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*
Division of Critical Care, Children’s Hospital Medical Center, Cincinnati, OH 45229; and
Department of Pathophysiology, University Medical School of Debrecen, Debrecen, Hungary
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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m)
in thymocytes, as determined by flow cytometry using the mitochondrial
potential sensitive dyes DiOC6(3) and JC-1. A time- and dose-dependent
increase in secondary reactive oxygen intermediate production and loss
of cardiolipin, an indicator of mitochondrial membrane damage, were
also observed, as measured by flow cytometry using the fluorescent dyes
dihydroethidine and nonyl-acridine orange, respectively. Inhibition of
PARS by 3-aminobenzamide or 5-iodo-6-amino-1,2-benzopyrone attenuated
peroxynitrite-induced m reduction, secondary reactive
oxygen intermediate generation, cardiolipin degradation, and
intracellular calcium mobilization. Furthermore, thymocytes from
PARS-deficient animals were protected against the peroxynitrite- and
hydrogen peroxide-induced functional and ultrastructural mitochondrial
alterations. In conclusion, mitochondrial perturbations during
oxidant-mediated cytotoxicity are, to a significant degree, related to
PARS activation rather than to direct effects of the oxidants on the
mitochondria. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, mitochondria emerged as key regulators of cell death (8, 9, 10, 11, 12, 13, 14). Intact mitochondria maintain a large (up to 180 mV) negative membrane potential across the mitochondrial inner membrane. A decrease in mitochondrial transmembrane potential followed by an intense reactive oxygen intermediate (ROI) production and a reduction of mitochondrial mass have been shown to occur in various models of cell death (10, 11, 12, 13). Although most work in this area focuses on the role of mitochondrial alterations during programmed cell death (apoptosis), mitochondrial alterations also play a role in the process of necrotic death (13, 14, 15). Oxidants, such as peroxynitrite and hydrogen peroxide, induce mitochondrial permeability transition and inhibit the mitochondrial respiratory chain (16, 17, 18).
Here we report that PARS activation mediates the mitochondrial injury in cells exposed to peroxynitrite or hydrogen peroxide. The data presented in the current study demonstrate that the changes in mitochondrial membrane potential, the mitochondrial permeability transition, the increase in ROI production, the increased calcium mobilization, and the destruction of mitochondrial structure are attenuated by inhibition of PARS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fluorescent dyes were purchased from Molecular Probes (Eugene, OR). INH2BP was a gift from Dr. E. Kun (State University of San Francisco, Tiburon, CA), and bongkrekic acid was generously donated by Dr. J. A. Duine (Technical University of Delft, Delft, The Netherlands). Annexin V-FITC was obtained from PharMingen (San Diego, CA). Peroxynitrite was a kind gift of Dr. H. Ischiropoulos (University of Pennsylvania, Philadelphia, PA). 3-Morpholinosidnonimine (SIN-1) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) were purchased from Calbiochem (San Diego, CA). All other regents were obtained from Sigma (St. Louis, MO).
Thymocyte preparation and treatment with oxidants
Thymi from wild-type (WT) and PARS-deficient mice (gift from Dr. Z. Q. Wang, Institute of Molecular Pathology, Vienna, Austria) were aseptically removed and placed into ice-cold RPMI (10% FCS, 10 mM glutamine, 10 mM HEPES, 100 U/ml penicillin, and 100 g/ml streptomycin) medium. Single-cell suspensions were prepared by sieving the organs through a stainless wire mesh. Cells isolated this way were routinely 95% viable, as assessed by trypan blue exclusion assay. Thymocytes were seeded in 24-well plates (0.5 ml/well) for cytofluorometry. Unless stated differently, inhibitors were applied as a pretreatment (15 min before the oxidants, except for bongkrekic acid, where a 30-min pretreatment was used).
Cells were treated with peroxynitrite (20 µM) diluted in PBS, pH 8.9, and incubated for 3 h at 37°C. Cells treated with decomposed peroxynitrite (incubated for 20 min in PBS, pH 7.2) were used as the vehicle control. Decomposed peroxynitrite had no effect on any parameter measured. Hydrogen peroxide was diluted in PBS, pH 7.4, and added to the cells at various concentrations. In addition to authentic peroxynitrite, we tested the effect of a 3-h exposure of the cells to SIN-1 (200 µM) and the combination of pyrogallol and SNAP (PG+SNAP; 100–100 µM) to investigate the effect of continuous peroxynitrite generation.
