FUNDAMENTAL AMD APPLIED TOXICOLOGY 3 8 , 2 3 - 3 7 (1997)
ARTICLE NO. FA972320
SYMPOSIUM OVERVIEW
Mitochondria-Mediated Cell Injury1
K. B. Wallace,* J. T. Eells.t V. M. C. Madeira,* G. Cortopassi,§ and D. P. Jones1
*Department of Biochemistry and Molecular Biology, University of Minnesota, Duluth, Minnesota 55812; f Department of Pharmacology
and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; tCenter for Neurosciences, University of Coimbra,
Coimbra, Portugal; ^Department of Molecular Biosciences, University of California, Davis, California 95616;
and ^Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322
Received April 11, 1997; accepted April 11, 1997
interference with mitochondrial respiration results in proMitochondria-Mediated Cell Injury. Wallace, K. B., Eells, J. T., found bioenergetic deficits leading to the loss of various
Madeira, V. M. C, Cortopassi, G., and Jones, D. P. (1997). Funfunctions vital to the survival of the cell and, ultimately, the
dam. Appl. Toxicol. 38, 23-37.
organism. An emerging sentiment is that mitochondria serve
Mitochondria have long been known to participate in the pro- as a common "target," either directly or indirectly, for
cess of cell injury associated with metabolic failure. Only recently, chemical-induced toxicities. This contrasts with mitochonhowever, have we come to appreciate the role of mitochondria as drial dysfunction being simply an epiphenomenon that modprimary intracellular targets in the initiation of cell dysfunction. ulates other forms of cytotoxicity. To emphasize this subject
In addition to ATP synthesis, mitochondria are also critical to
of rapidly growing interest, a symposium on mitochondriamodulation of cell redox status, osmotic regulation, pH control,
and cytosolic calcium homeostasis and cell signaling. Mitochon- mediated cell injury was organized for the 35th Annual
dria are susceptible to damage by oxidants, electrophiles, and Meeting of the Society of Toxicology. The following overlipophilic cations and weak acids. Chemical-induced mitochon- view provides a brief background on mitochondrial function
drial dysfunction may be manifested as diverse bioenergetic disor- and presents examples of major pathophysiological changes
ders and considerable effort is required to distinguish between in mitochondrial metabolism, respiration, ion transport, and
mechanisms involving critical mitochondrial targets and those in genetic material that occur as a consequence of chemical
which mitochondrial dysfunction is secondary and plays only a intoxication.
modulatory role in cell injury. The following paragraphs review
With the exception of erythrocytes, all cell types are vula few important examples of chemical-induced cytotoxic responses that are manifested as interference with mitochondrial nerable and the symptomology of sublethal mitochondrial
metabolism and bioenergetics, gene regulation, or signal transduc- poisoning is rather vague; however, certain degrees of organtion in the form of apoptosis and altered cell cycle control. Greater otropy are observed with more severe cases of poisoning.
understanding of the molecular mechanisms of mitochondrial bi- In addition to pharmacokinetic considerations, important deoenergetics, ion regulation, and genetics will lead to numerous terminants of any organ-specific effects include the number
additional examples of mitochondria-mediated cell injury, revealand size of the mitochondria within each cell type along
ing important new insight regarding the prediction, prevention,
diagnosis, and treatment of chemical-induced toxic tissue injury. with tissue-specific differences in mitochondrial enzyme
profiles and membrane transporter systems. Balanced against
C 1997 Sodcty of Toifcotogy.
the metabolic demand on the organ, these factors determine
what is referred to as a "bioenergetic reserve," which varies
among tissues and defines a threshold that each tissue is
Mitochondria are essential to supporting the energy-de- capable of tolerating. Another important factor is the reliance
pendent regulation of assorted cell functions, including inter- of each tissue on mitochondrial oxidative phosphorylation
mediary metabolism, ion regulation and other active trans- to supply the needed bioenergetic equivalents. Muscle and
port processes, cell motility, and cell proliferation. De- nervous tissue possess little glycolytic capability and rely
pending on the tissue type and metabolic state, cells derive almost exclusively on oxidative phosphorylation. Combined
up to 95% of their energy through oxidative phosphorylation; with their high bioenergetic demands, these tissues are particularly vulnerable to mitochondrial dysfunction. Common
early
signs and symptoms of mitochondrial poisoning reflect
1
Symposium held at the 35th Annual Meeting of the Society of Toxicolthis and include fatigue, cardiac arrhythmias and contractile
ogy, Anaheim, CA.
23
0272-0590/97 $25.00
Copyright O 1997 by the Society of Toxicology.
All rights of reproduction in any form reserved.
24
WALLACE ET AL.
failure, neuromuscular fasciculations, and central nervous
system (CNS) depression.
Classic and long-recognized examples of mitochondrial
poisons are the inhibitors (e.g., rotenone, antimycin A, and
cyanide) and uncouplers [e.g., 2,4-dinitrophenol and carbonyl cyanide (m-chloro)- or (/?-trifiuoromethoxy)-phenylhydrazone, CCCP and FCCP, respectively) of oxidative
phosphorylation. In each case, exposure causes depolarization of mitochondrial membrane potential and inhibition of
ATP synthesis. Hallmarks of poisonings include either hypoxemia or cytotoxic hypoxia, acceleration of anaerobic glycolysis, and lactic acidosis. If not resolved, interference with
mitochondrial respiration leads to a loss of cell ATP and
high-energy phosphates and, ultimately, to cell death (Lemasters et al, 1987).
One mode of mitochondria-mediated cell injury that has
received considerable attention and support is the liberation
of partially reduced species of molecular oxygen. Even under
well-coupled conditions, as much as 2 - 4 % of the reducing
equivalents escape the respiratory chain to liberate superoxide anion free radicals and hydrogen peroxide. There are
also numerous examples wherein alternate electron acceptors
(in the stead of dioxygen) are reduced on the respiratory
chain to form unstable intermediates that shuttle the unpaired
electrons to molecular oxygen to complete a redox cycle.
