
infected with AdNull, AdCat, or AdmCat adenovirus, followed by
treatment with different concentrations of AA (0, 10, 20, and 30 µM) plus 30 µM Fe-NTA. Similarly treated
uninfected E47 cells were used as a control. The culture medium was
collected to measure LDH activity as LDHout. Cells were
harvested by scraping, washed in PBS, suspended in 1 ml of PBS, and
sonicated for 10 s. The lactate dehydrogenase assay kit (Sigma)
was used for the quantitative kinetic determination of LDH activity.
The LDH activity of the cell suspension was measured as
LDHin. The percentage of LDH release was calculated by the formula LDHout/(LDHout + LDHin) × 100% and used to express the cytotoxicity.
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 rhodamine 123. E47 cells (5 × 105) were seeded onto 6-well plates and infected
with adenovirus. Forty-eight hours after infection, cells were
incubated with 20 µM AA for 16 h, followed by
incubation with 30 µM Fe-NTA for an additional 6 h.
The cells were then incubated with medium containing 5 µg/ml
rhodamine 123 for 1 h. Cells were harvested by trypsinization and
resuspended in 1 ml of minimal essential medium containing 5 µg of
PI. The intensity of fluorescence from PI and rhodamine 123 was
analyzed by flow cytometry.
Lipid Peroxidation Assay--
Lipid peroxidation of the cells
was measured with the thiobarbituric acid assay using minor
modifications of the method described by Niehaus and Samuelsson (32).
Briefly, C34 and uninfected E47 cells as well as E47 cells infected
with AdNull, AdCat, or AdmCat adenovirus were incubated in the absence
of any addition or treated with 20 µM AA for 16 h,
followed by incubation with 30 µM Fe-NTA for an
additional 6 h. Cells were washed twice in PBS, scraped from the
dishes, and then suspended in 0.5 ml of PBS containing 0.5 mM trolox
(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Cells
were centrifuged at 2000 rpm for 5 min. The supernatant was discarded,
and the cells were resuspended in 50 µl of PBS containing 0.5 mM trolox. Fifty microliters of cell suspension and 100 µl of 15% trichloroacetic acid, 0.375% thiobarbituric acid, and
0.25 N hydrochloric acid were mixed in a Vortex mixer and
incubated for 15 min at 100 °C, followed by centrifugation for 5 min
at 13,000 rpm. The absorbance of the supernatant was measured at 535 nm. The concentration of malondialdehyde (MDA) equivalents was
calculated using an extinction coefficient of 1.56 × 105 M
1
cm
1.
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RESULTS |
Overexpression of Catalase in the Cytosol and
Mitochondria--
To examine the capacity of AdCat and AdmCat to
enhance the expression and activity of catalase in cells, E47 cells
were infected with AdCat and AdmCat as well as the empty adenovirus
AdNull at m.o.i. = 100. Using an adenovirus containing the
lacZ gene to infect E47 cells followed by
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-gal)
assay, it was determined that >98% of the E47 cells were infected
with the adenovirus under these reaction conditions. After 48 h,
cell lysates were prepared and subjected to Western blot analysis. Fig.
1A shows the expression of
catalase in the total cell extract from these cells. Results from
densitometric analyses of the intensity of the various bands indicated
that the expression of catalase in total cell extracts of cells
infected with AdCat and AdmCat was 2-fold higher than that in
uninfected E47 cells or E47 cells infected with AdNull. Catalase levels
in the uninfected E47 cells were 2-fold higher than those in C34 cells;
this may reflect an up-regulation of catalase expression to remove ROS
generated from CYP2E1. Similar results were obtained by measuring
catalase activity (Fig. 1B). In total cell extracts of E47
cells infected with AdCat and AdmCat, catalase activity was 2-fold
higher than that in uninfected E47 cells or E47 cells infected with
AdNull virus and 4-fold higher than that in C34 cells.

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Fig. 1.
