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INTRODUCTION |
Polyunsaturated fatty acids
(PUFA)1 such as arachidonic
acid (AA) or its metabolites play an important role in a variety of biological processes, such as signal transduction, chemotaxis, and cell
proliferation and differentiation (1-3). PUFA also play an important
role in alcoholic liver injury (4-6). In the intragastric infusion
model of ethanol feeding, liver injury occurs when the rats consume
diets containing polyunsaturated fatty acid but not saturated fatty
acid (7, 8). This model is associated with induction of high levels of
CYP2E1 and greatly increased lipid peroxidation, which appear to
contribute to the liver injury. AA induced toxicity in HepG2 E47 cells,
a cell line that expresses CYP2E1 but not control HepG2 cells, which do
not express CYP2E1 (9). AA also induced toxicity in pyrazole-induced
rat hepatocytes with high levels of CYP2E1 but not saline control
hepatocytes (10). This AA toxicity was prevented by inhibitors of
CYP2E1 and by antioxidants (9, 10).
AA can activate mitogen-activated protein kinase (MAPK), a ubiquitous
group of serine/threonine kinases, which play a crucial role in
transmitting transmembrane signals required for cell growth, differentiation, and apoptosis (11-13). Members of the kinase family, originally found to be activated by mitogens, have now been found to be
activated by a wide variety of mitogenic and non-mitogenic agents via a
cascade of kinase/effector molecules which, in mammalian cells,
includes protein kinase C, p21, Raf-1, and MEK (MAPK/extracellular signal-regulated protein kinase, ERK) (14-16). AA or its metabolites can activate MAPK members such as ERKs and JNKs/SAPKs, suggesting an
important role for AA and its metabolites in mitogenic signaling events
(17, 18).
We recently reported that salicylate can potentiate the toxicity of AA
in CYP2E1-induced hepatocytes (19). Salicylate was found to increase
CYP2E1 levels, preventing its degradation, and this may be one
mechanism by which salicylate increases AA-induced toxicity. Sodium
salicylate and acetylsalicylic acid are non-steroidal anti-inflammatory
agents that prevent activation of nuclear factor
B by
inhibition of phosphorylation and subsequent degradation of I
B, or
by direct inhibition of I
B kinase (20- 23). Salicylate also
interferes with MAPK and other kinase-dependent signaling pathways (24-26) and can affect mitochondrial function (27, 28). Damage to mitochondria plays an important role in AA plus
CYP2E1-dependent toxicity (29). Whether MAPKs are involved
in the AA-induced toxicity, and if so, how MAPKs induce or regulate
this toxicity have not been reported, for cytochrome
P450-dependent processes in general, and specifically for
CYP2E1. In the present study, we characterized the possible role of the
MAPKs, p38 and ERK, and PI3K on AA or AA plus salicylate-induced
toxicity in pyrazole-induced rat hepatocytes, human hepatocyte
cultures, and HepG2 E47 cells. Effects of specific kinase inhibitors on
cellular toxicity, CYP2E1 levels, lipid peroxidation, mitochondrial
membrane potential, and NF-
B activation and the role of MAPKs in the
salicylate enhancement of AA-induced toxicity were evaluated. Results
show that AA or AA plus salicylate activated p38 MAPK but not ERK or
PI3K, and that p38 MAPK plays a role in AA-induced cytotoxicity in
CYP2E1-expressing cells.
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MATERIALS AND METHODS |
Hepatocyte Isolation and Cell Culture--
Rats received humane
care, and studies were carried out according to the criteria outline in
the Guide for the Care and Use of Laboratory Animals and Institutional
Animal Care and Use Committee approval. Male Sprague-Dawley
rats, 150-170 g body weight were injected intraperitoneally with
pyrazole, 200 mg/kg body weight, once a day for 2 days to induce
CYP2E1. After overnight fasting, rat hepatocytes were isolated by a
two-step collagenase perfusion method (30). Induction of CYP2E1 was
validated by Western blot analysis and catalytic activity with
p-nitrophenol. Cell viability was generally about 90%.
Hepatocytes were seeded onto 100-mm culture dishes, which were coated
with the basement membrane Matrigel (BD Biosciences) and cultured in
serum-free HeptoZYME-SFM medium (Invitrogen) containing 1% penicillin
and streptomycin. One to two hours after seeding, the medium was
changed, unattached cells were gently washed out, and the cell culture
experiments were initiated. Human hepatocyte cultures plated on
rat tail collagen-coated flasks (T-25) in serum-supplemented media were
obtained from the Liver Tissue Procurement and Distribution System
(University of Minnesota, Minneapolis, MN).
E47 cells are HepG2 cells that were transfected with a human CYP2E1
cDNA in the sense orientation and constitutively express CYP2E1.
C34 cells are HepG2 cells that were transfected with the pCI vector
only; these cells do not express CYP2E1 (9). The HepG2 cells were
cultured in minimal essential medium supplemented with 10% fetal
bovine serum plus 100 units/ml penicillin plus 100 µg/ml streptomycin
in 5% CO2 at 37 °C.
Toxicity Determination--
Hepatocyte or HepG2 cell cultures
were treated with arachidonic acid (AA, 60 µM), with or
without salicylate (sal, 5 mM) in the presence or absence
of 10 µM SB203580 (a specific p38 MAPK inhibitor), 10 µM PD98059 (a specific ERK inhibitor) (Calbiochem Inc.),
or 0.1-10 µM wortmannin (a PI3K inhibitor, Sigma
Chemical Co.) for 24 h. Inhibitors were dissolved in
Me2SO, and controls were incubated with
Me2SO (0.6% v/v final concentration). As a non-CYP2E1-dependent control, hepatocytes were treated with
15 ng/ml tumor necrosis factor
plus 40 µM
cycloheximide in the presence or absence of MAPK or PI3K
inhibitors. Cytotoxicity was determined by assays of either trypan blue
exclusion or reduction of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide as
described previously (31).