Flow cytometry
After 3 h of incubation, thymocytes were stained with 40 nM
DiOC6(3), 1 µM JC-1, 2 µM hydroethidine (HE), and 100 nM
10-N nonyl-acridine orange (NAO) for 15 min or with fura red (5
µM) and Oregon green BAPTA-AM (3 µM) for 30 min at 37°C,
washed once with PBS, and analyzed with a FacsCalibur flow
cytometer (Becton-Dickinson, San Jose, CA). Forward and side
scatters were gated on the major population of normal size cells. In
control experiments cells were pretreated (1 h, 37°C) with 50 µM
carbonyl cyanide m-chlorophenyl hydrazone, a protonophore
that completely de-energizes mitochondria by dissipating
m.
Samples processed for annexin V-FITC/propidium iodide staining (19) were washed in PBS, and 105 cells (in 100 µl) were stained with 5 µl annexin V-FITC and 5 µg/ml propidium iodide in annexin binding buffer (10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2) at room temperature. After 15 min, 400 µl annexin binding buffer was added to the samples, which were then immediately analyzed with flow cytometry.
Measurement of mitochondrial membrane potential
The mitochondrial membrane potential was quantitated by the flow
cytometric analysis of DiOC6(3)-stained cells (12). Lipophylic cations
such as the fluorescent dyes DiOC6(3), JC-1, or rhodamine are
transported into the mitochondria by the negative mitochondrial
membrane potential and thus concentrated within the mitochondrial
matrix. Since under certain conditions, DiOC6(3) fluorescence may be
influenced by the cell membrane potential, we confirmed our results
obtained with DiOC6(3) by using JC-1, a fluorescent dye that forms
fluorescent aggregates when m decreases (20), a
process unaffected by the changes in cell membrane potential.
Determination of secondary ROI generation and mitochondrial membrane damage
Intramitochondrial generation of ROI was determined using a previously established flow cytometry technique based on the superoxide-induced conversion of the oxidant-sensitive dye, hydroethidine (HE), to ethidium (21). Mitochondrial membrane damage was determined by measuring the concentration of cardiolipin, the cellular distribution of which is restricted to mitochondria. The assay uses the fluorochrome NAO, which stoichiometrically interacts with cardiolipin (1:2); this interaction is not influenced by the mitochondrial state (12).
Electron microscopy
Thymocytes were treated with 20 µM peroxynitrite and then incubated for 3 h at 37°C. Cells were then washed with PBS and fixed with 2% glutaraldehyde in 0.1 M cacodilate buffer, pH 7.3, for 2 h at 4°C. Fixed cells were washed in 0.1 M cacodilate buffer (twice, 10 min each time) and postfixed in 1% OsO4 in 0.1 M cacodylate for 1 h. After osmication and washings, samples were incubated in 2% uranyl acetate (1 h) and then dehydrated with graded ethanols and embedded in epoxy resin. Sections (0.8 nm) were stained with lead citrate and uranyl acetate and examined with a Hitachi transmission electron microscope (Hialeah, FL).
Statistical analysis
All values in the figures and text are expressed as the
mean ± SEM of n observations (n 
4).
Datasets were examined by analysis of variance, and individual group
means were then compared with Bonferroni’s post-hoc test;
p < 0.05 was considered statistically significant.
When the results are presented as representative flow cytometric
analyses, results identical with the ones shown were obtained in at
least three different experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A characteristic feature of necrotic cell death is the loss of
plasma membrane integrity, whereas early during apoptosis
phosphatidylserine translocates from the inner to the outer plasma
membrane layer (22). Exposure of WT thymocytes to 20 µM peroxynitrite
results in phosphatidylserine exposure, as determined by annexin V-FITC
binding, and in the loss of plasma membrane integrity, as indicated by
propidium iodide uptake (Fig. 1
).