Expression of catalase in E47 cells infected
with adenovirus. E47 cells were infected with AdCat, AdmCat, or
AdNull virus at m.o.i. = 100. Forty-eight hours later, a cell extract
was prepared. Cell extracts from C34 cells and uninfected E47
cells were also prepared as controls. Ten micrograms of protein was
loaded onto each lane for 10% SDS-polyacrylamide gel electrophorsis,
followed by Western blot analysis with polyclonal rabbit anti-human
catalase antibody as described under "Materials and Methods"
(A). Lane 1, C34 cells; lane
2, E47 cells; lane 3, E47 cells infected with AdNull
virus; lane 4, E47 cells infected with AdCat virus;
lane 5, E47 cells infected with AdmCat virus. Ten micrograms
of protein from cell extracts was used to measure the catalytic
activity of catalase (B). Specific catalase units (units/mg
of protein/min) were calculated as described under "Materials and
Methods." First bar, C34 cells; second bar, E47
cells; third bar, E47 cells infected with AdNull virus;
fourth bar, E47 cells infected with AdCat virus; fifth
bar, E47 cells infected with AdmCat virus. Data are the means ± S.D. of triplicate experiments.
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To localize where the catalase is found inside the cells,
immunofluorescence microscopy was performed with detection by confocal microscopy (Fig. 2). Catalase in cells
was labeled by green fluorescence, whereas the mitochondria were
stained red using a mitochondrion-specific dye,
MitoTracker® Red. Catalase in E47 cells infected with
AdNull was located in the peroxisome, as shown by the punctate staining
that did not overlap with the MitoTracker (Fig. 2, upper
panels). This likely reflects the localization of the endogenous
catalase. Catalase in cells infected with AdmCat was located in
mitochondria, as shown by the overlay (yellow) of the green
fluorescence color of fluorescein isothiocyanate with the red color
from MitoTracker® Red in mitochondria, with no green
fluorescence in the cytosolic fraction (Fig. 2, lower
panels). This validates that the manganese-superoxide dismutase
mitochondrial signal peptide successfully transported catalase into the
mitochondria. Cells infected with AdCat showed a high intensity of
green fluorescence in the cytosolic fraction with more diffuse
staining. There was no overlay with the MitoTracker (Fig. 2,
middle panels). These results indicate that the increased expression of catalase in E47 cells infected with AdmCat was localized to the mitochondria, whereas catalase in E47 cells infected with AdCat
was localized to the cytosol.

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Fig. 2.
Immunofluorescence images of E47 cells
infected with AdNull, AdCat, or AdmCat virus. Presented are
catalase immunoreactive protein (left panels with
green color), MitoTracker® Red (middle
panels with red color), and superimposed images
(right panels; overlay) showing the distribution of
immunoreactive catalase and MitoTracker® Red in E47 cells
infected with AdNull (upper panels), AdCat (middle
panels), or AdmCat (lower panels) virus.
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Suppression of Arachidonic Acid-plus Iron-induced
Cytotoxicity--
Arachidonic acid plus iron can induce cytotoxicity
in HepG2 cells expressing CYP2E1 by a process involving increased lipid peroxidation (20, 21). To determine whether mitochondrial or cytosolic
catalase can protect against AA- plus iron-induced cytotoxicity, E47
cells were infected with AdCat, AdmCat, and AdNull adenovirus at m.o.i. = 100 for 48 h, followed by incubation with different
concentrations of AA (0, 10, 20, and 30 µM) for 16 h
and 30 µM Fe-NTA for an additional 8 h. The
viability of the cells was determined by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and
by LDH release. The percentage of surviving cells decreased in an AA
concentration-dependent manner, and this decrease was
similar for uninfected E47 cells and E47 cells infected with AdNull
virus (Fig. 3A). E47 cells
infected with AdCat or AdmCat were more resistant to the cytotoxic
effects of AA plus iron than were uninfected E47 cells or E47 cells
infected with AdNull virus (Fig. 3A). There was no
significant difference between E47 cells infected with AdCat or
AdmCat in protecting against the toxicity induced by AA plus Fe-NTA.