Immunoblot Analysis--
To study whether SB203580 modulates the
CYP2E1 level, Pyrazole-induced rat hepatocytes were incubated with
either 10 µM SB203580 (or Me2SO solvent
control) or 5 mM sal for 1 or 2 days. Microsomes were
prepared by differential centrifugation, and SDS-PAGE was carried out
using 10 µg of microsomal protein. The blotted membranes were
incubated with a monospecific polyclonal CYP2E1 antibody, followed by
incubation with goat anti-rabbit antibody conjugated with horseradish
peroxidase. Fluorescence was developed using the enhanced
chemiluminescence immunoblot-detecting agent (ECL, Amersham
Biosciences). The arbitrary unit of density of each band was scanned
with a computer software program (UN-SCAN-IT, Automated Digitizing
System, version 5.1, Silk Scientific Corp.).
Lipid Peroxidation Analysis--
Cells were treated with AA or
AA plus sal in the presence or absence of 10 µM SB203580,
10 µM PD98059, 10 µM wortmannin, or Me2SO (solvent for the kinase inhibitors) for 24 h.
Cells were harvested and sonicated in PBS for 10 s in an ice bath
using a W-375 sonicator (50% duty cycle, output at 4 watts).
The cellular lysate was collected, and cell extract containing 0.2 mg
of protein was incubated with 0.2 ml of trichloroacetic
acid-TBA-HCl solution in a 100 °C water bath for 1 h as
previously described (19, 29). The formation of thiobarbituric
acid-reactive products (TBARS) was determined by measuring absorbance
at 535 nm and using an extinction coefficient of 1.56 × 105 M/cm to calculate malondialdehyde
equivalents. As a control, the potential antioxidant activity of
SB203580 was evaluated using isolated rat liver microsomes incubated
with iron-ADP plus NADPH as previously described (32).
Activation of p38 or ERK MAPK or PI3K--
Cells were treated
with AA or AA plus sal in the presence or absence of 10 µM SB203580, 10 µM PD98059, or 10 µM wortmannin for 10, 20, or 30 min. The cells were
harvested and sonicated as described above. The cellular extract (15 µg of protein) was subjected to SDS-PAGE using an 8% gel. The
blotted membranes were incubated with either polyclonal p38 or ERK MAPK
antibodies or AKT polyclonal antibody to detect the total content of
these kinases or incubated with the appropriate phosphorylated
monoclonal antibodies, respectively (Santa Cruz), to determine the
content of the activated, phosphorylated kinase. The blots were then
incubated with either anti-rabbit IgG or anti-mouse IgG conjugated with
horseradish peroxidase and fluorescence developed, and results were
analyzed as described above.
Immunohistochemical Localization of the Translocation of
NF-
B--
Freshly isolated rat hepatocytes were grown on a glass
slide placed in culture dishes. The cells were treated as described above. At the end of treatment, the cells on the glass slide were fixed
with 4% paraformaldehyde, washed with PBS, and incubated with NF-
B
antibody (Santa Cruz Biotechnology) for 2 h. After rinsing several
times in PBS, the slides were incubated with anti-IgG antibody
conjugated with fluorescein isothiocyanate. The slides were rinsed
several times with PBS and mounted onto a microscopy glass slide with
mounting medium for fluorescence (Vector Laboratories, Inc.,
Burlingame, CA). The localization of NF-
B in the nucleus or the
cytosol was observed under a fluorescence microscope.
Electrophoretic Mobility Shift Assay--
To determine the
effect of AA or AA plus sal on NF-
B DNA binding activity, cells were
treated with AA or AA plus sal in the presence or absence of 10 µM SB203580, 10 µM PD98059, or 10 µM wortmannin for 30 min or for 24 h. Cells were
harvested and lysed with a lysis solution containing 0.32 M
sucrose, 2 mM CaCl2, 2 mM
MgCl2, 0.1 mM EDTA, 0.5% Triton X-100, 1 mM dithiothreitol, 20 mM Hepes, pH 7.5, and 0.1 mM phenylmethylsulfonyl fluoride for 15 min. After
centrifugation at 2500 rpm for 10 min, the pellet was collected and
resuspended in 1 ml of lysis solution and centrifuged at 6000 rpm for
10 min. The pellet was resuspended in a buffer containing 0.42 M NaCl, 20 mM Hepes, pH 7.5, 25% glycerol, 2 mM MgCl2, 0.2 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 15 µg/ml antipain, 2.5 µg/ml aprotinin, 2.5 µg/ml
leupeptin, 2.5 µg/ml pepstatin, and 2.5 µg/ml bestatin, incubated
for 10 min and centrifuged at 15,000 rpm for 15 min. The supernatant containing nuclear extract was collected, and 10 µg of nuclear extract was used to carry out the electrophoretic mobility shift assay
with a kit (Promega) according the protocol offered by the company. The
NF-
B oligonucleotide in the kit was labeled with [32P]ATP, and bands on the gel were visualized by
exposure to the Kodak film and then developed.