Inhibition of PARS by 3-AB and INH2BP or the absence of PARS protected
cells from the increased membrane permeability, as shown by the shift
of the annexin V-FITC/PI double-positive population toward an annexin
V-FITC single-positive population (Fig. 1). This finding coupled with
our previous observation demonstrating that PARS inhibition causes an
increased DNA fragmentation of peroxynitrite-treated thymocytes (23)
indicate that PARS inhibition diverts a subpopulation of the cells from
the necrotic toward the apoptotic pathway. Furthermore, in
peroxynitrite-treated cells in the absence of functional PARS, the
proportion of cells exhibiting neither increased annexin V-FITC binding
nor increased propidium iodide also increases consistently with
complete rescue of a smaller subpopulation of the cells from
oxidant-induced death (Fig. 1).
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m reduction is mediated by
PARS activation
Since mitochondrial dysfunction has been proposed to represent a
point of no return during cell death (12), we hypothesized that PARS
activation-related changes may act proximal to the disruption of
mitochondrial membrane potential. Thymocytes exposed to peroxynitrite
or hydrogen peroxide displayed a dose- and time-dependent decrease in
mitochondrial transmembrane potential (m; Figs. 2 and 3).
Using 3-AB (1 mM) and INH2BP (100 µM), cellular inhibitors of PARS
(24, 25), we found that both pharmacologic inhibitors of PARS abrogated
the peroxynitrite-induced m reduction (Figs. 2 and 3). Similarly, cells from PARS knockout mice were resistant to changes
in m in response to hydrogen peroxide or
peroxynitrite (Figs. 2 and 3). However, when using agents that do not
induce DNA single-strand breakage (which is the obligatory trigger of
PARS activation), such as dexamethasone, m reduction
was unaffected by inhibition of PARS (data not shown), indicating the
specific role of DNA single-strand break-induced PARS activation in
aggravating cell death.
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m reduction was
significantly (p < 0.01) reduced by 50 µM
bongkrekic acid (64.68 ± 4.9% in the absence of bongkrekic acid
vs 45.9 ± 2.1% in the presence of the inhibitor). This finding
indicates that PARS activation-induced m reduction is
mediated by pore formation to only a minor extent. Secondary ROI production and mitochondrial membrane damage are attenuated by PARS inhibition
Disruption of the mitochondrial transmembrane potential is usually
followed by increased mitochondrial ROI production and mitochondrial
membrane damage (10). We have found that peroxynitrite and hydrogen
peroxide induced a dose- and time-dependent increase in ROI production
(Figs. 4 and
5) and triggered a loss of mitochondrial
cardiolipin (Fig. 6). Both effects were
blocked by the PARS inhibitors 3-AB and INH2BP or by the PARS-negative
phenotype (
Figs. 5–7). The loss of
cardiolipin may result from the secondary ROI production and is
unlikely to be related to a direct effect of peroxynitrite, since
treatment of the cells (1 h after peroxynitrite exposure) with the
antioxidants glutathione (10 mM) and N-acetyl-cysteine (10
mM) significantly reduced secondary ROI production and the loss of
cardiolipin content (Fig. 7). Since at physiologic pH, the half-life of
peroxynitrite is <1 s, this inhibition cannot be attributed to the
scavenging of peroxynitrite itself by the antioxidants. Moreover,
antioxidant treatment also inhibited m reduction to a
small extent (Fig. 7C). Although ROI production is
considered to be the consequence of m reduction, our
finding is in line with the concept proposed by Kroemer and colleagues
that mitochondrial perturbations trigger self-amplifying vicious
circles. L-NG-methyl arginine (1
mM), an inhibitor of nitric oxide synthase, did not inhibit the
peroxynitrite-induced progressive mitochondrial membrane damage (Fig. 7), indicating that endogenous formation of nitric oxide or
peroxynitrite does not play a role in the peroxynitrite-induced
progressive mitochondrial alterations.
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). Similar to the findings with
authentic peroxynitrite, mitochondrial m reduction,
ROI production, and cardiolipin loss were reduced in thymocytes from
PARS-/- animals compared with the response in WT cells
(Table I).
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Since disruption of mitochondrial function and subsequent
oxidative stress are often followed by an elevated intracellular
Ca2+ level, we have investigated whether Ca2+
is mobilized in the cells exposed to the oxidants. Peroxynitrite caused
a dose-dependent increase in cytosolic free Ca2+ as
indicated by the decreased fluorescence of the
Ca2+-sensitive dye fura red (Fig. 8). (Similar results were obtained using
another Ca2+ sensitive dye, Oregon green BAPTA-AM,
which shows an increased fluorescence upon binding to
Ca2+.) PARS-deficient thymocytes and cells treated with the
PARS inhibitors mobilized significantly less Ca2+,
indicating the crucial role of PARS activation-induced changes in the
induction of Ca2+ efflux (Fig. 8).
