Bioscience Reports, Vol. 17, No. 3, 1997
REVIEW
Mitochondrial Oxygen Radical Formation
during Reductive and Oxidative Stress to
Intact Hepatocytes
John J. Lemasters2,3 and Anna-Liisa Nieminen1
Received January 6, 1997; accepted January 30, 1997
After simple respiratory inhibition, glycolytic substrates prevent cell death by providing an alternate
source of cellular ATP. When mitochondrial uncoupling ensues, the uncoupler-stimulated mitochondrial ATPase hydrolyzes ATP formed by glycolysis and protection is lost. Electron transfer
components abnormally reduced by respiratory inhibition, especially ubisemiquinone, react directly
with oxygen to form toxic radicals. Mitochondria also generate reactive oxygen species after exposure
to oxidant chemicals. A consequence is onset of the mitochondrial permeability transition, which leads
to uncoupling, cellular ATP depletion and loss of viability. Thus, mitochondria are both a source and
a target of toxic oxygen radicals in cell injury.
KEY WORDS: Hepatocyte; oxygen radical; oxidative stress; reductive stress
ABBREVIATIONS: MPT; mitochondrial permeability transition; t-BuOOH, tert-butylhydro
peroxide; and TMRM, tetramethylrhodamine methylester.
INTRODUCTION
Tissue damage from anoxia and ischemia is a major cause of human disease. Such
injury may be aggravated by reoxygenation, and much evidence supports the
concept that reactive oxygen species (e.g., H2O2, OH' and O2) aggravate cell
injury after reoxygenation and reperfusion (1-3). A knowledge of the subcellular
sources of these reactive oxygen species is therefore of considerable importance
for understanding the pathophysiology of ischemia/reperfusion injury and for
developing strategies to combat this injury.
Anoxia and reoxygenation in aerobic tissues present contrasting extremes of
Department of Anatomy, Case Western Reserve University, Cleveland, Ohio.
Laboratories for Cell Biology, Department of Cell Biology & Anatomy, CB#7090, 236 Taylor Hall,
University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.
3 To whom correspondence should be addressed.
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2
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0144-8463/97/0600-028l$12.50/0©
1997 Plenum Publishing Corporation
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oxidation and reduction of electron transfer components. During hypoxia and
ischemia, cellular oxidation-reduction components that are normally highly
oxidized in the aerobic state, such as NAD + , ubiquinone and the respiratory
cytochromes, become virtually completely reduced. This condition of "reductive
stress" may promote otherwise forbidden reactions between molecular oxygen
and reduced electron transfer components to generate reactive oxygen species
when tissues are reoxygenated or reperfused. Subsequent to reoxygenation, a
contrasting oxidative stress may ensure that is characterized by abnormal
oxidation of NAD(P)H, glutathione and other compounds needed for the
reductive detoxification of reactive oxygen species and their toxic reaction
products. Here, we review recent evidence showing a role for mitochondria as a
source and target of reactive oxygen generation in cell injury during reductive and
oxidative stress.
REDUCTIVE STRESS AND ACCELERATED CELL KILLING AT THE
BORDER OF ANOXIC AND HYPOXIC TISSUE
Although ischemia is often defined as an absence of tissue blood flow, a
better definition is a decrease of blood flow to the point that oxygen delivery no
longer meets tissue oxygen demand. At this point, the clinical signs and symptoms
of ischemia occur and the danger of infarction (cell death) develops. During
low-flow ischemia, tissue oxygen concentration can be quite heterogeneous. Cells
nearest arterioles bringing in fresh blood have adequate oxygen to maintain
normal mitochondrial function and ATP formation. Tissue further removed from
the arterioles are anoxic and lose mitochondrial ATP formation by oxidative
phosphorylation.
In the liver during perfusion at low flow rates, discrete circumscribed areas of
anoxia develop in downstream perivenular (pericentral) regions of the liver lobule
(4). Cells in these regions rapidly develop changes characteristic of hypoxic stress,
such as surface bleb formation, and ultimately lose viability if low flow is
maintained for a long enough time (4-6), By contrast, upstream periportal
regions of the liver lobule remain normoxic, do not develop structural abnormalities, and retain viability.