The ultimate products of this redox cycling are oxygen free
radicals. Regardless of the electron acceptor, the limiting
factor for free radical generation is the availability of reducing equivalents on the respiratory chain (Boveris and
Chance, 1973). Accordingly, the more reduced are the respective electron transport proteins, the greater is the rate of
generation of oxygen free radicals. Examples of agents that
enhance the redox potential are inhibitors of the respiratory
chain, such as rotenone, antimycin A, and cyanide. Adding
these to respiring mitochondria results in the stimulation of
free radical generation, which is oftentimes implicated in the
mechanism of oxidative cell injury caused by these agents.
The paradigm for poisonings by respiratory inhibitors and
uncouplers of oxidative phosphorylation is well established;
however, only recently have we come to appreciate that
mitochondrial bioenergetics is far more complex and that
mitochondria contribute more to cell sustenance than simply
ATP synthesis. The multifaceted role of mitochondria in cell
homeostasis is rooted in the protonmotive force ( A / J H + ) .
Active extrusion of protons from the mitochondrial matrix,
which is coupled to the transport of electrons down a gradient
of univalent redox potentials, creates both a chemical (pH)
and electrical ( A * ) potential available to perform bioenergetic work. In addition to providing the driving force for
ATP synthesis, this electrochemical potential also provides
the energy for phosphate uptake; supports the transmembrane exchange of a number of intermediary metabolites,
including reducing substrates, fatty acids, and amino acid
NADH
\
Electron Transport Chain
FADH.
H2O
/
AuH+sAT-ZApH
ATPsynthase
ATP/ADP exchange
Ca 2+ uptake
protein import
GluH/Asp
Pi uptake
Ca2+/2H -i
Na+/2H +
K+/H •
FIG. 1. The mitochondrial protonmotive force supports a variety of
both bioenergetic and membrane transport processes.
intermediates for aerobic metabolism, the exchange of matrix ATP for cytosolic ADP; and supplies the energy required
for regulating mitochondrial calcium concentration and for
importing proteins encoded by the nuclear genome (Fig. 1).
The full significance of this shared bioenergetics is manifested at several levels: (1) The transmembrane movement
of metabolites, ions, or proteins represents a depolarizing
current that competes with the ATP synthase for the protonmotive force. Accordingly, the spectrum of mitochondrial
poisons is much more broad than simply those classic agents
that interfere directly with the electron transport process or
inhibit ATP synthase. Interference with these intricately regulated membrane transport and homeostatic processes is implicated in the manifestation of mitochondrial dysfunction
and cell injury caused by a number of xenobiotic agents
(Jones and Lash, 1993). (2) The competitive electrophoretic
transport serves as the basis for implicating mitochondria as
important transducers in bioenergetic cell signaling pathways. For example, it is widely speculated that mitochondria
participate in the mechanism of calcium signaling. Transient
increases in cytosolic calcium are mirrored by increases in
mitochondrial calcium and episodic depolarizations of mitochondria. Associated with this are oscillating rates of oxidative phosphorylation. Denton and McCormack (1990) provide compelling kinetic evidence for the regulation of mitochondrial respiration by transient changes in cytosolic
calcium, which is manifest as altered activities of calciumdependent rate-limiting dehydrogenase enzymes within the
matrix. A comparable paradigm for an important influence
of cytosolic pH on mitochondrial bioenergetics can also be
postulated (Nieminen et al., 1990).
Finally, since tightly coupled and efficient respiration relies on the physical integrity of the electron transport chain
and associated membrane transport proteins, mitochondrial
25
MITOCHONDRIA-MEDIATED CELL INJURY
dysfunction may also reflect improper assembly or orientation of the individual components within the inner and outer
membranes. Not only is mitochondrial respiration susceptible to changes in the lipid phase, but it also relies on the
effective transcription and translation of both the nuclear
and mitochondrial genomes. All of the membrane transport
proteins as well as all but 13 of the more than 80 components
of the electron transport chain are nuclear transcripts. Accordingly, it is essential that the mechanisms consisting of
leader sequences and chaperone molecules that function in
the transport, insertion, and assembly of nuclear transcripts
within the mitochondria are fully operational. Furthermore,
since the mitochondrial transcripts are exclusive to the mitochondrial genome, mutations to the mitochondrial DNA run
a high risk for infidelity of the electron transport chain. There
is a rapidly growing literature describing the accumulation
of both deletions and point mutations to the mitochondrial
DNA that are associated with the aging process. From this
has evolved a theory for "mitochondrial aging" wherein
mutations of the mitochondrial DNA result in dysfunctional
transcripts and, thus, infidelity to the respiratory chain (Shigenaga et al., 1994). The dysfunctional electron transport,
in turn, leads to greater rates of free radical liberation and
accelerated rates of mitochondrial mutations, resulting in a
futile, feed-forward homicidal cycle of events that many
authors have associated with the progressive deterioration of
the tissues with aging. The numerous accounts of xenobioticinduced accumulation of mitochondrial mutations alerts us
to the fact that this mitochondrial aging is also influenced
by the exposure history of the individual, risking the occurrence of chemical-induced premature "mitochondrial senility," which is manifested as an accelerated decline in tissue
bioenergetics and loss of cell function.
It becomes obvious that mitochondrial bioenergetics is a
complex and highly integrated process and that mitochondria
serve a very important, if not central, role in the mechanisms
of toxic tissue injury. The following examples illustrate major pathways of mitochondria-mediated toxicity, namely, the
direct inhibition of energy production by formate in methanol toxicity, the uncoupling of oxidative phosphorylation by
phenolic herbicides, activation of the catastrophic permeability transition by oxidants, and disruption of the mitochondrial genome by the accumulation of deletions and point
mutations. Although these examples are not comprehensive,
they illustrate the central role of mitochondria in mechanisms
of chemical-induced toxic tissue injury.