Mitochondrial Membrane Potential Analysis--
Mitochondrial
membrane potential (MMP) was analyzed from the accumulation of
rhodamine 123. Hepatocytes were seeded onto six-well dishes for 1-2 h,
and unattached hepatocytes were gently removed and replaced with fresh
medium. The cells were treated with AA, or AA plus sal with or without
10 µM SB203580, 10 µM PD98059, or 10 µM wortmannin for 20 h. One hour before ending the
treatment, rhodamine 123 (5 µg/ml) was added to the medium. Cells
were harvested by trypsinization and resuspended in 0.5-0.8 ml of PBS.
The intensity of the fluorescence from rhodamine 123 was determined
with a Fas-Scan flow cytometer as previously described (29).
Statistics--
One-way ANOVA (ANOVA with subsequent post hoc
comparisons by Sheffe) was performed (Version 10.0, SPSS, Chicago, IL).
p values of less than 0.05 were considered statistically
significant; values reflect means ± S.E., and the number of
experiments are given in the figure legends.
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RESULTS |
SB203580 Prevents AA or AA Plus Salicylate-induced Toxicity in E47
Cells--
HepG2 E47 cells were treated with 60 µM AA or
60 µM AA plus 5 mM sal for 12, 24, or 36 h in the presence or absence of 10 µM SB203580, an
inhibitor of p38 MAPK, and cell viability (trypan blue exclusion) was
determined. As shown in Fig. 1, AA or AA
plus sal induced significant toxicity in E47 cells in a
time-dependent manner. Salicylate, which was not toxic by
itself, increased the toxicity by AA as described previously (19).
SB203580 by itself was not toxic to the E47 cells, however, SB203580
lowered the AA or AA plus sal-induced toxicity e.g. from 60 or 80% at 24 h in the absences of SB203580 to toxicity values of
20 or 30% (p < 0.05) in the presence of SB203580.
Similar protection by SB203580 was observed in AA concentration
dependence experiments, e.g. in the absence of SB203580,
toxicity by 15, 30, or 60 µM AA at 24 h was 24 ± 3, 34 ± 7, and 60 ± 6%, respectively, whereas the toxicity in the presence of 10 µM SB203580 was lowered to
values of 18 ± 2, 20 ± 3, and 22 ± 7%
(p < 0.05) at 15, 30, or 60 µM AA,
respectively (data not shown). C34 cells, which do not express CYP2E1,
only exhibited a small response to AA or AA plus sal (Table I) as compared with the E47 cells, which
was not altered by SB203580. The effect of SB203580 on another model of
toxicity, independent of CYP2E1, was evaluated. In contrast to results
with AA or AA plus salicylate in E47 cells, the toxicity by tumor
necrosis factor
plus cycloheximide was similar in E47 and C34
cells, and this toxicity was not altered by SB203580 (Table I). Thus,
there appears to be some specificity in the protective actions of
SB203580.

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Fig. 1.
SB203580 prevents AA- or AA plus
salicylate-induced toxicity in HepG2 E47 cells. HepG2 E47
cells, which express CYP2E1, were treated with AA (60 µM)
or AA plus sal (5 mM) in the absence or presence of 10 µM SB203580 for 12, 24, or 36 h. Cytotoxicity was
determined from the percentage of cells staining with trypan blue. *,
significantly different (p < 0.05) compared with
AA-treated groups for the same time course period. **, significantly
different (p < 0.05) compared with AA plus sal groups
for the same time period. Results are from four preparations.
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Table I
The inhibition of P38 MAPK prevents AA and AA plus salicylate-induced
cytotoxicity in HepG2 E47 cells
HepG2 E47 cells, which express CYP2E1, and control HepG2 cells, which
do not express CYP2E1 (C34 cells), were incubated with the indicated
treatments, 5 mM sodium salicylate, 60 µM AA,
AA plus salicylate, 10 µM SB203580 alone or with AA or
with AA plus salicylate. Other groups were treated with 20 ng/ml TNF
in the absence or presence of 40 µM cycloheximide (CHX).
The cytotoxicity index refers to the percent cells staining with trypan
blue. Results are from three experiments.
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SB203580 and PD98059 Lower AA or AA Plus Salicylate Induced
Toxicity in Pyrazole Rat Hepatocytes and Human Hepatocytes--
To
extend the results with the HepG2 cells to primary liver cells, rats
were treated with pyrazole (PY) to increase hepatic CYP2E1 levels.
Hepatocytes from PY-induced rats were treated with AA or AA plus sal,
and cell viability was assayed by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction.
Cell viability was decreased to 44 ± 5 or 31 ± 10% after
24 h of culture with 60 µM AA or 60 µM
AA plus 5 mM sal, respectively (data not shown). Adding
SB203580 a p38 MAPK inhibitor or PD98059, an ERK MAPK inhibitor,
lowered this toxicity. In the presence of SB203580, cell viability
increased to 84 ± 4 (AA) and 75 ± 6% (AA plus sal)
(p < 0.05), whereas in the presence of PD98059, cell
viability increased to 63 ± 6 (AA) and 78 ± 7% (AA plus
sal) (p < 0.05), respectively. Wortmannin, a PI3K
inhibitor used at concentrations ranging from 0.1 to 10 µM, failed to prevent AA- or AA plus sal-induced
toxicity. SB203580, PD98059, or wortmannin had no effect on cell
viability; neither did the Me2SO (0.6%) solvent control.
Human hepatocyte cultures were treated with AA or AA plus sal in the
presence or absence of SB203580, PD98059, or wortmannin for 24 h.
