1 Department of Medicine, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461; and 2 IDUN Pharmaceuticals, Incorporated, La Jolla, California 92037
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ABSTRACT |
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Reactive oxygen intermediates (ROI) have
been implicated as mediators of hepatocyte death resulting from a
variety of forms of liver injury. To delineate the mechanisms that
underlie ROI-induced apoptosis, the roles of caspase activation and
nuclear factor-B (NF-
B) signaling were determined in the rat
hepatocyte cell line RALA255-10G after treatment with
H2O2 or the superoxide generator menadione. By
8 h, H2O2 and menadione caused 26% and 33%
cell death, respectively. Death from both ROI occurred by apoptosis as
indicated by morphology under fluorescence microscopy, the induction of
caspase activation and DNA fragmentation, and the cleavage of
poly(ADP-ribose) polymerase. Despite the presence of caspase activation
in both forms of apoptosis, caspase inhibition blocked
H2O2- but not menadione-induced apoptosis. In
contrast, inhibition of NF-
B activation decreased cell death from
both ROI. Different ROI, therefore, induce distinct apoptotic pathways in RALA hepatocytes that are both caspase dependent and independent. In
contrast to the known protective effect of NF-
B activation in tumor
necrosis factor-
-induced hepatocyte apoptosis, NF-
B promotes
hepatocellular death from ROI in these cells.
reactive oxygen intermediates; liver; menadione
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INTRODUCTION |
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A NUMBER OF CELLULAR STIMULI, including mitogens, cytokines, and toxins, trigger the intracellular generation of reactive oxygen intermediates (ROI) (13, 34). During cellular injury in vivo, cells are also exposed to high levels of exogenous ROI produced by neutrophils and macrophages activated during the accompanying inflammatory response (34). The ultimate effect of ROI on cells is dependent on a number of factors, including the level of oxidant stress generated, the nature of the intracellular signaling pathways activated, and the state of cellular antioxidant defenses. However, it is clear that high concentrations of ROI cause death in nonhepatic cells and hepatocytes (15, 31), and the generation of ROI may be a common mechanism by which a variety of liver injuries ultimately result in hepatocyte death (34).
The mechanism of ROI-induced injury in hepatocytes is incompletely
understood. ROI can damage cells by direct effects on cellular macromolecules through lipid peroxidation, the cross-linking and degradation of proteins, and DNA nicking (25, 29). Cell death from ROI
commonly occurs by apoptosis, although high concentrations of ROI can
convert the cell death response to necrosis (11, 16). Apoptosis is
usually mediated by the proteolytic actions of a family of cysteine
proteases termed caspases (5), but the involvement and regulation of
caspase activation in ROI-induced hepatocyte death have not been
examined. Recent studies of apoptosis induced by the cytokine tumor
necrosis factor- (TNF-
) have demonstrated that the induction of
caspase-dependent apoptosis in hepatocytes may be regulated by the
state of activation of the transcription factor nuclear factor-
B
(NF-
B) (2, 38). When NF-
B is activated, hepatocytes are resistant
to TNF-
cytotoxicity, and TNF-
treatment induces a proliferative
response (38). However, if NF-
B activation is inhibited, TNF-
induces caspase-dependent hepatocyte apoptosis (2, 38). Whether
NF-
B-mediated cell signaling regulates caspase activation in
additional forms of hepatocyte apoptosis remains to be determined. The
known ability of ROI to activate NF-
B (22), and reports of caspase
involvement in ROI-mediated death in nonhepatic cells (15, 16),
suggested that in ROI-induced hepatocyte death NF-
B may regulate
caspase activation.
To test this hypothesis, the mechanism of cell death from two forms of
ROI, H2O2 and superoxide, was examined in a rat
hepatocyte cell line. Specifically, it was determined whether ROI cause
hepatocyte death from apoptosis that is mediated by caspase activation.
In addition, the effect of NF-B activation on ROI-induced apoptosis was examined. Surprisingly, it was demonstrated that the two types of
oxidative stress initiate distinct cell death pathways that differ in
their dependence on caspases but are both stimulated by NF-
B activation.