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To provide morphologic evidence of mitochondrial destruction
indicated by NAO staining, we conducted electron microscopic
examinations on peroxynitrite-treated (15 µM) cells. WT thymocytes
challenged with the oxidants displayed a typical necrotic morphology
(swollen cytoplasm and organelles and decreased electron density), with
mitochondrial damage signs ranging from broken cristae to high
amplitude mitochondrial swelling, total disruption of ultrastructure,
and appearance of flocculent matrix densities. In comparison,
mitochondria of the PARS-deficient cells showed no or minor changes
(Fig. 9). Gross morphologic changes in
the mitochondria have been thought to be characteristic of necrosis.
However, recently, severe mitochondrial damage has also been found to
occur during apoptosis (26).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Disrupted mitochondrial membrane potential followed by increased ROI generation, loss of mitochondrial cardiolipin, and increased intracellular Ca2+ level have recently been described as common features of the apoptotic process. The current results demonstrate that these changes can also occur during oxidant-induced necrotic cell death. Although peroxynitrite can cause delayed apoptosis (39, 40), peroxynitrite-induced apoptosis is not attenuated by PARS inhibitors (5, 23, 41, 42). On the contrary, PARS inhibition of peroxynitrite-treated thymocytes can shift the necrotic cell death toward apoptosis, as indicated by an increased output of apoptotic parameters (DNA fragmentation and phosphatidylserine exposure) (23). In hydrogen peroxide-treated human myeloid leukemia U937 cells, inhibition of PARS has been shown to reduce necrosis, but increase apoptosis (43), whereas in a human epithelial cell line, the suppression of hydrogen peroxide-induced necrosis by 3-AB was not associated with increased apoptosis (44).
The data presented in the present study put the mode of oxidant-induced
cell death into a new perspective (Fig. 10). 1) The current data, contrary to
the previously held view, demonstrate that oxidant-induced
mitochondrial alterations are not due to a direct effect of the
oxidants on the mitochondria, but are related to an indirect mechanism
governed by PARS. 2) It is a widely held view that that necrosis is a
process that cannot be influenced by pharmacologic means, and apoptosis
is the process that is under the control of a sophisticated cellular
machinery. The present data, demonstrating protection against
mitochondrial injury by inhibition of PARS, support the view that
cellular necrosis and related mitochondrial injury indeed are governed
by an endogenous regulatory mechanism, i.e., PARS. 3) The role of PARS
in the process of apoptotic cell death is generally viewed as a
terminal effector step, whereby PARS acts as a "death substrate"
for caspases (45, 46, 47, 48). The present data demonstrate that during
oxidant-induced cell necrosis, PARS mediates an early, rather than a
delayed, effector mechanism, at a level proximal to mitochondrial
alterations.
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Necrotic cell death is an important pathway of cell death, which has direct relevance for various forms of reperfusion injury and for various forms of inflammation. Under such conditions, overwhelming oxidant production can occur, and cells die via the necrotic route. In recent in vivo experiments, pharmacologic inhibition or inactivation of PARS protected against stroke, myocardial reperfusion injury, shock, and inflammation (4, 5, 6, 7, 31). Based on the current work, we propose that prevention of mitochondrial injury and consequent cell necrosis is one of the mechanisms by which PARS inhibitors exert beneficial effects in various pathophysiologic conditions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Csaba Szabó, Division of Critical Care, Children’s Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229. E-mail address:
3 Abbreviations used in this paper: PARS, poly(ADP-ribose) synthetase; ROI, reactive oxygen intermediates; INH2BP, 5-iodo-6-amino-1,2-benzopyrone; SIN-1, 3-morpholinosidnonimine; SNAP, S-nitroso-N-acetyl-D,L-penicillamine; PG, pyrogallol; DiOC6(3), 3,3'-dihexyloxacarbocyanine iodide; HE, hydroethidine; NAO, 10-N nonyl-acridine orange; 3-AB, 3-aminobenzamide; WT, wild-type.
Received for publication March 5, 1998. Accepted for publication June 3, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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