AA (60 µM) or AA plus 5 mM sal caused a
50 ± 4 or 84 ± 1% loss of cell viability, respectively
(Fig. 2). SB203580 lowered toxicity to
23 ± 7 or 50 ± 2% (p < 0.05), whereas
PD98059 lowered toxicity to 36 ± 4 or 63 ± 10%
(p < 0.05), respectively. Wortmannin failed to prevent
AA- or AA plus sal-induced toxicity in human hepatocyte cultures
(p > 0.05, Fig. 2). Generally, with both PY rat
hepatocytes and the human hepatocytes, SB203580 was more effective than
PD98059 in preventing the CYP2E1 plus AA toxicity.

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Fig. 2.
SB203580 and PD98059 but not wortmannin
decrease AA- or AA plus salicylate-induced toxicity in human hepatocyte
cultures. Human hepatocyte cultures were treated with 60 µM AA or AA plus 5 mM sal in the absence or
presence of 10 µM SB203580, 10 µM PD98059,
or 10 µM wortmannin for 24 h. At the end of
treatment, trypan blue exclusion was carried out to determine the
percent toxicity. *, significantly different (p < 0.05) compared with control group; **, significantly different
(p < 0.05) compared with the AA-treated group; ***,
significantly different (p < 0.05) compared with the
AA plus sal-treated group. Results are from four experiments.
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Effect of SB203580 on CYP2E1 Protein Levels--
Changes in the
level of CYP2E1 could be one mechanism by which SB203580 protects
against AA toxicity in the hepatocytes or E47 cells. Pyrazole-induced
rat hepatocytes were incubated with 10 µM SB203580 for 1 or 2 days, and levels of CYP2E1 were determined by Western blot
analysis. The immunoblots showed the expected decrease in CYP2E1 levels
when hepatocytes are placed in culture (30) (Fig.
3, lanes 1, 2, and
6). SB203580 did not lower CYP2E1 protein levels as compared
with control incubations (Fig. 3, lanes 4 and 8);
actually, CYP2E1 levels were increased, but this increase was due to
the Me2SO solvent used to solubilize the SB203580 (Fig. 3,
lanes 5 and 9). Me2SO is a CYP2E1
ligand that protects against CYP2E1 degradation. This suggests that
SB203580 prevention of AA- or AA plus sal-induced toxicity in
hepatocytes is not mediated via lowering the levels of CYP2E1. As
previously shown (19), salicylate helped to partially maintain CYP2E1
levels at 24 h in the tissue culture (Fig. 3, compare lanes
3 and 7 with lanes 2 and 6), and
this maintenance was not altered by SB203580 (not shown).

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Fig. 3.
SB203580 does not lower the level of CYP2E1
in rat hepatocyte cultures. Pyrazole-induced rat hepatocytes were
incubated with or without 5 mM sal or 10 µM
SB203580 or 0.6% Me2SO (solvent control for SB203580) for
1 or 2 days. At the end of treatment the cells were harvested,
microsomes were prepared, and levels of CYP2E1 were determined by
Western blot analysis. The immunoblots were carried out by using
anti-CYP2E1 antibody. A typical Western blot from three independent
experiments is shown. The bands were scanned and the arbitrary unit
values from the experiment are shown above the blots.
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Effect of SB203580 on Lipid Peroxidation--
Treating the cells
with AA increased lipid peroxidation in hepatocytes about 2-fold, and
this was further elevated in the presence of salicylate (Fig.
4A). Salicylate alone, as
previously shown (19), had no effect on lipid peroxidation.
Antioxidants such as Trolox, which prevent the AA-induced lipid
peroxidation, were previously shown to protect against the AA toxicity
(29); hence, if SB203580 had any antioxidant action, its ability to prevent lipid peroxidation could explain its protective effects against
AA-induced toxicity. However, SB203580, PD98059, or wortmannin (or
Me2SO) did not reduce lipid peroxidation induced by AA or AA plus sal (Fig. 4A). To further exclude a possible
antioxidant effect of SB203580, we incubated rat liver microsomes with
Me2SO, Me2SO plus SB203580, or, as a positive
control, the antioxidant Trolox (100 µM) and determined
lipid peroxidation (Fig. 4B). SB203580 did not affect lipid
peroxidation of rat microsomes compared with its Me2SO
solvent control. In contrast, Trolox completely inhibited the lipid
peroxidation (Fig. 4B). These results suggest that the prevention by SB203580 of AA- or AA plus sal-induced toxicity is not
mediated by an antioxidant action that lowers lipid peroxidation.

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Fig. 4.
MAPK or PI3K inhibitors do not affect AA- or
AA plus salicylate-induced lipid peroxidation. A,
pyrazole-induced rat hepatocytes were treated with 60 µM
AA or AA plus 5 mM sal in the presence or absence of 10 µM SB203580, 10 µM PD98059, or 10 µM wortmannin (or 0.6% Me2SO) for 24 h,
respectively. Cells were harvested, and a cellular lysate was prepared
by sonication. Cell extract containing 0.2 mg of protein was incubated
with a trichloroacetic acid-TBA-HCl solution to carry out the lipid
peroxidation analysis. The production of TBARS (malondialdehyde) was
measured at 535 nm. *, significantly different (p < 0.05) compared with control. There was no significant effect of
SB203580, PD98059, or wortmannin on the AA- or AA plus sal-induced
increase in lipid peroxidation. Results are from four experiments.
B, rat liver microsomes (0.2 mg) were incubated with
Me2SO (0.6%), SB203580 (10 µM), in
Me2SO (0.6%) or Trolox (100 µM) for the
indicated time periods. Lipid peroxidation was determined by assay of
TBARS production, measuring absorbance at 532 nm.