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MATERIALS AND METHODS |
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Cells and culture conditions. The rat hepatocyte cell line RALA255-10G (4) was cultured in DMEM (GIBCO BRL, Grand Island, NY) supplemented with 4% fetal bovine serum (HyClone, Logan, UT), 2 mM glutamine, and antibiotics (GIBCO BRL), as previously described (4, 38). These cells are conditionally transformed with a temperature-sensitive T antigen. At the permissive temperature of 33°C, the cells express T antigen, remain undifferentiated, and proliferate. Culture at the restrictive temperature of 37°C suppresses T antigen expression, slows growth, and allows differentiated hepatocyte gene expression (4, 9). For all experiments, the cells were cultured at 33°C until confluent, trypsinized, and replated at 0.65 × 106 cells/dish on 35-mm plastic dishes (Falcon, Becton Dickinson, Lincoln Park, NJ). After 24 h, the medium was changed to DMEM supplemented with 2% fetal bovine serum, glutamine, antibiotics, and 1 µM dexamethasone, and the cells were placed at 37°C. After 3 days of culture at 37°C, the cells received fresh serum-free medium containing dexamethasone. Medium was supplemented with dexamethasone to optimize hepatocyte differentiation as previously described (4). Some cells were treated with H2O2 or menadione 20 h later.
To inhibit caspase activity, cells were pretreated for 1 h before the addition of ROI with the following caspase inhibitors dissolved in DMSO: 100 µM Val-Ala-Asp-fluoromethylketone (ZVAD) (Bachem, Torrance, CA), 50 µM N-[(indole-2-carbonyl)-alaninyl]-3-amino-4-oxo-5-fluoropentanoic acid (IDN-1529), or N-[(1,3-dimethylindole-2-carbonyl)-valinyl]-3-amino-4-oxo-5-fluoropentanoic acid (IDN-1965) (IDUN Pharmaceuticals, La Jolla, CA). IDN-1529 and IDN-1965 have broad anti-caspase activity, inhibiting caspase-1, caspase-3, caspase-6, and caspase-8 (J. Wu, personal communication).MTT assay. The amount of cell death was determined by examining cell number with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (8), as previously described (38). The percent cell survival was calculated by taking the optical density (OD) reading of cells given a particular treatment, dividing that number by the OD reading for the untreated control cells, and then multiplying by 100.
Microscopic determination of apoptosis. The presence of apoptosis or necrosis was determined by fluorescence microscopy as previously described (37). The relative numbers of apoptotic and necrotic cells were determined by fluorescence microscopy after costaining with acridine orange and ethidium bromide (10). The percentage of cells with apoptotic morphology (nuclear and cytoplasmic condensation, nuclear fragmentation, membrane blebbing, and apoptotic body formation) was determined by examining >400 cells/dish. Necrosis was determined by the presence of ethidium bromide staining.
Protein isolation and Western blot analysis. For protein isolation, cells were scraped in the medium and centrifuged. The cell pellet was resuspended in lysis buffer containing 10 mM HEPES (pH 7.4), 42 mM MgCl2, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM dithiothreitol, and 2 µg/ml pepstatin A, leupeptin, and aprotinin and mixed at 4°C for 30 min. After centrifugation, the supernatant was collected and the protein concentration determined by the Bio-Rad protein assay (Hercules, CA).
Fifty micrograms of protein were resolved on 10% or 12% SDS-PAGE gels as previously described (38). Membranes were stained with Ponceau red to ensure equivalent amounts of protein loading and electrophoretic transfer among samples. Rabbit anti-caspase-2 polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-caspase-3 polyclonal IgG, rabbit anti-caspase-7 polyclonal IgG, or rabbit anti-caspase-8 polyclonal IgG (IDUN Pharmaceuticals) were used as primary antibodies at 1:2,000, 1:4,000, 1:1,000, and 1:4,000 dilutions, respectively. A goat anti-rabbit IgG conjugated with horseradish peroxidase (GIBCO BRL) was used as a secondary antibody at a 1:10,000 dilution. Proteins were visualized by chemiluminescence (Supersignal Ultra, Pierce, Rockford, IL). For Western immunoblots of poly(ADP-ribose) polymerase (PARP), centrifuged cells were suspended in lysis buffer composed of 20 mM Tris, pH 7.5, 1% SDS, 2 mM EDTA, 2 mM EGTA, 6 mMQuantification of DNA hypoploidy by flow cytometry.