Disruption of Retinal Mitochondrial Function by Formate
in Methanol Toxicity (J. T. Eells)
Methanol is an important public health and environmental
concern because of its selective neurotoxic actions on the
retina and optic nerve. Blindness or serious visual impairment is a well-documented effect of methanol intoxication
Methanol Metabolism to Formic Acid
Y
Formic Acid Accumulation
Y
Inhibition of Cytochrome Oxidase
Y
ATP Depletion
Y
Disruption of ATP-Dependent Processes
Y
Neuronal and Glial Cell Dysfunction
Y
Neuronal and Glial Cell Death
FIG. 2. Postulated mechanism of formic acid toxicity.
(Roe, 1955). Although methanol has been recognized as a
human neurotoxin for more than a century, the mechanisms
responsible for the toxic actions of this agent on retinal
and optic nerve function are not understood (Tephly and
McMartin, 1984; Eells, 1992). Methanol is one of the most
commonly used organic solvents and is accessible to the
public in a variety of products. Furthermore, methanol is
being developed as an alternative fuel and energy source
throughout the world, greatly expanding the potential for
accidental exposure and underscoring the importance of understanding its toxicity (Kavet and Nauss, 1990).
Formic acid is the toxic metabolite of methanol responsible for the visual impairment and blindness associated with
methanol poisoning (Jacobsen and McMartin, 1986; Hayreh
et ai, 1980; Eells, 1991; Murray et al., 1991). Formic acid
has been hypothesized to produce retinal and optic nerve
toxicity by disrupting mitochondrial energy production (Fig.
2) (Martin-Amat et al, 1977; Sharpe et al., 1982). Formic
acid has been shown in vitro to inhibit the activity of cytochrome oxidase, a vital component of the mitochondrial electron transport chain involved in ATP synthesis (Nicholls,
1975). Inhibition occurs subsequent to the binding of formic
acid with the ferric heme iron of cytochrome oxidase, the
apparent inhibition constant being between 5 and 30 rrtM
(Nicholls, 1975). Concentrations of formate present in the
blood and tissues of methanol-intoxicated humans, nonhuman primates, and rodent models of methanol intoxication
are within this range (Sejersted et al., 1983; Martin-Amat
etal, 1911; Eells, 1991).
Studies conducted in methanol-sensitive rodent models
have revealed abnormalities in retinal and optic nerve function and morphology, consistent with the hypothesis that
formate acts as a mitochondrial toxin (Fig. 3). In these ani-
2ft
WALLACE ET AL.
Formic Acid
FIG. 3. Bioenergetic, neurofunctional, and morphologic manifestations of formic acid toxicity. (A) Diagram of a retinal photoreceptor cell depicting
the inhibition of cytochrome oxidase by formic acid. (B) Reduction in the electroretinogram produced by formic acid in methanol-intoxicated rats. (C)
Swollen and disrupted mitochondria (arrows) in photoreceptor cells in human methanol intoxication.
mal models, formate oxidation has been selectively inhibited
by dietary (Lee, et al, 1994) or chemical (Eells et al, 1981)
depletion of folate coenzymes, thus allowing formate to accumulate to toxic concentrations following methanol administration. Methanol-intoxicated rats developed formic acidemia, metabolic acidosis, and visual toxicity analogous to
the human methanol poisoning syndrome (Eells, 1991; Murray et al, 1991; Lee et al, 1994). Visual dysfunction was
measured as reductions in the flash-evoked cortical potential
(FEP) and electroretinogram (ERG). The FEP is a measure
of the functional integrity of the primary visual pathway
from the retina to the visual cortex and the ERG is a global
measure of retinal function in response to illumination (Creel
et al, 1970; Dowling, 1987). The FEP was progressively
diminished in methanol-intoxicated rats, indicative of a disruption of neuronal conduction along the primary visual
pathway (Eells, 1991). ERG analysis in methanol intoxicated
rats revealed a significant early deficit in b-wave amplitude,
followed by a temporally delayed lesser reduction in a-wave
amplitude (Fig. 3B) (Murray et al, 1991). The b wave of
the ERG is generated by depolarization of the MUller glial
cells and reflects synaptic activity at the level of the bipolar
cells (Dowling, 1987). The b wave of the ERG is extremely
sensitive to conditions that interfere with retinal energy metabolism and is reduced or abolished following brief ischemia or the administration of metabolic poisons (Bresnick,
1989; Dowling, 1987). Both FEP and ERG alterations occurred coincident with the accumulation of blood formate,
indicative of a causal relationship between formate-induced
metabolic and visual disturbances. Similar ERG reductions
have been reported in methanol-intoxicated primates (Inge-
mansson, 1983) and in human methanol intoxication (Ruedeman, 1961; Murray et al, 1996).
In addition to neurofunctional changes, bioenergetic and
morphological alterations indicative of formate-induced disruption of retinal energy metabolism have been documented
in experimental methanol intoxication (Murray et al, 1991;
Eells et al, 1995; Gamer et al, 1995). Morphological studies, coupled with cytochrome oxidase histochemistry, revealed generalized retinal edema, photoreceptor and retinal
pigment epithelium vacuolation, mitochondrial swelling, and
a reduction in cytochrome oxidase activity in photoreceptor
mitochondria from methanol-intoxicated rats (Murray et al,
1991; Eells et al, 1995, 1996b). The most striking structural
alterations observed in the retinas of methanol-intoxicated
rats was vacuolation and mitochondrial swelling in inner
segments of the photoreceptor cells (Murray et al, 1991).