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Effect of Kinase Inhibitors on AA- or AA Plus Salicylate-induced
Reduction of the MMP--
Mitochondria appear to be a critical target
for damage by AA as well as a major organelle for the production of
reactive oxygen species. AA was previously shown to decrease MMP, a
decrease that was intensified by salicylate; moreover, cyclosporin
A, an inhibitor of the mitochondrial permeability transition
protected against AA or AA plus salicylate toxicity (29). Therefore, it
was important to study whether the inhibition of p38 MAPK by SB203580
could protect against the AA or AA plus sal reduction of the MMP. Cells were treated with AA or AA plus sal in the presence or absence of
kinase inhibitors for 24 h, and the cells were then treated with
Rh123 for 1 h. Flow cytometry was carried out to determine the
extent of Rh123 fluorescence, an index of the MMP. The percentage of
cells with low Rh123 fluorescence (M1 population; reflective of low
MMP) was increased from 12 to 31% by AA or to 49% by AA plus sal
(Fig. 5). However, in the presence of
SB203580, the decline of MMP was prevented as the cells with low MMP
decreased to 10% (AA) or 16% (AA plus sal) (p < 0.05, Fig. 5). PD98059 showed only a modest prevention effect (21% or
33% M1 cells), whereas wortmannin had no protective effect against the
decline in MMP produced by AA or AA plus salicylate (Fig. 5).

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Fig. 5.
SB203580 prevents AA- or AA plus
salicylate-induced reduction of MMP. Pyrazole-induced rat
hepatocyte cultures were treated with 60 µM AA or AA plus
5 mM sal in the presence or absence of 10 µM
SB203580, 10 µM PD98059, or 10 µM
wortmannin for 20 h, respectively. One hour before ending of
treatment, Rh123 (5 µg/ml) was added. Cells were then harvested, and
the MMP was analyzed by flow cytometry. M1 and
M2 refer to two cell populations with low or high
membrane potential. The percentage of cells in the M1 zone
is indicated in the cytogram.
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Activation of p38 MAPK Phosphorylation by AA or AA Plus
Salicylate--
The prevention of AA toxicity by SB203580 suggests a
role for p38 MAPK in the overall toxicity mechanism of AA. One method to assess activation of p38 MAPK is by determining its extent of
phosphorylation. Treatment of rat hepatocytes with AA, sal, or AA plus
sal significantly increased the level of phosphorylated p38 MAPK. After
20-min treatment with AA, sal, or AA plus sal, the extent
of p38 MAPK phosphorylation increased 2.6-, 2.9-, or 5.2-fold,
respectively (Fig. 6A,
lanes 4, 2, and 5, compared with lane 1, respectively), whereas the extent of phosphorylation
increased 3.1-, 3.7-, or 3.9-fold, respectively, after 30-min
incubation (Fig. 6A, lanes 4, 2, and
5 compared with lane 1, respectively). AA or AA
plus sal did not activate p38 MAPK phosphorylation activity after
30-min treatment in saline hepatocytes with lower levels of CYP2E1
(data not shown). AA, sal, or AA plus sal also induced p38 MAPK
phosphorylation in human hepatocyte cultures (Fig. 6B, top panels, lanes 4, 2, and
5 compared with lane 1, respectively). The ratio
of phosphorylated p38 MAPK to total p38 MAPK was increased to values of
2.7, 2.1, and 4.2 after 30-min treatment with salicylate, AA, or AA
plus salicylate, respectively, over the control values of 1.0. In
contrast to p38 MAPK, AA or AA plus salicylate did not promote the
phosphorylation of either ERK or PI3K in PY hepatocytes (data not
shown) or human hepatocytes (Fig. 6B, bottom
panels, ERK ratios of phosphorylated ERK to total ERK between 0.9 to 1.2). SB203580 lowered the level of phosphorylated p38 MAPK induced by AA or AA plus salicylate in the pyrazole hepatocytes (lanes 6 and 7 compared with lanes 4 and
5 in Fig. 6A) and the human hepatocytes
(lanes 6 and 8 compared with lanes 4 and 5, Fig. 6B, top panels),
validating the ability of this compound to inhibit p38 MAPK activity
(ratios of phosphorylated p38 MAPK to total MAPK of 0.5 to 0.7 in the
presence of AA or AA plus salicylate plus SB203580).

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Fig. 6.
Effect of AA or AA plus salicylate on
activation of p38 MAPK in rat hepatocytes (A) or human
hepatocytes (B). A, pyrazole-induced
rat hepatocytes were treated with AA (60 µM) or AA plus
sal (5 mM) in the presence or absence of SB203580 (10 µM) for 10, 20, or 30 min. The cells were harvested and
sonicated, the cellular lysate was collected, and 15 µg of cellular
extract was electrophoresed using SDS-PAGE (8% gel). The membranes
were incubated with either p38 MAPK polyclonal antibody or
phosphorylated p38 MAPK monoclonal antibody. Results shown are one blot
from three independent experiments. B, human hepatocyte
cultures were treated with AA (60 µM) or AA plus sal (5 mM) in the presence or absence of SB203580 (10 µM) or PD98059 (10 µM) for 30 min,
respectively. Cells were harvested and lysed by sonication. The cell
extract (15 ug) was used to carry out SDS-PAGE (8% gel).
Immunoblot analysis was carried out using p38 and ERK
antibodies or phosphorylated p38 and ERK antibodies,
respectively.