Essentially similar results were obtained using the LDH release assay
to determine the cell viability in the same treatment protocol as
described above (Fig. 3B). Large amounts of LDH were
released from untreated E47 cells or E47 cells infected with AdNull
virus into the medium when cells were treated with different
concentrations of AA plus 30 µM Fe-NTA. However, little
LDH was detected in the medium of E47 cells infected with AdCat or
AdmCat. The morphology of uninfected E47 cells and E47 cells infected
with AdNull, AdCat, or AdmCat adenovirus in the absence and presence
of treatment with different concentrations of AA (0, 10, 20, and 30 µM) for 16 h, followed by incubation with 30 µM Fe-NTA for 8 h and 24 h, was recorded by
visualizing cells under a light microscope. Fig.
4 shows the results obtained with control
cells (a, c, e, and g)
and cells treated with 30 µM AA plus Fe-NTA for 24 h
(b, d, f, and h). Almost
all of the uninfected E47 cells and E47 cells infected with AdNull
lost normal morphology when treated with AA plus Fe-NTA, whereas most
of the E47 cells infected with AdCat or AdmCat retained their shape and structure (Fig. 4).

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Fig. 3.
Effect of catalase overexpression in the
cytosolic or mitochondrial compartment of E47 cells on arachidonic
acid- and iron-induced cytotoxicity. E47 cells were infected with
AdNull, AdCat, or AdmCat adenovirus at m.o.i. = 100. After 48 h,
cells were treated with different concentrations of arachidonic acid
(0, 10, 20, and 30 µM) for 16 h, followed by
incubation with 30 µM Fe-NTA for an additional 8 h.
Uninfected E47 cells treated in the same manner were used as a control.
Cell viability was evaluated by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(A) and LDH release (B) assays. Data are the
means ± S.D. of triplicate experiments.
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Fig. 4.
Morphology of uninfected E47 cells and
E47 cells infected with AdNull, AdCat, or AdmCat adenovirus in the
absence and presence of arachidonic acid plus iron. E47 cells were
infected with AdNull, AdCat, or AdmCat adenovirus at m.o.i. = 100. After 48 h, cells were treated with AA plus Fe-NTA. Cell
morphology was visualized under a light microscope. Shown is the
morphology of uninfected E47 cells (a and b) and
E47 cells infected with AdNull (c and d), AdCat
(e and f), and AdmCat (g and
h) adenovirus in the absence (a, c,
e, and g) and presence (b,
d, f, and h) of 30 µM
arachidonic acid for 16 h, followed by incubation with 30 µM Fe-NTA for 24 h.
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Mitochondrial Membrane Potential (
) and Integrity of the
Plasma Membrane--
Forty-eight hours after infection with
adenovirus, E47 cells were incubated with or without 20 µM AA for 16 h and then incubated with 30 µM Fe-NTA for an additional 6 h. Cells not treated
with AA plus Fe-NTA were predominantly located in the PI-negative and high 
(strong rhodamine 123 fluorescence) field (PI(
)-
high), reflective of viable, intact cells (Fig.
5, a, c,
e, and g and respective insets). A
small percentage of cells were located in the PI(+)-
low field,
reflective of damaged cells (Fig. 5, a, c,
e, and g). Uninfected E47 cells or E47 cells
infected with AdNull that were treated with AA plus Fe-NTA moved to the
PI(
)-
low and PI(+)-
low fields, and 67.3 and 82.5% of
the cells displayed low rhodamine 123 intensity, respectively (Fig. 5,
b and d, M1), compared with 30.8% of
uninfected E47 cells and 40.4% of E47 cells infected with
AdNull, but not treated with AA plus Fe-NTA (Fig. 5,
a and c). However, after treatment with AA plus
Fe-NTA, less cells were in the PI(+) field, and only 40.2 and
32.4% of E47 cells infected with AdCat and AdmCat were present in the

low field, respectively (Fig. 5, f and h).
These results indicate that both cytosolic catalase and mitochondrial
catalase protected the cells from loss of mitochondrial membrane
potential and loss of membrane integrity caused by AA plus iron.

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Fig. 5.