Photoreceptor mitochondria from methanol-intoxicated rats
exhibited expanded or disrupted cristae and no evidence of
the cytochrome oxidase reaction product. In contrast, photoreceptor mitochondria from control animals showed normal
morphology with well-defined cristae and were moderately
reactive for cytochrome oxidase reaction product. These
findings are consistent with disruption of ionic homeostasis
in the photoreceptors, secondary to inhibition of mitochondrial function. Similar retinal histopathological changes have
been reported in human methanol intoxication (Fig. 3c)
(Murray et al, 1996); in patients with mitochondrial diseases
that inhibit electron transport (McKechnie et al, 1985;
McKelvie et al, 1991); and in certain forms of light-induced
retinal degeneration in which inactivation of cytochrome oxidase is postulated to play a role in the pathology (Rapp et al.,
MITOCHONDRIA-MEDIATED CELL INJURY
1990). Biochemical measurements also showed a significant
reduction in retinal and brain cytochrome oxidase activity
and ATP concentrations in methanol-intoxicated rats relative
to control animals (Eells et al., 1995). Surprisingly, no differences from control values were observed in hepatic, renal,
or cardiac cytochrome oxidase activity or ATP concentrations in methanol-intoxicated rats. The reduction in retinal
function, inhibition of retinal, optic nerve, and brain cytochrome oxidase activity, depletion of retinal and brain ATP
concentrations, and mitochondria] disruption produced in
methanol-intoxicated rats are consistent with the hypothesis
that formate acts as a mitochondrial toxin with selectivity
for the retina and brain.
Studies measuring ATP synthesis in mitochondria isolated
from bovine retina and bovine heart have provided additional
evidence for a tissue-selective action of formate (Eells et
al., 1996a). In these studies, mitochondrial ATP synthesis
was measured in the presence of different metabolic substrates. Formate selectively inhibited ATP synthesis in mitochondria isolated from bovine retina in the presence of metabolic substrates supplying electrons at the level of complex I,
complex II, and complex IV in the mitochondrial respiratory
chain. The inhibitory effect of formate on retinal mitochondrial ATP synthesis was concentration dependent, with significant reductions in ATP synthesis produced at 10 rriM
formate and K, values for inhibition ranging from 30 to 50
IHM formate. Comparative studies conducted in mitochondria
isolated from bovine heart showed little or no inhibition of
ATP synthesis at formate concentrations up to 50 mM. These
findings provide direct evidence that formate acts as retinal
mitochondrial toxin and suggest that one component of the
retinotoxic actions of formate may be due to tissue-specific
differences in mitochondrial transport mechanisms or in mitochondrial metabolism.
The apparent selective vulnerability of the retina and optic
nerve to the toxic actions of formate in methanol poisoning
has been the subject of considerable speculation (Sharpe et
al, 1982; Roe, 1955; Jacobsen and McMartin, 1986). Although methanol intoxication is known to disrupt brain function and severe intoxication results in coma and death, the
most common permanent consequence of methanol intoxication is blindness (Roe, 1955). Several factors may contribute
to the unique vulnerability of the retina and optic nerve to
the cytotoxic actions of formate. One component of this
selectivity is related to the differences in the distribution of
formate in the eye and the brain. Formate concentrations
measured in the vitreous humor and retinas of methanolintoxicated rats (Eells, 1991; Eells et ai, 1996b) and monkeys (Eells and Tephly, 1979) were equivalent to or greater
than corresponding blood formate concentrations. In contrast, the concentrations of formate in the brain were significantly lower than blood formate concentrations. These data
suggest that the toxic actions of methanol on the visual sys-
27
tem may be due to the selective accumulation of formate in
the vitreous humor and the retina as compared with other
regions of the CNS. Second, the retina has a very limited
metabolic capacity to oxidize and thus detoxify formate
(Eells et al., 1994). Third, cytochrome oxidase activity and
ATP concentrations have been shown to be selectively reduced in the retina, optic nerve, and brain in methanol-intoxicated rats, suggesting that there may be tissue- and cellspecific differences in mitochondrial populations and in the
actions of formate on mitochondrial function (Eells et al.,
1995). Finally, in vitro studies in isolated retinal and cardiac
mitochondria have shown that formate selectively inhibits
retinal mitochondrial ATP synthesis (Eells et al., 1996a).
These findings support the hypothesis that formate acts as a
selective mitochondrial toxin in the retina and establish a
link between the effects of formate in vitro and the retinal
toxicity associated with formate accumulation in methanol
intoxication.
Toxicity Testing for Hepatocyte Mitochondrial
Bioenergetics as Affected by Phenyl Herbicides
(V. M. C. Madeira)
The use of industrial compounds in pharmacology and
agrochemistry is of increasing concern since most compounds can cause severe toxic injuries to humans and to
useful animals and plants (Hayes, 1975; Metcalf, 1971). In
the last decade, an increasing interest in in vitro toxicity
testing has developed to predict acute toxicities of industrial
xenobiotics. For this purpose, we have used isolated hepatocytes with an emphasis on bioenergetic changes as a powerful parameter for in vitro evaluation of chemical toxicity.
Our initial studies employed three widely used herbicides
in agrotechnology representative of three different chemical
families: (1) a quaternary nitrogen bipyridinium compound,
paraquat; (2) a chlorophenoxy herbicide, 2,4-D; and (3) a
dinitrophenol compound, dinoseb.
Paraquat and 2,4-D caused a dose- and time-dependent
cell death (Fig. 4) accompanied by depletion of intracellular
reduced glutathione (GSH) and a corresponding increase in
oxidized glutathione (GSSG) (Palmeira et al., 1994a). Dinoseb, the most cytotoxic of the three compounds, exhibited
moderate effects on the levels of GSH at concentrations that
severely depress cell viability (Fig. 4). These limited effects
are at variance with the pronounced effects of dinoseb on
the adenine and pyridine nucleotide contents (Palmeira etai,
1994a); ATP and NADH were rapidly depleted on dinoseb
addition in the micromolar range. Similar depletions were
observed for paraquat and 2,4-D, but in the millimolar range
(1000-fold higher than for dinoseb). As compared with 2,4D, paraquat is more effective, as expected from the redox
cycling of the herbicide metabolism (Fig. 5). The depletion
of ATP correlates temporally with the of onset of cell death.
Furthermore, paraquat and 2,4-D may initiate cell death by
28
WALLACE ET AL.
depressing GSH. Additionally, paraquat and 2,4-D induce
lipid peroxidation at concentrations that effect cell viability.