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Antioxidants Prevent AA or AA Plus Salicylate
Activation of p38 MAPK--
In E47 cells or pyrazole hepatocytes with
high levels of CYP2E1, AA produces radical stress and lipid
peroxidation (10, 19, 29). Oxidative stress is known to modulate signal
transduction pathways (33). To test whether oxidative stress produced
by AA treatment is related to the activation of p38 MAPK,
pyrazole-induced hepatocytes were preincubated with the antioxidants
Trolox or catalase, and then the cells were treated with AA or AA plus
salicylate for 30 or 60 min. The p38 MAPK phosphorylation level was
determined by Western blot using the phosphorylated p38 MAPK antibody.
AA increased p38 phosphorylation at 30 min but not after 60 min; salicylate potentiated the AA-induced increase in p38 MAPK at 30 or 60 min (Fig. 7, lanes 4 and
7 compared with lane 1). Both antioxidants
blocked AA (lanes 5 and 6)- or AA plus salicylate (lanes 8 and 9)-induced activation of p38 MAPK to
the control levels (Fig. 7). SB203580, as a positive control, also
blocked the activation of p38 MAPK (lanes 10-12). These
results suggest that AA-induced oxidative stress and not AA per
se played a role in the activation of p38 MAPK in hepatocytes with
high levels of CYP2E1.

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Fig. 7.
Antioxidants prevent AA or AA plus salicylate
activation of p38 MAPK. Pyrazole-induced rat hepatocyte cultures
were treated with 60 µM AA or AA plus 5 mM
sal in the presence or absence of Trolox (100 µM),
catalase (500 units/ml), or SB203580 (10 µM) for 30 or 60 min, respectively. Cells were harvested and lysed by sonication.
Immunoblot analysis using phosphorylated p38 MAPK antibody was carried
out on 15 µg of cell extract. The blot shown is one from three
independent experiments.
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Effect of AA, Salicylate, or SB203580 on the Translocation of
NF-
B--
The activation of NF-
B may inhibit apoptosis by
increasing NF-
B-dependent gene expression to produce
products that prevent cellular toxicity. Inhibition of NF-
B
activation enhances toxicity in various models, including hepatocytes
(33, 34). In hepatocytes, p38 MAPK can inhibit the activation of
NF-
B, and it was therefore considered that such effects might
contribute to the role of p38 MAPK in AA and AA plus salicylate
toxicity and in the protection afforded by SB203580. The possibility
that AA could, at least initially (prior to the onset of significant
toxicity), activate NF-
B by promoting its translocation into the
nucleus was therefore evaluated. Pyrazole-induced rat hepatocytes were
treated with AA, sal, or SB203580 for 24 h, and the translocation
of NF-
B into the nucleus was detected by immunohistochemistry. In
control, non-treated cells, little or no NF-
B was detected in the
nucleus in the absence or presence of salicylate (Fig.
8, A and B). After 24-h treatment, AA induced translocation of NF-
B into the nucleus (Fig. 8C). Salicylate inhibited this
translocation of NF-
B (Fig. 8D), which may contribute to
the salicylate enhancement of AA toxicity. In the presence of
salicylate plus SB203580, the translocation of NF-
B was restored
(Fig. 8E), which may play a role in the protective effects
of SB203580. SB203580 alone has no significant effect on the
translocation of NF-
B (Fig. 8F). An electrophoretic mobility shift assay was carried out to evaluate the effect of AA, sal,
or SB203580 on NF-
B DNA binding activity. Control NF-
B binding
activity in hepatocytes in the absence of additions was low and
assigned a value of 100 arbitrary densitometric units (Fig.
9, lane 5). Treatment with 60 µM AA for 24 h increased NF-
B binding with DNA to
360 ± 20 units (Fig. 9, lane 8). Salicylate inhibited
this increased binding produced by AA to levels of 100 ± 15 units
(lanes 10 and 11). SB203580 partially restored
the NF-
B binding level in the presence of AA plus salicylate to
200 ± 20 units (lanes 15 and 16). AA did
not increase the NF-
B binding level after a 30-min treatment in
contrast to the increase found after the 24-h treatment (data not
shown).

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Fig. 8.
The effect of AA or AA plus salicylate on the
translocation of NF- B to the nucleus in rat
hepatocyte cultures. Immunohistological location of the
translocation of NF- B in intact hepatocytes. Pyrazole-induced rat
hepatocytes were grown on a glass slide in a culture disk. The cells
were treated for 24 h as follows: A, control;
B, sal (5 mM); C, AA (60 µM); D, AA plus sal; E, AA plus sal
plus 10 µM SB203580; F, SB203580 (10 µM). The slides were fixed with 4% paraformaldehyde and
incubated with NF- B antibody for 2 h. After washing several
times in PBS, the slides were incubated with anti-IgG antibody
conjugated with fluorescein isothiocyanate and washed again in PBS. The
translocation of NF- B was observed under a fluorescence
microscope.
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Fig. 9.
Gel shift assay to determine the effect of AA
or AA plus salicylate on the DNA binding activity of
NF- B. Pyrazole-induced rat hepatocyte
cultures were treated with AA or AA plus sal in the presence or absence
of SB203580 or PD98059 for 24 h, respectively. Cells were
harvested, and the nuclear extract was prepared. Gel shifts were
carried out by using 10 µg of nuclear protein incubated with a gel
shift kit containing a NF- B oligonucleotide probe, which was labeled
with [32P]ATP. Lanes 2-4 contained the HeLa
cell extract used as a positive control. Excess unlabeled NF- B
(lane 3) and AP2 (lane 4) probes were used as
controls to validate that the band detected is composed of NF- B
subunits. Top panel, one gel shift result from three
independent experiments. The lanes refer to the
corresponding bars shown in the bottom panel,
which summarizes the results of the three experiments. P65
bands were scanned, and the average arbitrary units from three
experiments are shown.