Surprisingly, loss of cell viability during low-flow hypoxia does not occur first
in cells most downstream from the oxygen source. Rather, more upstream
midzonal cells are the first to lose viability. Even when livers are perfused in a
retrograde fashion, midzonal cells are still first to lose viability. Subsequent to
midzonal cell killing, loss of cell viability progresses into the downstream
regions—pericental regions during anterograde perfusions and periportal regions
during retrograde perfusions. Thus, the regions most vulnerable to injury during
low-flow ischemia reside at the border of anoxic and normoxic tissue.
Early midzonal cell killing is retarded by antioxidants, such as allopurinol (an
inhibitor of xanthine oxidase), cyanidanol (a hydroxyl radical scavenger) and
desferal (an inhibitor of iron-catalyzed hydroxyl radical formation from superoxide and hydrogen peroxide) (6,7). This finding suggests that reactive oxygen
Mitochondrial Oxygen Radical Formation
283
species are involved in midzonal hypoxic liver injury during low-flow states. A
model was proposed based on principles outlined by McCord (8) to explain this
injury: (1) Low-flow ischemia to liver leads to formation of anoxic zones around
the central veins (terminal hepatic venules) that are contiguous with normoxic
regions around the portal tracts. (2) In the anoxic pericentral regions, respiration
ceases, electron transfer components becomes maximally reduced, and ATP
levels decline. Additionally, hypoxanthine accumulates from adenine nucleotide
degradation. (3) At the border zone between anoxic pericentral tissue and
normoxic periportal cells, enough oxygen may be present to support superoxide
formation by the xanthine oxidase reaction or by reaction of molecular oxygen
with abnormally reduced electron transfer components. Additionally, the anoxic
border may oscillate due to local microcirculatory disturbances, causing cycles of
reoxygenation in the border zone. (4) As cells die, the anoxic border progresses
downstream to expose new tissue to this oxygen-dependent injury. Although
anoxia is sufficient to kill cells without involvement of reactive oxygen species,
toxic oxygen radicals generated uniquely at the border of anoxic and normoxic
tissue accelerate the progression of lethal cell injury. Thus, antioxidants have a
partially protective effect, delaying but not preventing cell death. Generation of
reactive oxygen species at anoxic border regions may explain autopsy findings of
midzonal hepatic necrosis in patients succumbing to congestive heart failure (9).
This mechanism may also explain why subendocardial regions of the heart are
most vulnerable to myocardial infarction. During myocardial ischemia, subendocardial regions are the border between totally anoxic midmyocardium and
endocardial regions that remain aerobic by diffusion of oxygen from blood
contained in the heart chambers. Thus, oxygen radicals are likely formed in the
subendocardial regions, accelerating the progression to infarction.
MITOCHONDRIA AS A SOURCE OF REACTIVE OXYGEN SPECIES
DURING CHEMICAL HYPOXIA IN HEPATOCYTES
In anoxia, respiration and ATP formation by oxidative phosphorylation are
inhibited due to the lack of oxygen. As a consequence, mitochondrial electron
transfer components become maximally reduced, and cellular ATP becomes
profoundly depleted. To simulate this reductive stress and ATP depletion, we
have extensively used a model of "chemical hypoxia" that employs cyanide to
inhibit cytochrome c oxidase (Complex IV), the same enzyme whose activity is
lost in oxygen deprivation (10). Additionally, we inhibit glycolysis with iodoacetate to prevent glycolytic ATP formation, since endogenous glycolytic substrates,
such as glycogen and various sugars, are rapidly depleted in ischemia. During
chemical hypoxia, ATP is depleted by more than 95% within 5 minutes, and
hepatocytes lose viability over a time course that parallels cell death in the anoxic
perfused liver (11). The advantage of chemical hypoxia is that it circumvents the
severe technical requirements for maintining strict anaerobiosis during cell
manipulations that would otherwise cause reoxygenation. In most respects, but
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not all, cell injury from chemical hypoxia closely resembles that caused by anoxia
(12).
An important difference between true anoxia and chemical hypoxia is the
fact that oxygen remains present during chemical hypoxia, although it no longer
participates in normal mitochondrial respiration. As a consequence, peroxidation
occurs during chemical hypoxia (7,13). This peroxidation is documented by the
reaction of organic peroxides with nonfluorescent dichlorofluoresciVz to form
highly fluorescent dichlorofluorescein, which is easily measured fluorometrically.
Peroxidation during chemical hypoxia is virtually completely eliminated by the
removal of oxygen. Importantly, this generation of reactive oxygen species
actually contributes to the rate of cell killing, since anoxia and antioxidants like
mannitol, desferal and cyanidanol delay onset of cell death during chemical
hypoxia (7,13).