The effects of dinoseb are relatively moderate (Palmeira et
al., 1995a). It should be emphasized that the toxicity of
paraquat is difficult to appreciate precisely by this "shortterm" methodology, since the effects may be dependent
on the reaction time. In fact, the redox cycling provides
a continuous catalytic source for free radical generation.
Therefore, the effects of the compound are long-lasting,
which may explain the irreversibility of injuries responsible
for the large number of human fatalities despite the moderate
acute toxicity (Hayes, 1975).
Since the three herbicides are effective in cell energy
NAD +
Fe2+ + H,0 o + O
'2^2
FIG. 5. Redox cycle of paraquat (Ross and Moldeus, 1991) and putative
oxidation effects on the proton uncoupling protein. It is proposed that
oxidation of critical thiols induces opening of the pore, an effect partially
counteracted by synthesized ATP during phosphorylation. Extensive damage of the protein causes loss of ATP sensitivity.
100
60
120
90
150
180
Time (mini
100
Dinoseb
d
Control
0
•
Sj/M
10/jM
f
120
150
180
r
f
120
Cl—(
100
I"
•
l«0
£-1 40
20
NADH
-
'
Control \
1mM
5mM
^—OCHSCOOH
/
N
lOmM
J
30
f
H
\\ ii
i l l ii
60
90
i
1
ill I: ,11k i l l ;
120
150
180
Time (mini
FIG. 4. Viability of isolated hepatocytes estimated by lactic dehydrogenase leakage as a function of the incubation time with the listed herbicides.
Note that the concentration range for dinoseb is 1000-fold lower as compared with those for paraquat and 2,4-D.
depletion, we identified their effects on bioenergetic functions of isolated mitochondria, namely, A * , oxidative phosphorylation, ATP synthase, ATPase, respiration, and activities of redox complexes. Dinoseb, in the nanomolar range,
strongly increases the rate of state 4 O2 consumption. Additionally, it depresses A * , stimulates the ATPase, and induces permeabilization of mitochondrial membrane to H +
(Palmeira et al., 1994c). These data characterize dinoseb as
a classical and potent proton uncoupler. The activities of
redox complexes and uncoupled respiration by FCCP are
not affected.
The chlorophenoxy compound, 2,4-D, in the micromolar
range (1000-fold higher as compared with dinoseb) decreases A * as a function of concentration. State 3 respiration
and uncoupled respiration are depressed to the same extent
(60%), indicating that the phosphorylation assembly is not
affected (Palmeira et al, 1994c). The effects on respiration
are a consequence of the strong inhibition of succinate dehydrogenase (complex II) and cytochrome c reductase (complex III). The herbicide also uncouples mitochondria! respiration at concentrations 1000-fold higher than those required
for a similar dinoseb effect (Palmeira et al., 1994c). These
data support the suggestion that the cell damage induced by
dinoseb and 2,4-D is preceded by alteration of mitochondrial
bioenergetic functions.
Paraquat, in the millimolar range, increases state 4 respiration, indicating uncoupling effects. Additionally, state 3 respiration and uncoupled respiration are depressed and the
ATP synthase is partially inhibited. Depression of respiration
is mediated by partial inhibition of mitochondrial complexes
m and IV (Palmeira et al., 1995b). Paraquat also depresses
A * as a function of concentration. Additionally, the depolarization on ADP addition is decreased, and A * on repolariza-
MITOCHONDRIA-MEDIATED CELL INJURY
tion resumes at a level consistently higher than the initial
level before ADP addition. This effect is tentatively ascribed
to the generation of hydroxyl radicals as a consequence of
the oxidative cycling of paraquat radicals (Fig. 5) to oxidize
critical SH groups, leading to the opening of membrane
pores (Castilho et al, 1995). Oxidation of these critical thiols
induces high permeability of a proton pore, explaining the
uncoupling promoted by paraquat. This effect is partially
counteracted by ATP synthesized during phosphorylation,
since added ATP increases A * . The process may take place
at the level of the uncoupling protein (Vercesi et al, 1995),
explaining the observed repolarization to a higher A * . This
effect of ATP diminishes as the protein becomes progressively oxidized, probably as consequence of alterations promoted by the oxidation extended to the ATP interaction sites.
Statistical evaluation of correlations relating cell viability
with herbicide suggests that the biochemical processes initiating cell damage may be a consequence of the reduced
available energy. The correlations of decreased GSH are also
very high for paraquat and 2,4-D, suggesting the decreased
GSH/GSSG ratio is involved in the induction of irreversible
cell injury.
Quinone-Induced Interference with Mitochondrial Calcium
Regulation and Bioenergetics (K. B. Wallace)
Exposure of mitochondria in vitro to chemicals or conditions that evoke an oxidative stress causes the mitochondria
to undergo a dramatic and reversible swelling response. Associated with this is the depolarization of membrane potential and inhibition of oxidative phosphorylation (for reviews,
see Gunter and Pfeiffer, 1990; Gunteretai, 1994). Although
it has been suggested that this swelling reflects a general
change in organization of the membrane lipoproteins, recent
data indicate a much more specific response, perhaps reflecting the transformation of a single specific channel or a
specific complex of proteins that collectively constitute a
regulated channel within the inner membrane (for recent
reviews, see Bemardi et al., 1994; Zoratti and Szabo, 1995).