Identification of apoptotic cells by detection of DNA loss after
controlled extraction of low-molecular-weight DNA was performed as
previously described (6). Cells were trypsinized, washed in Hanks'
buffered saline solution (HBSS), and centrifuged. The cell pellets were
resuspended, fixed in 70% ethanol, and stored at 20°C for
up to 1 wk. After the pellets were washed twice in HBSS, 1 ml HBSS cell
suspensions were incubated with 0.5 ml of phosphate-citric acid buffer
(0.2 M Na2HPO4, 0.1 M citric acid, pH 7.8) for
5 min to extract low-molecular-weight DNA from apoptotic cells.
Subsequently, the cells were centrifuged, and the pellet was
resuspended in 0.5 ml of HBSS containing 20 µg/µl propidium iodide
and RNase (100 µg/ml). After a 30-min incubation at room temperature,
cells were analyzed by fluorescence-activated cell sorting (FACS)
(FACScan, Becton Dickinson Immunocytometry Systems, San Jose, CA) at an
excitation of 488 nm. DNA fluorescence pulse processing
was used to discriminate between single cells and aggregates of cells
(doublet discrimination) by evaluating the FL2-width vs. FL2-area
scatter plot. Light scatter gating was used to eliminate smaller debris
from analysis. An analysis gate was set to limit the measurement of
hypoploidy to an area of 10-fold loss of DNA content (6).
Electrophoretic mobility shift assays.
Nuclear proteins were isolated by the method of Schreiber et al. (30),
modified as previously described (37). Electrophoretic mobility shift
assays (EMSA) were performed on 5 µg of protein with a
32P-end-labeled oligonucleotide for the NF-B consensus
sequence (Santa Cruz Biotechnology). The DNA binding reaction was
performed as previously described (39), and the samples were resolved on a 4% polyacrylamide gel, dried, and subjected to autoradiography.
Adenovirus infection.
To inhibit NF-B activation, the recombinant replication-deficient
adenovirus Ad5I
B was employed as previously described (38). This
mutant I
B cannot be phosphorylated and therefore irreversibly binds
NF-
B, preventing its activation. In addition, the adenovirus
Ad5LacZ, which contains the Escherichia coli
-galactosidase gene, was used as a control. Both viruses
were grown in 293 cells and purified by banding twice on CsCl gradients
as previously described (38). Cells were infected with 5 × 109 particles of the appropriate virus per 35-mm culture
dish (~1.5 × 103 particles/cell or 5-15
plaque-forming units/cell), for 3 h before the final medium change.
Statistical analysis. All numerical results are reported as means ± SE and represent data from a minimum of three independent experiments.
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RESULTS |
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ROI induce RALA hepatocyte cell death by apoptosis.
Experiments were performed in the RALA hepatocyte cell line cultured at
37°C. At this temperature, the adult rat hepatocyte cell line is
nontransformed and maintains a differentiated hepatocyte phenotype (4).
The use of these cells avoids the potential problems that exist in
studying cell death pathways in primary rat hepatocytes, which in
culture spontaneously undergo apoptosis (17) and significant changes in
their expression of cell death modifiers such as AP-1 (23, 24), NF-B
(26, 36), and glutathione (1). RALA hepatocytes were exposed to a range
of concentrations of H2O2 and the superoxide
generator menadione to establish concentrations of each ROI that would
lead to moderate and equivalent degrees of cell death. The addition of
H2O2 at 1.25 µmol/106 cells and
27.5 µM menadione to the culture medium resulted in 26% and 33%
cell death, respectively, at 8 h (Fig. 1).
A further increase in cell death was seen from both ROI at 24 h (Fig.
1). These concentrations of H2O2 and menadione
were selected for use in all subsequent experiments.
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H2O2 and menadione cytotoxicity are
associated with caspase activation.
Cell death from apoptosis usually results from the actions of a family
of proteolytic enzymes termed caspases (5). To assess the potential
role of various caspase family members in ROI-mediated RALA hepatocyte
apoptosis, caspase processing was analyzed by Western immunoblotting.
At 8 and 24 h after H2O2 and menadione treatment, protein levels of procaspase-2, procaspase-3, procaspase-7, and procaspase-8 were all substantially reduced, indicating that cleavage and activation of the inactive proenzymes had occurred (Fig.
3). Also indicative of caspase activation
was the presence at 8 h of the cleaved p20 and p17 subunits of
caspase-3 after H2O2 and menadione treatments
(Fig. 3).
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ROI induce PARP degradation and internucleosomal DNA cleavage.