Lipid peroxidation also occurs when azide, another Complex IV inhibitor, is
employed to inhibit respiration (13). Myxothiazol, an inhibitor of the cytochrome
bcl complex (Comples III), decreases peroxidation stimulated by cyanide and
azide. Myxothiazol also delays the onset of cell death induced by these Complex
IV inhibitors.
Myxothiazol blocks oxidation of ubiquinol to ubisemiquinone by the Rieske
iron-sulfur central of Complex III. Ubisemiquinone formed by this reaction can
react with molecular oxygen to form superoxide (14,15). Since myxothiazol
decreased cell killing and peroxidation in hepatocytes exposed to the Complex IV
inhibitors, we conclude that Complex III is a significant source of reactive oxygen
species during reductive stress. Isolated mitochondria have long been known to
generate reactive oxygen (14-17). However, these experiments with hepatocytes
are the first to show that reactive oxygen species are formed by mitochondria
within intact cells and that these mitochondrially derived oxygen radicals actually
contribute to cell injury.
Inhibition of Complex III with myxothiazol does not completely inhibit
formation of reactive oxygen species, and some hydroperoxides continue to form
even in the presence of myxothiazol. Thus, other sources of reactive oxygen
species also exist. Possible sources include Complex I of the respiratory chain,
cytosolic enzymes such as cycloxygenase and xanthine oxidase, and microsomal
cytochrome P-450.
For mitochondria to become a source of reactive oxygen species during
hypoxic injury, special conditions are necessary. In absolute anoxia, electron
transfer components are reduced, but oxygen radical formation is not possible,
since oxygen is absent. After reperfusion, mitochondrial oxygen radical formation
may ensue (18), but will end rapidly as the electron carriers of the respiratory
sequence are reoxidized. this may limit total oxygen radical generation and
ensuing injury after reperfusion. However, under conditions of low-flow or
intermittent ischemia, cycles of anoxia and reoxygenation may occur repeatedly,
leading to increased formation of reactive oxygen species and more aggravated
tissue injury. Coincident damage to the respiratory complexes, especially
Complex III, may also predispose tissue mitochondria to oxygen radical formation (19).
Mitochondrial Oxygen Radical Formation
285
ROLE OF MITOCHONDRIAL FAILURE IN CYTOTOXICITY
The importance of mitochondrial dysfunction in cytotoxicity can be assessed
experimentally by the ability of glycolytic substrates to rescue cells from loss of
viability. Glycolysis is an alternative ATP source that can partially replace ATP
production lost after mitochondrial injury and prevent cell killing. Glucose and
endogenous glycogen are excellent glycolytic substrates for most cells that prevent
anoxic cell killing. In liver, however, glucose is poorly metabolized because of the
absence of hexokinase. Fructose is a much superior glycolytic substrate, and
fructose but not glucose prevents hepatocellular death after anoxia, cyanide, and
oligomycin (11,20-22). In aerobic hepatocytes, fructose at high concentrations
causes ATP to decrease becaue of ATP consumption in the fructokinase reaction.
However, in anoxic hepatocytes, fructose increases ATP (Fig. 1) (20,22). ATP
needs not rise to normal levels to achieve protection, since only a fraction of
normal ATP is sufficient to prevent onset of irreversible cell death. Fructose also
prevents lethal toxic injury to hepatocytes caused by various oxidant chemicals
(21). These findings implicate mitochondria as a target of toxicity in oxidative
stress.
When toxic stress causes uncoupling of oxidative phosphorylation, then
glycolysis alone is insufficient to protect against cell killing, because the
uncoupler-stimulated mitochondrial ATPase consumes the ATP generated by
Fig. 1. Role of mitochondrial inhibition and uncoupling in
cytotoxicity—Anoxia and respiratory inhibition with cyanide cause
mitochondrial depolarization and cellular ATP depletion. Glycolytic
substrates, such as fructose, reverse ATP depletion and restore
mitochondrial membrane potential (AW), rescuing cells from lethal
injury. Uncoupling of mitochondria by the MPT or with a protonophoric uncoupler like CCCP collapses &*V, stimulates the mitochondrial F]F0-ATPase and abolishes fructose cyto-protection.