The significance of the induction of the mitochondrial
permeability transition (MPT) to the manifestation of toxicity has been demonstrated in cell culture for numerous chemicals. The list of compounds for which the MPT has been
implicated includes hydroperoxides, rotenone, l-methyl-4phenylpyridinium (MPP + ), 1,2-dichlorovinyl-l-cysteine, 3,5dimethyl-jV-acetyl-p-benzoquinone imine, and doxorubicin
(Adriamycin) (Broekemeier et al., 1992; Chacon and Acosta,
1991; Henry and Wallace, 1996; Kass etai, 1992; Nieminen
et al., 1995; Pastorino et al., 1993; Saxena et al., 1995;
Snyder et al., 1992; Solem et al., 1994, 1996; van de Water
et al, 1994). Adding compounds that are known to prevent
the induction of the permeability transition in isolated mitochondria protects cells in culture against the cytotoxic effect
of these agents. Since cell killing is also prevented by adding
29
excess fructose, the cytoprotection afforded by inhibitors of
the MPT is believed to reflect prevention of the inhibition
of oxidative phosphorylation and loss of cytosolic ATP
(Henry and Wallace, 1996; Imberti et al., 1993; Nieminen
et al, 1990, 1994; Snyder et al, 1993; Toxopeus et al,
1993, 1994; Wu et al, 1990).
Induction of this MPT is characterized by the rendering
of the inner membrane nonselectively permeable to solutes
up to 1500 Da. It has a strict requirement for a threshold
concentration of calcium within the matrix space coupled
to the presence of any one of numerous "inducing agents"
(Petronilli et al, 1993). The permeability transition is also
influenced by the transmembrane electrical potential, resembling a voltage-gated channel wherein depolarization
increases the probability for calcium-dependent mitochondrial swelling (Bernardi, 1992; Petronilli etai, 1994). All
three regulatory parameters are interrelated; the critical
concentration of calcium required to elicit the transition is
dependent on both membrane potential and the sum of the
concentrations of inducing agents. Conversely, the concentration of inducing agent required to trigger the MPT is
inversely related to the matrix calcium concentration
(Henry et al, 1995).
The list of inducing agents reflects a large array of structurally dissimilar chemicals, many of which are important
environmental, industrial, or pharmaceutical agents. It includes free radical-generating systems (xanthine oxidase-catalyzed), various hydroperoxides, heavy metals, fatty acids
and other weak carboxylic acids, and xenobiotics that are
known to undergo redox cycling to liberate oxygen free
radicals in biological systems (Gunter and Pfeiffer, 1990).
Examples of the latter group include various quinones and
naphthoquinones, MPP + , doxorubicin, and paraquat. Inhibitors and uncouplers of mitochondrial respiration most likely
increase the probability for induction of the MPT by altering
membrane potential. Inhibitors of the MPT constitute an
equally diverse collection of compounds, the most specific
of which is the immunosuppressant drug cyclosporine A
(Broekemeier et al, 1989). Other inhibitors include Mg 2+ ,
reduced pyridine nucleotides, polyamines, carnitine, and
sulfhydryl reducing agents. The fact that atractyloside induces the MPT and that ADP and bongkrekic acid inhibit
the transition implicates an important role for the adenine
nucleotide transporter (Halestrap and Davidson, 1990). Zoratti and Szabo (Szabo and Zoratti, 1992; Zoratti and Szabo,
1995) suggest that the adenine nucleotide transporter is one
of several key components that constitute the mitochondrial
megachannel, which is synonymous with the mitochondrial
permeability transition pore (MPTP).
Despite the diversity in chemical structure, a common
characteristic shared by most chemical agents that induce
the MPT in vitro is the sulfhydryl reactivity. The majority
of inducing agents deplete cellular glutathione and protein
30
WALLACE ET AL.
thiols either via oxidation to the corresponding disulfide or
nucleophilic substitution to form the corresponding thioether. This observation, combined with the fact that sulfhydryl reducing agents are effective in preventing the MPT,
provided the initial basis for implicating the redox state of
thiols as being critical to regulating the open/closed state of
the MPTP (Fagian et al., 1990; Rizzuto et al., 1987). The
well-documented importance of mitochondrial glutathione
in combating oxidative stress (Meredith and Reed, 1982;
1983; Reed, 1990) lends credence to the inferred importance
of the MPT in the manifestation of oxidative cell injury.
Bernardi and associates have elaborated on the molecular
mechanism of regulation of the MPT by protein thiols, implicating vicinal cysteine residues in the voltage-sensing element of the pore (Petronilli et al., 1994; Constantini et al.,
1995). Oxidation increases the voltage gating potential,
whereas sulfhydryl reducing agents and monofunctional alkylating agents, such as A'-ethylmaleimide (NEM), decrease
the probability of pore opening. Bifunctional alkylating
agents are potent inducers of the MPT. A model has been
proposed for thiol-dependent regulation of the mitochondrial
permeability pore that accounts for the opposing effects of
oxidants and alkylating agents (Petronilli et al., 1994). The
model depicts a scenario to explain how monofunctional
alkylating agents protect against induction of the MPT by
oxidants.
We found this model for the thiol-dependent regulation
of the MPTP helpful in explaining the distinct mechanisms
by which naphthoquinones of varying chemical reactivities
interfere with mitochondrial bioenergetics (Henry and Wallace, 1995). Menadione (2-methyl-l,4-naphthoquinone) has
long been known to be an inducer of the MPT in vitro;
however, the mechanism by which menadione inhibits mitochondrial bioenergetics and causes lethal cell injury remains
controversial because it possesses both redox cycling and
arylating chemical reactivities (Gant et al, 1988; Henry and
Wallace, 1996; Miller et al., 1986; O'Brien, 1991; Ross et
al., 1986; Rossi et al., 1986). In isolated hepatic mitochondria, we found that substituted naphthoquinones induced a
calcium-dependent depolarization of mitochondrial membrane potential that is inhibited by cyclosporine A (Henry
and Wallace, 1995). The rank-order potency for induction
of the MPT correlates with the rates of redox cycling and
oxygen radical generation in submitochondrial particles,
suggesting a direct role of oxygen free radicals in the manifestation of the MPT. Conversely, unsubstituted 1,4-naphthoquinone caused the calcium-induced calcium release and
depolarization of mitochondrial membranes at concentrations far below that required for redox cycling. Unsubstituted
p-benzoquinone, which is a potent arylator but does not
redox cycle, caused a calcium-independent depolarization of
mitochondria that was not inhibited by cyclosporine A. It
was concluded that electrophilic quinones interfere with mi-
Naphthoquinone
ROS
Semiquinone
O2
DTT
Benzoquinone
Menadione
FIG. 6. Schematic representation of the thiol-dependent regulation of
the mitochondrial permeability transition pore (as adapted from Petronilli
el al., 1994) to reflect the putative interaction between alkylating and redox
cycling quinone compounds (Palmeira and Wallace, 1997).
tochondrial bioenergetics directly by inhibiting electron
transport, whereas the redox cycling naphthoquinones elicit
an oxygen radical-mediated induction of the MPT (Henry
and Wallace, 1995).