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DISCUSSION |
Mitogen-activated protein kinases (MAPKs) are regulated by
distinct signal transduction pathways that control many aspects of
mammalian cellular physiology, including cell proliferation, differentiation, inflammation, and apoptosis (35, 36). The activation
of MAPK also plays a role in cell toxicity effects (37-39). We are not
aware of any studies on the possible role of MAPKs in cytochrome
P450-dependent toxicities, especially with respect to
CYP2E1-dependent actions.
Arachidonic acid has been shown to induce the phosphorylation of MAPKs
(40-42), but whether such activation can be related to AA toxicity and
the mechanism through which MAPK plays a role in AA toxicity have not
been addressed. Exogenous polyunsaturated fatty acids (PUFAs) such as
AA exert a wide range of effects on cells of diverse origin, such as
regulation of gap junctions, permeability between adherent cells,
neutrophil secretion and migration, NADPH oxidase activities,
expression of cell-surface receptors, gene transcription, cytotoxic T
cell function, and modulation of the activities of components of
intracellular signaling. PUFAs such as AA also play an important role
in chronic alcohol-induced liver injury, where correlations between
injury, lipid peroxidation, and CYP2E1 have been reported (4-7). Our
previous work has demonstrated that AA can induce apoptosis and
necrosis in pyrazole-induced rat hepatocytes with high levels of CYP2E1
to a much greater extent than in saline control rat hepatocytes, or in
E47 cells as compared with non-CYP2E1-expressing HepG2 cells (19, 29).
Salicylate was recently found to have a synergistic effect in
enhancement of AA toxicity and AA-induced lipid peroxidation (19).
In the current study, AA or AA plus salicylate was shown to induce the
phosphorylation of p38 MAPK 2.0- to 5.6-fold as compared with
non-treated controls in pyrazole-induced rat hepatocyte or human
hepatocyte cultures. SB203580, a specific p38 MAPK inhibitor, lowered
the AA or AA plus salicylate-induced toxicity. These results suggest
that p38 MAPK plays a role in AA- and AA plus salicylate-induced toxicity. Although PD98059, an ERK inhibitor, also lowered AA- or AA
plus salicylate-induced toxicity, generally, this compound was not as
effective as SB203580, and ERK phosphorylation activity was not induced
by AA or AA plus salicylate. Thus, activation of ERK does not appear to
play an important role in AA toxicity in hepatocyte cultures, although
why PD98059 is somewhat protective remains unclear. AA or
AA plus salicylate did not increase AKT phosphorylation, and the PI3K
inhibitor wortmannin over a wide concentration range did not block AA-
or AA plus salicylate-induced toxicity, indicating that PI3K does not
play a role in this toxicity process. p38 MAPK inhibitors such as
SB203580 or SB202190 are pyridinyl imidazole compounds that inhibit p38
MAPK function by binding to the ATP-substrate binding pocket. In the
present study, SB203580 alone had no effect on CYP2E1 levels nor did it
act as an antioxidant. Importantly, SB203580 did not affect AA- or AA plus salicylate-induced lipid peroxidation suggesting that the inhibitor was functioning downstream from the generation of lipid peroxidation, which results from the interaction of CYP2E1 with AA.
It was reported that AA alone, or its epoxygenase or
-hydroxylase products, can activate the MAPK superfamily
members C-Jun NH2-terminal kinase (JNK) and extracellular
signal-regulated kinase (ERK) in rabbit proximal tubule cells (43, 44).
In the present study, AA or AA plus salicylate activated p38 MAPK in
pyrazole-induced rat hepatocytes, which contain a 3- to
4-fold higher CYP2E1 level than normal rat hepatocytes; AA or AA plus
salicylate did not activate p38 MAPK in saline control rat hepatocytes,
which contain very low levels of CYP2E1. Catalase and Trolox block the
activation of p38 MAPK by AA in the pyrazole-induced rat hepatocytes.
These results suggest that the activation of p38 MAPK by AA or AA plus salicylate is CYP2E1-dependent, and ROS and lipid
peroxidation metabolites, but not AA itself, promote the
phosphorylation of p38 MAPK. Further studies to evaluate how
CYP2E1-dependent ROS production activates p38 MAPK,
e.g. the role of upstream regulators such as Ras, are in
progress. Interestingly, the antioxidants Trolox and catalase did not
prevent the activation of p38 MAPK by salicylate, indicating that ROS
and lipid peroxidation did not appear to play a role in the salicylate
activation. This is consistent with the lack of effect of salicylate on
lipid peroxidation. The mechanism by which salicylate activates p38
MAPK is unknown (21, 24).
How does activation of p38 MAPK contribute to the CYP2E1 plus AA
toxicity? Activation of p38 MAPK has been shown to cause apoptosis and
cell toxicity (37-39). This may be mediated by downstream mediators
such as c-Jun NH2-terminal kinase; preliminary studies have
shown that a JNK II inhibitor could partially protect against the AA or
AA plus salicylate toxicity in pyrazole hepatocytes, and further
studies to identify p38 MAPK downstream mediators are in progress.