Oligomycin inhibits the mitochondrial ATPase and blocks futile
uncoupler-stimulated ATP hydrolysis, restoring cellular ATP and
rescuing cells without recovery of A1*. Adapted from (22).
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glycolysis (Fig. 1). When uncoupling is involved, inhibition of the mitochondrial
ATPase with oligomycin has the paradoxical effect of protecting cells from
cytotoxicity (21,22). In the absence of a glycolytic substrate, oligomycin is toxic to
hepatocytes. By contrast in the presence of glycolytic substrate, oligomycin
prevents cytotoxicity caused by uncouplers of oxidative phosphorylation (Fig. 1).
The synergism of oligomycin and glycolytic substrate shows that the cytotoxicity
of classical protonophoric uncouplers like CCCP and FCCP is mediated by
activation of the mitochondrial ATPase. Other ionophores cause injury by the
same mechanism. In particular, the calcium ionophore A23187, often used as a
model of calcium-dependent cytotoxicity, causes lethal hepatocellular injury that
is prevented by fructose in combination with oligomycin (21,23).
MITOCHONDRIAL DYSFUNCTION IN OXIDATIVE STRESS
ferf-Butylhydroperoxide (r-BuOOH) is a short chain analog of the lipid
hydroperoxides formed from peroxidation reactions during reductive stress (see
above), oxidative stress, ischemia/reperfusion and normal metabolism. f-BuOOH
is detoxified by glutathione peroxidase to yield f-butanol and oxidized glutathione
(24,25). In the presence of excess f-BuOOH, NADPH and NADH become
secondarily oxidized via action of glutathione reductase and the mitochondrial
NADPH-NAD + transhydrogenase. Oxidation of glutathione and pyridine nucleotides constitutes a condition of oxidative stress that promotes the accumulation
of lipid peroxides and possibly other toxic metabolites.
Mitochondria are a major target of the cytotoxicity of f-BuOOH (21,26).
The mechanism underlying mitochondrial injury in hepatocytes exposed to
/-BuOOH depends on the dose of /-BuOOH used. At the lowest concentrations
causing acute cytotoxicity (25-50 piM), fructose prevents cell killing almost
completely. Rescue by fructose implies that f-BuOOH at these concentrations
inhibits mitochondrial ATP formation. At higher concentrations of f-BuOOH
(100-300/>,M), fructose becomes poorly effective. However, fructose plus oligomycin improves viability, implying that the higher concentration of t-BuOOH
causes mitochondrial uncoupling (see Fig. 1). At still higher concentrations of
t-BuOOH (1 mM), glycolysis becomes strongly inhibited. Thus at very high
t-BuOOH concentrations, all significant cellular sources for ATP production
become blocked, and no combination of glycolytic substrate and ATPase inhibitor
can prevent cell killing.
ROLE OF THE MITOCHONDRIAL PERMEABILITY TRANSITION IN
TOXICITY DURING OXIDATIVE STRESS
In isolated mitochondria, a wide variety of oxidant chemicals induce greatly
increased non-specific permeability of the inner mitochondrial membrane to ions
and solutes of molecular weight less than about 1500 Daltons. This mitochondrial
permeability transition (MPT) causes mitochondrial swelling, membrane depolarization and uncoupling of oxidative phosphorylation (reviewed in 27). The
opening of a high conductance permeability transition pore causes the MPT.
Mitochondrial Oxygen Radical Formation
287
Involvement of the MPT in the toxicity of t-BuOOH was first suggested by
the observation that cyclosporin A and trifluoperazine, inhibitors of the MPT,
delay lethal injury to hepatocytes caused by 100 /xM t-BuOOH (28,29). Onset the
MPT after r-BuOOH was then directly demonstrated in single intact hepatocytes
by laser scanning confocal microscopy (30). Calcein is a fluorophore with a
molecular weight of 623 that can be loaded into the cytosol but not the
mitochondria of cultured hepatocytes. Confocal fluoresence images of greenfluorescing calcein in these cells show a diffuse cellular fluoresence containing
small round and oblong voids that correspond exactly to mitochondria labeled
with red-fluorescing tetramethylrhodamine methylester (TMRM), a cationic
fluorophore that accumulates electrophoretically into mitochondria in response to
their negative membrane potential. After exposure to t-BuOOH, mitochondria in
hepatocytes rapidly lose their TMRM fluorescence, indicating depolarization.