In view of the proposed model for the thiol-dependent
regulation of the MPTP (Petronilli et al., 1994), we hypothesized that unsubstituted p-benzoquinone, because of its potent arylating chemical reactivity, will mimic NEM in preventing induction of the MPT by menadione. Indeed, preincubation of isolated hepatic mitochondria for 90 sec with pbenzoquinone prevented not only the mitochondrial swelling
and depolarization of membrane potential caused by menadione, but also that caused by each of the other redox cycling
naphthoquinones examined (Palmeira and Wallace, 1997).
From these results, a modified version of the model was
proposed that is specific to the interaction of p-benzoquinone
and the redox cycling naphthoquinones (Fig. 6). It is proposed that naphthoquinones redox cycle on the mitochondrial electron transport chain to liberate reactive oxygen species, which are responsible for inducing the MPT by oxidizing critical thiol groups associated with the voltage-sensing
element of the pore. This shift in voltage gating potential is
reversed by sulfhydryl reducing agents and prevented by
forming the monofunctional thioether adduct of benzoquinone, which prevents formation of the disulfide that is critical
to induction of the MPTP.
It is concluded that induction of the MPT by oxidant
chemicals is important in the manifestation of toxic cell
injury and that mitochondria! thiols serve an essential protective role. Of particular significance is the demonstration of
MITOCHONDRIA-MEDIATED CELL INJURY
discrete mechanisms of mitochondrial dysfunction among
structurally similar compounds. The fact that redox cycling
and arylating naphthoquinones both interfere with mitochondrial bioenergetics, but by very distinct and noncomplementary mechanisms, alerts us to the importance of distinguishing between physicochemical properties and chemical reactivities in discriminating between vastly different modes of
toxic tissue injury. This may be of particular significance
when contemplating the mechanism of toxicity of substances, such as menadione, that possess "mixed" or complex chemical reactivities.
Mitochondrial Genetic Damage Occurs in Aging Human
Cells and Tissues: Distribution, Frequency, and
Potential Mechanisms (G. A. Cortopassi)
Evidence is accumulating that mitochondrial damage and
dysfunction increase with advancing age, which may contribute to age-related disease and sensitivities to mitochondrial toxins. The invention of the polymerase chain reaction
(PCR) has facilitated the development of a new class of
assays to quantify human somatic mutations, based on genotypic selection of mutants at the DNA level rather than phenotypic selection of mutants at the cell level. Use of these
assays has provided new perspectives on the timing, spectrum, and distribution of somatic mutagenesis in mitochondrial genes in the aging human body. Mutations of mitochondrial genes rise rapidly with age to frequencies a 1000-fold
higher than those of nuclear genes on a per-gene basis. Genotypic selection analysis has revealed that mitochondrial mutations accumulate predominantly in nonmitotic excitable
cells whose age-dependent loss is associated with pathology.
DNA replication is required for most kinds of mutagenesis
(Loeb et al, 1986). But most cells in the human body are
nonmitotic, and little if any replication of nuclear DNA occurs in nonmitotic cells except for replication associated with
DNA repair. In contrast to the nonreplicative state of the
nucleus, the mitochondrial DNA (mtDNA) undergoes replication in nonmitotic cells about once per month in the mitochondrial matrix, an active site of oxygen free radical generation (Chance et al., 1979).
Deletions and point mutations of the mitochondrial genome that cause genetic diseases (for recent reviews, see
DiMauro and Wallace, 1993) occur in normal aging people.
The level of mitochondrial deletions and point mutations
rises strikingly with age. Mitochondrial DNA deletions rise
by more than 1,000-fold in aging human tissues, and the
rate of rise appears to be exponential (Cortopassi and Arnheim, 1990; Simonetti et al., 1992; Corral-Debrinski et al.,
1991). The mutation frequency per gene of a single mitochondria] mutation, the 4977 deletion, ranges from undetectable levels to as high as a few percent of mitochondrial
genomes in some aging tissues (Soong et al., 1992; CorralDebrinski et al, 1992).
31
In humans, mtDNA mutations accumulate with age from
10- to 100-fold higher levels in nonmitotic tissues of brain,
muscle, and heart than the mitotic tissues of skin, spleen, and
lung (Cortopassi et al., 1992). In mice, mtDNA mutations
accumulate preferentially in brain (Edris et al., 1994; Tanhauser and Laipis, 1995; Wang and Cortopassi, submitted).
The reasons why mtDNA mutations accumulate preferentially in postmitotic, oxidatively active excitable cells that
make up muscle and nerve have yet to be proven; several
speculative explanations are possible, however. First, postmitotic cells turn over slowly or not at all; thus they are
more likely to reflect the cumulative burden of mitochondria]
damage. Second, DNA-damaging reactive oxygen species
(ROS) are produced as a simple consequence of aerobic
metabolism, and thus oxidatively active cells are more likely
to receive mtDNA damage than cells that metabolize less
oxygen. Third, excitable cells experience transient Ca 2+
fluxes that trigger opening of the MPT, resulting in transient
mitochondrial uncoupling; this should result in an increased
generation of reactive oxygen species per mole ATP generated (Fig. 7).
One may estimate mitochondrial mutation frequency in
the brain by subdivision and assay of particular brain areas.