Flow cytometry analysis of the
mitochondrial membrane potential. Uninfected E47 cells as well as
E47 cells infected with AdNull, AdCat, or AdmCat adenovirus were
incubated in the absence of any addition or treated with 20 µM AA for 16 h, followed by incubation with 30 µM Fe-NTA for an additional 6 h. The mitochondrial
membrane potential was measured by flow cytometry as described under
"Materials and Methods." a, c, e,
and g show uninfected E47 cells and E47 cells infected
with AdNull, AdCat, and AdmCat adenovirus, respectively, in the
absence of any treatment. b, d, f, and
h show uninfected E47 cells and E47 cells infected with
AdNull, AdCat, and AdmCat adenovirus, respectively, in the presence of
AA plus Fe-NTA. M1 refers to a population of cells with low
rhodamine 123 (Rh) fluorescence intensity, and the percent
cells with low rhodamine 123 fluorescence is shown in each panel. The
figure is one representative experiment out of three.
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Suppression of Lipid Peroxidation--
Uninfected E47 cells and
E47 cells infected with AdCat, AdmCat, and AdNull virus were treated
with 20 µM AA for 16 h, followed by incubation with
30 µM Fe-NTA for 6 h, and lipid peroxidation was
measured by detecting the concentration of MDA. C34 cells without
CYP2E1 expression were used as a control. There was no significant
difference in MDA production among all these cells in the absence of AA
plus Fe-NTA treatment. The MDA concentration in E47 cells infected with
AdCat or AdmCat was significantly lower than that in uninfected E47
cells or E47 cells infected with AdNull virus when cells were treated
with AA plus Fe-NTA (p < 0.01, AdCat compared with E47
or AdNull; p < 0.05, AdmCat compared with E47 or
AdNull). Lipid peroxidation in C34 cells was lower than that in E47
cells (p < 0.05) or E47 cells infected with AdNull
(p < 0.01) due to the lack of CYP2E1. There was no
significant difference in MDA production between E47 cells infected
with AdCat or AdmCat (Fig. 6).

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Fig. 6.
Effect of catalase overexpression in the
cytosol and mitochondria of E47 cells on lipid peroxidation induced by
arachidonic acid and iron. Cells were either incubated in the
absence of any addition (control) or treated with 20 µM AA for 16 h, followed by incubation with 30 µM Fe-NTA for an additional 6 h. Lipid peroxidation
was measured as described under "Materials and Methods." Data are
the means ± S.D. of triplicate experiments. Student's
t test was used for statistical analysis.
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DISCUSSION |
Using adenovirus-mediated gene transfer, high levels of catalase
could be expressed in E47 cells as determined by Western blot and
catalase activity assays. Immunofluorescence studies confirmed that
catalase with a manganese-superoxide dismutase mitochondrial signal
peptide, which was encoded by the recombinant adenovirus AdmCat,
localized to the mitochondria. Thus, the manganese-superoxide dismutase
leader sequence could be used to successfully import catalase into
mitochondria. Catalase in AdCat-infected E47 cells was localized in the
cytosol in a more diffused manner. Why the cytosolic catalase
distributes in the whole cytosol, but not the peroxisome as the
endogenous catalase does, is not clear, but might be due to
accumulation of catalase in the cytosol in excess of available
peroxisomal content.
Alcohol liver disease has been associated with oxidative stress (14,
15). Although CYP2E1 is not the only primary source of ROS, CYP2E1 is a
major producer of ROS, including superoxide and hydrogen peroxide.
HepG2 cell lines overexpressing CYP2E1 were developed in our laboratory
by stably inducing or transfecting human CYP2E1 cDNA into HepG2
cells (30, 33). Compared with the HepG2 cells line transfected with
empty plasmid vector, HepG2 cells overexpressing CYP2E1 are more
sensitive to arachidonic acid-, L-buthionine sulfoximine
(which lowers cellular GSH levels)-, iron-, and ethanol-induced
toxicity and apoptosis (19-21, 30). Ascorbic acid, the iron chelator
desferrioxamine, and several typical inhibitors of lipid peroxidation
produce efficient protection against arachidonic acid toxicity in
CYP2E1-expressing HepG2 cells (20) probably by quenching or preventing
formation of powerful oxidants such as hydroxyl radical or other
initiators of lipid peroxidation. These studies indicate that ROS are
responsible for the CYP2E1-dependent cytotoxicity.