Clearly, activation of p38 MAPK alone is not sufficient to account for
the AA-induced toxicity, because salicylate, which activated p38 MAPK
to an equal or even greater extent than did AA, was not toxic under
conditions in which AA was toxic. AA-induced lipid peroxidation and ROS
generation are critical for the developing toxicity, and other cell
targets besides p38 MAPK must play a role in the AA-induced toxicity,
although activation of this kinase potentiates or is permissive for the
developing toxicity. We have recently shown that damage to mitochondria
plays an important role in the AA- or AA plus salicylate-induced
toxicity in pyrazole hepatocytes, because there was an early reduction of mitochondrial membrane potential prior to toxicity, and cyclosporin A, an inhibitor of the mitochondrial permeability transition, partially prevented the toxicity (29). The decrease in mitochondrial membrane potential produced by AA (but not salicylate) was confirmed in
the current study (Fig. 5). Importantly, SB203580 prevented the AA- or
AA plus salicylate-induced lowering of the mitochondrial membrane
potential. We hypothesize that mitochondrial membrane potential is
sensitive to the combined actions of activated p38 MAPK coupled to ROS
and lipid peroxidation-induced oxidative stress. The AA and the
salicylate potentiation of AA toxicity were shown to be a mixed mode of
cell death, because both necrosis and apoptosis could be
observed (19, 29). Damage to the mitochondria and a decrease in
membrane potential would result in a decline in ATP levels, which would
promote a necrotic mode of cell death. AA induced a membrane
permeability transition, which could result in release of
apoptotic-inducing factors such as cytochrome c and
activation of caspase-3. These events have been shown to occur in E47
cells and in pyrazole hepatocytes treated with AA or AA plus salicylate
(19, 29). Hence, the prevention by SB203580 of the decline in
mitochondrial membrane potential and, ultimately, cellular toxicity
induced by AA suggests that a critical target of activated p38 MAPK are
the mitochondria.
A second target of activated p38 MAPK may be the NF-
B system. p38
MAPK is one of the more important regulators in the cellular response
to inflammation. p38 MAPK can inhibit the degradation of I
B
kinase (45, 46) and, thereby, inhibit the translocation of NF-
B into
the nucleus. NF-
B is a key component of innate immunity (47),
promoting the expression of a set of genes involved in host defense
(48, 49). NF-
B is normally constitutively present in the cytosol,
because the interaction with inhibitory I
B masks the nuclear
localization domain of the complex (50). NF-
B-dependent
gene transcription requires the phosphorylation of I
B by IKK2, which
then releases this inhibitory component from the dimer of Rel proteins,
followed by degradation of the phospho-I
B by the proteasome (51,
52). Inhibiting the degradation of I
B will prevent NF-
B
translocation into the nucleus, and this generally increases
susceptibility of the hepatocyte to cytotoxicity by various agents,
including pro-oxidants. Sodium salicylate and acetylsalicylic acid
(aspirin) inhibit the activation of NF-
B by preventing the
phosphorylation of I
B and its degradation by the
ubiquitin-proteasome pathway (20, 53, 54). It has been shown that
sodium salicylate prevents the phosphorylation of I
B through
activation of the p38 MAPK pathway (21). AA did not activate NF-
B
when incubated with pyrazole hepatocytes for short time periods,
e.g. 15 min. This time point is associated with maximum
activation of p38 MAPK (Fig. 6), which may prevent any potential
activation of NF-
B by AA. At longer time periods, e.g. 24 h, AA did activate NF-
B translocation into the nucleus and increased DNA binding activity (Figs. 8 and 9). This may reflect an
attempt to protect the hepatocyte against the ensuing oxidant stress
and toxicity. Salicylate, which itself induced the
activation of p38 MAPK, blocked this AA-induced NF-
B translocation
and decreased the NF-
B DNA binding activity (Figs. 8 and 9). This
may contribute to the enhancement of AA toxicity by salicylate.
SB203580 counters these actions of salicylate and restores NF-
B
translocation and DNA binding activity by inhibiting p38 MAPK activity,
and this results in partial protection against toxicity. These results suggest that the activation of p38 MAPK may eventually modulate the
activation of NF-
B and affect NF-
B-dependent gene
expression, which subsequently weakens response of the cell to stress
and oxidative toxicity induced by AA.
In summary, the CYP2E1-dependent toxicity of AA in HepG2
cells expressing CYP2E1 and in pyrazole hepatocytes is mediated in part, via activation of p38 MAPK. A model to accommodate current and
previous results is shown in Fig. 10.
This activation is due to CYP2E1 plus AA production of ROS and lipid
peroxidation metabolites, because antioxidants block the activation of
p38 MAPK by AA, and the ensuing toxicity. Enhancement of AA toxicity by
salicylate, previously shown to be due in part to salicylate increasing
the half-life of CYP2E1 by decreasing its turnover, is likely also due
to salicylate activation of p38 MAPK. Activated p38 MAPK alone is not
likely to promote significant cellular toxicity, because salicylate
alone was not toxic. However, the combination of activated p38 MAPK and
oxidative stress synergize to promote cellular toxicity, perhaps by
effects on the mitochondrial membrane potential, and may prevent
activation of NF-
B and production of cellular protective agents.
Although further studies are necessary to identify upstream and
downstream mediators involved in the activation of p38 MAPK by AA, and
in the toxic actions associated with activated p38 MAPK, this is the
first report on the role of stress-associated MAPK in
CYP2E1-dependent toxicity. In view of the critical role of
polyunsaturated fatty acids in alcohol-induced liver injury, the
possible therapeutic effectiveness of MAPK inhibitors such as SB203580
against CYP2E1-dependent toxicity and
alcohol-dependent toxicity may be worthwhile to
consider.

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Fig. 10.
Proposed model for the role of p38 MAPK
in CYP2E1 plus AA-induced toxicity. Please see
"Discussion."
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