Simultaneously, calcein fluorescence enters the dark voids and equilibrates
between the cytosolic and mitochondrial spaces (Fig. 2). Since calcein is an
organic polyanion that is impermeable to mitochondria under normal conditions,
Fig. 2. Onset of the mitochondrial permeability transition in cultured hepatocytes induced by
t-BuOOH—A cultured hepatocyte was loaded with TMRM and calcein. The red fluorescence of
TMRM and green fluorescence of calcein were imaged by laser scanning confocal microscopy. In the
baseline image (A), note that TMRM-labeled mitochondria correspond to dark voids in the calcein
fluorescence. After addition of 100 ^M t-BuOOH, calcein redistributed from the cytosol into the
mitochondria and TMRM fluorescence was lost from the mitochondria (B, C), events signifying the
onset of the MPT. These changes preceded cell death, documented by loss of cytosolic calcein
fluorescence (D). Adapted from (30).
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Fig. 3. Scheme of mitochondrial events leading to
onset of the mitochondrial permeability transition during oxidative stress—t-Butylhydroperoxide stimulates
oxidation of mitochondrial NAD(P)H, formation of
reactive oxygen species (ROS) in mitochondria and an
increase of mitochondrial free Ca2+, events well documented in isolated mitochondria to promote onset of
the MPT. Onset of the MPT causes loss of mitochondrial membrane potential (AW), cellular ATP depletion
and cell death, since trifluoperazine (TFZ) blocks onset
of the MPT, mitochondrial depolarization and cell
killing. NAD(P)H oxidation, formation of reactive oxygen species and increased Ca2* all contribute to onset
of the MPT, because cell killing is delayed or prevented
by /3-hydroxybutyrate (BHB), antioxidants (DPPD and
desferal) and intracellular Ca2* chelation with BAPTA.
the abrupt movement of calcein into mitochondria signifies onset of the MPT.
Trifluoperazine, which blocks onset of the MPT in isolated micochondria, also
blocks the MPT in situ after t-BuOOH and prevents the ensuing mitochondrial
depolarization, ATP depletion and cell death (26,30).
Factors that cause onset of the MPT in isolated mitochondria also appear to
promote the MPT in intact hepatocytes after t-BuOOH. Specifically, oxidative
stress induced by t-BuOOH causes rapid oxidation of mitochondrial pyridine
nucleotides, followed by an increase of mitochondrial free Ca2+ and generation of
mitochondrial reactive oxygen species (31,32). Subsequently, onset of the MPT
occurs, the mitochondria uncouple, and the hepatocytes lose viability (Fig. 3).
Several experimental manipulations retard onset of the MPT and subsequent cell
death. /3-Hydroxybutyrate increases the content of mitochondrial NADH and
delays t-BuOOH-induced cell killing. The antioxidants, diphenylphenylenediamine and desferal, block NAD(P)H oxidation, formation of mitochondrial
oxygen radicals, onset of the MPT, and subsequent mitochondrial depolarization
and cell killing. The intracellular Ca2+ chelators, BAPTA-AM and Quin 2-AM,
also prevent lethal injury to hepatocytes from t-BuOOH. Thus, mitochondrial
NAD(P)H oxidation, mitochondrial oxygen radical formation and increased
mitochondrial free Ca2+ not only precede the MPT but actually promote onset of
the MPT in hepatocytes exposed to t-BuOOH, leading ultimately to oxidative cell
killing (Fig. 3).
CONCLUSION
In conclusion, mitochondrial dysfunction is a critical feature of toxic, hypoxic
and reperfusion injury. Under conditions of reductive and oxidative stress,
mitochondria become both a source and target of reactive oxygen species. A
Mitochondria] Oxygen Radical Formation
289
novel mechanism causing mitochondrial injury is onset of the MPT. Although
beyond the scope of this short review, the mitochondrial permeability transition is
now implicated in a range of other pathophysiological states, including
ischemia/reperfusion injury, calcium ionophore toxicity, ethanol hepatotoxicity,
bite acid toxicity, doxorubicin cardiotoxicity, excitotoxicity, Reye's syndrome, and
apoptosis (23,33-40). Indeed, mitochondrial dysfunction and onset of the
mitochondrial permeability transition may be a typical rather than an exceptional
event in the progression to loss of cell viability in necrosis and apoptosis. These
recent developments illustrate how knowledge of basic mitochondrial mechanisms
leads to a better understanding of pathophysiologic processes.
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