Activated caspases cleave known proteins such as PARP. To further
evaluate whether processed caspases were functionally active in
H2O2- and menadione-treated RALA hepatocytes,
we assessed PARP cleavage by Western blotting. PARP cleavage occurred
at 8, 12, and 24 h after H2O2 and menadione
treatment, as indicated by the presence of the 85-kDa fragment at these
times (Fig. 5).
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Caspase inhibition decreases H2O2- but not
menadione-induced apoptosis.
Having established that caspase activation occurred in hepatocytes
subjected to two forms of ROI-induced cytotoxicity, we investigated the
mechanistic involvement of caspases in these forms of cell death. The
effects of the pan-caspase inhibitors ZVAD, IDN-1529, and IDN-1965 on
ROI-induced cell death were determined. Caspase inhibitors blocked
H2O2-induced cell death with IDN-1529 and
IDN-1965 reducing cell death by ~60% (Fig.
7). In contrast, caspase inhibition did not
prevent menadione-induced apoptosis in RALA hepatoctyes (Fig. 7).
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NF-B inactivation inhibits
H2O2- and menadione-induced apoptosis.
Oxidative stress is a known activator of NF-
B (22), and NF-
B
activation induced by TNF-
is critical for cellular resistance to
apoptosis caused by this cytokine (2, 38). To determine whether NF-
B
has a similar function in ROI-mediated apoptosis, the role of NF-
B
in ROI-induced death was examined. Initially, the effects of
H2O2 and menadione on RALA hepatocyte NF-
B
activity were investigated. Treatment with H2O2
or menadione led to NF-
B activation within 2 h that persisted for 8 h, as reflected by increased DNA binding measured by EMSA (Fig.
8). NF-
B activation was slightly greater
at 2 and 4 h with H2O2 compared with menadione treatment (Fig. 8).
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DISCUSSION |
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In liver injury, hepatocytes are subjected to oxidative stress from both ROI generated intracellularly in response to cytokines and hepatotoxins and ROI produced extracellularly by inflammatory cells. Endogenously produced ROI are known to act as intermediates in apoptotic signaling based on the findings that 1) ROI generation can be detected in cells undergoing apoptosis (32); 2) antioxidants inhibit several forms of apoptosis (11, 21); and 3) decreases in intracellular antioxidants can induce apoptosis in some cells (27). Exposure of cells to low concentrations of exogenous ROI also induces apoptotic cell death, whereas higher amounts can cause necrosis (11, 16). During liver injury, the exposure of hepatocytes to endogenously and exogenously generated ROI may therefore initiate apoptotic death pathways, and ROI have been implicated as mediators of hepatocellular death from a variety of stimuli (34). Because apoptosis requires active cellular signaling, understanding the pathways by which ROI induce hepatocyte apoptosis may suggest new therapeutic avenues for the prevention of liver injury.
The present study examined two forms of ROI-induced cell death in a hepatocyte cell line. H2O2 freely diffuses across the cell membrane and in the presence of cellular iron can be metabolized to the toxic hydroxyl free radical (14). Menadione is a quinone that on reduction enters the redox cycle with molecular oxygen, leading to the generation of superoxide radicals in hepatocytes (33). The hydroxyl and superoxide radicals generated by H2O2 and menadione can lead to necrotic cell injury through damaging interactions with cellular DNA, protein, or lipids, which disrupt critical cellular macromolecules and energy production. Alternatively, ROI activate a number of intracellular signaling pathways, and lower concentrations of ROI may trigger apoptosis through the induction of pro-apoptotic signals. However, there may not be such a distinct separation between the mechanisms of necrosis and apoptosis (18), and we have previously described (39) the involvement of AP-1 signaling in the promotion of H2O2-induced necrotic cell death in a hepatoma cell line.
The concentrations of H2O2 and menadione used
to induce RALA hepatocyte death in this study caused an apoptotic cell
death as indicated by the presence of morphological changes of
apoptosis, DNA hypoploidy, and PARP degradation. Associated with the
biochemical parameters of PARP degradation and internucleosomal DNA
cleavage was the activation of caspases as demonstrated by Western
immunoblotting and enzyme activity. Activation of caspase-3-like
proteases has been reported after H2O2
treatment of T cells (15), and xanthine/xanthine oxidase treatment of
myelogenous leukemia ML-1a cells (16). Our studies extend these
findings to hepatocytes and for the first time show that
H2O2 and superoxide activate upstream
(caspase-2 and caspase-8) as well as downstream (caspase-3 and
caspase-7) caspases. Although the mechanism by which these ROI lead to
caspase activation is unknown, these data suggest the possibility that ROI may induce apoptosis by initially triggering the activation of
upstream caspases such as caspase-8. Caspase-8 may then ultimately lead
to activation of downstream effector caspases such as caspase-3, similar to the events that occur with TNF--induced cell death. Alternatively, upstream caspases may become processed as a secondary phenomenon that follows the primary activation of downstream effector caspases by ROI.