The overexpression of catalase in the cytosol or mitochondria protected
the E47 cells from cytotoxicity caused by AA and iron. The cytosolic
catalase and mitochondrial catalase appear to be equally effective in
protecting the cells from cytotoxicity induced by AA plus iron. Because
CYP2E1 is present in the endoplasmic reticulum, CYP2E1-derived
H2O2 would be generated in the endoplasmic reticulum and diffuse into the cytosol. Therefore, cytosolic catalase might be anticipated to readily perform its function to reduce H2O2. Since catalase in mitochondria was
equally protective against CYP2E1-dependent AA- plus
iron-induced toxicity, it is likely that some of the
H2O2 diffuses into the mitochondria, and
perhaps damage to the mitochondria may be an important factor
contributing to AA- plus iron-induced toxicity. Catalase in
mitochondria might act as a sink for H2O2 and
promote H2O2 movement down its concentration gradient, thereby decreasing lipid peroxidation in the cells and protecting mitochondria from damage.
Mitochondrial permeability transition and mitochondrial membrane
potential are markers for mitochondrial damage and dysfunction (34-36). Mitochondrial dysfunction caused by ROS, especially
H2O2, can lead not only to necrosis by
depleting ATP, but also to apoptosis by inducing the release of
cytochrome c, which activates caspases together with other
mitochondrial factors such as Apaf-1 (37-39). We determined the
mitochondrial membrane potential in cells treated with 20 µM AA and incubated with 30 µM Fe-NTA. The
decline in 
caused by these agents was much less in E47 cells
infected with AdCat or AdmCat than in uninfected E47 cells or E47 cells infected with AdNull. These results suggest that both cytosolic catalase and mitochondrial catalase protected cells from
oxidant-induced loss of mitochondrial membrane potential, which may
play an important role in the overall protection against
oxidant-induced cytotoxicity.
The mechanism of AA- plus iron-induced cytotoxicity is thought to be
due to lipid peroxidation as a consequence of CYP2E1-derived ROS such
as superoxide and especially H2O2. Addition of
AA increases the content of polyunsaturated fatty acid. Supplementation
of iron enhances the Fenton reaction, by which superoxide radical and
H2O2 are transformed into the more toxic
hydroxyl radical or other ferryl-type oxidants that catalyze the
peroxidation of polyunsaturated fatty acid. Although human CYP2E1 has
been shown to metabolize arachidonic acid to
1-hydroxyarachidonic acid (40, 41), a CYP2E1 inhibitor
(4-methylpyrazole) could not prevent the toxicity of arachidonic acid
at concentrations that prevented toxicity of ethanol, CCl4,
and acetaminophen (19, 20, 42), and CYP2E1 substrates such as ethanol
did not effectively inhibit the arachidonic acid toxicity (20). These
results indicate that the direct metabolism of arachidonic acid to
potentially toxic products by CYP2E1 does not contribute significantly
to the polyunsaturated fatty acid toxicity, but rather metabolism of AA
by CYP2E1-derived oxidants is likely to be responsible for the ensuing
lipid peroxidation and loss of cell viability. This explains the
effectiveness of H2O2-degrading enzymes such as
catalase in protecting the E47 cells from the AA- plus Fe-NTA-induced
toxicity. Indeed, the lipid peroxidation assays showed that, after
treatment with AA plus iron, the concentration of MDA in E47 cells
infected with AdCat or AdmCat was significantly lower than that in
uninfected E47 cells or E47 cells infected with AdNull. The catalase
expression in E47 cells lowered MDA levels to those in C34 cells.
In summary, this study suggests that mitochondria are an important
target for oxidative damage and that both catalase in the cytosol and
catalase in mitochondria are capable of protecting HepG2 cells against
CYP2E1-dependent cytotoxicity induced by arachidonic acid
plus iron. It is hoped that these adenoviral constructs may prove to be
effective in preventing oxidative damage caused by alcohol or other
xenobiotics under in vivo conditions, and future studies
evaluating the in vivo expression of these constructs have
been initiated.