Despite the presence of caspase activation in both forms of ROI-induced cell death, caspase inhibition provided protection from H2O2-induced injury, but not menadione-induced apoptosis. Prior studies of H2O2-induced T cell apoptosis failed to examine whether inhibition of caspase activation prevented cell death (15). Caspase inhibition blocked DNA degradation in xanthine/xanthine oxidase-induced ML-1a cell apoptosis but failed to prevent membrane blebbing (16), suggesting that cell death may in fact not have been blocked in these cells. Our data indicate that despite the ability of both ROI to cause caspase activation, caspases mediate cell death after H2O2 but not superoxide exposure in RALA hepatocytes. In our experiments, H2O2 may have been a more potent activator of caspases than superoxide. In support of this possibility is the finding that caspase-3-like activity at 8 h was increased less in menadione-treated cells than in cells treated with H2O2. However, menadione-induced caspase activation was sufficient to lead to PARP and DNA degradation. With the cleavage of known caspase substrates, superoxide-induced apoptosis was clearly associated with caspase activation but cell death occurred independently of caspase action. Different injurious forms of molecular oxygen therefore trigger distinct pathways of apoptosis. The ability of superoxide to cause caspase-independent apoptosis also adds to the recent reports of other forms of apoptosis that do not rely on caspase activity (7, 35).
Inhibition of NF-B activation decreased rather than increased
apoptosis caused by H2O2 and menadione. NF-
B
suppression by means of I
B overexpression has been previously
reported to increase menadione toxicity in the HepG2 hepatoma cell line
(3). Our finding was surprising in light of this report and prior
studies that NF-
B activation prevents caspase-dependent,
TNF-
-induced apoptosis in RALA hepatocytes (38) and primary rat
hepatocytes (2). NF-
B activation has been reported to promote other
forms of apoptosis in nonhepatic cells (19). However, this report is
the first to our knowledge to demonstrate that in the same cell type,
NF-
B can promote or inhibit apoptosis depending on the apoptotic
stimulus. The present data suggest that in RALA hepatocytes NF-
B
activation may regulate a protein(s) that specifically blocks the
TNF-
death pathway but is not involved in the ROI death pathway. The
mechanism by which NF-
B activation promotes ROI-induced death is
unknown. One possible explanation of the ability of NF-
B to both
inhibit and promote apoptosis is that nitric oxide (NO) may have
differential effects in the two models. NF-
B activation upregulates
inducible NO synthetase, leading to NO production. Although NO inhibits
TNF-
-mediated RALA hepatocyte apoptosis (38), NO may be detrimental
during ROI injury because of the ability of NO to be converted to toxic
peroxynitrite in the presence of oxygen radicals (20). NF-
B may
therefore promote some forms of ROI-mediated liver injury in vivo, and
recently inhibition of NF-
B activation has been shown to prevent
cell death from brain ischemia (28).
In hepatocytes, ROI can initiate caspase-dependent and -independent
apoptosis depending on the molecular form of ROI causing the injury.
The use of caspase inhibitors therapeutically may offer cytoprotection
in some, but not all, forms of ROI-induced liver injury. In addition,
although efforts to enhance NF-B activation in hepatocytes may
prevent TNF-
-mediated cell death, such strategies may increase
ROI-induced death.
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ACKNOWLEDGEMENTS |
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We thank Amelia Bobe for secretarial assistance, Dr. David Brenner
for providing the Ad5LacZ and Ad5IB adenoviruses, Dr. Janice Chou
for providing the RALA255-10G cells, and David Gebhard for
assistance with the FACS analysis.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-44234 (M. J. Czaja), an Australian National Health and Medical Council Research Scholarship (B. E. Jones), and an American Digestive Health Foundation Astra/Merck Fellowship/Faculty Transition Award (B. E. Jones).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. J. Czaja, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: czaja{at}aecom.yu.edu).
Received 11 November 1999; accepted in final form 21 December 1999.
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