A 1000-fold range in mitochondrial mutation frequency exists in aging brain (Soong et al., 1992; Corral-Debrinski et
al., 1992). The substantia nigra of some aged persons may
contain 1000-fold more mitochondrial deletion mutations
than the cerebellum. Dopaminergic cells of the substantia
nigra are the first to degenerate in parkinsonism. Such cells
contain high levels of the neurotransmitter dopamine, the
breakdown of which produces hydrogen peroxide at the
mitochondrial inner membrane (Soong et al., 1992). Thus
genotypic selection of somatic mitochondrial mutations is
consistent with the view that dopaminergic neurons of the
substantia nigra are under heavier oxidative attack than
other brain areas.
Whether or not the deleterious mtDNA mutations produce
a phenotypic effect and how are still controversial. The level
of the 4977 deletion approaches 1-10% of mitochondrial
genomes in nigrostriatal cells (Soong et al., 1992; CorralDebrinski et al., 1992), and there are about 1000 mtDNAs
per cell. It is not clear if this level of defective mitochondrial
genomes is sufficient for pathology. But many other mutations, deletions and point mutations (Munscher et al., 1993;
Reynier et al., 1995; Melov et al., 1994), accumulate simultaneously in aging humans in parallel with the 4977 mutation, and the total mitochondrial mutational burden is yet to
be determined. The 4977 deletion has been likened to the
"tip of the mutational iceberg," a frequent mutation that
indicates tissues of high mitochondrial damage of many
types (Arnheim and Cortopassi, 1992). There is a need for
new assays that define the total mutational burden of
mtDNA. When such assays are in hand, the issue of whether
32
WALLACE ET AL.
neurogeruc
stimulation
t
plasma
membrane
[Ca++]
PTP
uncoupling
uniporter
FIG. 7. Speculative hypothetical model to explain the differential accumulation of mitochondrial mutations in excitable, postmitotic cells. PTP,
permeability transition pore; ROS, reactive oxygen species; mtDNA, mitochondrial DNA. For simplicity only the inner membrane of a single mitochondrion
is depicted.
the total burden of mitochondrial mutation is sufficient to mals with an inhibitor of Complex I produces massive cell
cause cell and tissue pathology can be critically addressed. loss in the brain, while treatment of littermate controls does
The distribution of mutations in cells is also of critical not (Cortopassi and Wang, 1994). The simplest explanation
significance for testing the somatic mutation model of of the inhibitability of cell death by MnSOD is that such
aging. Currently, mutations are assayed in homogenized death is mediated through an increase in mitochondrial sutissue rather than on a cell-by-cell basis. Thus the finding peroxide concentration.
that the 4977 deletion occurs in 1% of total mitochondrial
In summary, deletion and point mutations of the mitogenomes in 100 dopaminergic cells could reflect that every chondrial genome accumulate in all aging human tissues,
cell had about 990 normal mitochondrial genomes and about and reach their highest level in nonmitotic oxidatively active
10 mutant ones; or it could reflect that 10 dopaminergic cells, an example of which are dopaminergic cells of the
cells had 90% or more mutant mtDNAs, and 90 cells were substantia nigra which undergo early degeneration and death
completely normal, a highly toxic situation for those 10 in parkinsonism. This patterning of mutation has led to a
mutant cells. Because such mitochondrial mutations are oc- specific testable hypothesis of parkinsonism that is the result
curring somatically and independently by a completely ran- of cumulative damage to the mitochondrial genome, Comdom process, the second possibility is much more likely. plex I deficiency, superoxide generation, and cell death.
Recent data are consistent with an uneven distribution of
mitochondrial mutations with respect to cells (Wang and Conclusions and Perspectives
Cortopassi, submitted).
Because mitochondrial mutations occur somatically, they
Detailed studies of formate effects on bioenergetics and
must arise independently in particular mitochondria in par- retinal function, developments of improved in vitro toxicity
ticular cells. Because the mitochondrial genome encodes screening, delineation of the characteristics of the MPT, and
mostly NADH dehydrogenase (Complex I), random muta- development of techniques for study of the mitochondrial
genesis is more likely to affect Complex I activity than any genome are among the important contributions that lead to
other mitochondrial enzyme (Cortopassi and Wang, 1994). the emerging picture that mitochondria have a central role
Inhibition of Complex I is known to induce the formation in chemical toxicity. Of particular importance, the heterogeof mitochondria] superoxide, a potential cellular toxin (Tur- neous characteristics of mitochondria in different tissues are
rens and Boveris, 1980; Cleeter et ai, 1992). We have re- revealing a basis for tissue-specific injury mediated by this
cently described mice that underexpress the sole enzymatic organelle. Unique transport characteristics for formate conscavenger of mitochondrial superoxide, MnSOD (Wang and tribute to the selective retinal toxicity of methanol, and
Cortopassi, 1994). Treatment of these MnSOD-deficient ani- unique accumulation of mitochondria! DNA mutations con-
33
MITOCHONDRIA-MEDIATED CELL INJURY
tributes to the selective vulnerability of nonmitotic excitable
tissues. Specialized transport characteristics also appear to
contribute to the selective mitochondrial toxicities of aminoglycoside antibiotics and cisplatin in kidney. A relatively
high bioactivating enzyme activity in kidney is thought to
at least partially explain the mitochondrial toxicity of 1,2dichlorovinyl cysteine. Because mitochondria are phenotypically different in various cells types and tissues, one can
anticipate that additional examples of tissue-specific mitochondria-mediated injury will be described.
In addition to these important developments covered in
this symposium, findings in other laboratories show that mitochondria can have a very specific role in toxicity because
they can function in signaling apoptotic cell death. Although
apoptosis is activated by a range of signals, the common
sequence of cytoskeletal and membrane changes, chromatin
condensation, and DNA fragmentation appears to occur because the multiple activating events converge on afinalcommon signaling pathway for execution of cell death (Hengartner and Horvitz, 1994; Reed, 1994). Central among these
events is the activation of members of a cysteine protease
family (ICE-like proteases; caspases; Kumar and Harvey,
1995) which share sequence similarity or substrate specificity with the product of the Caenorhabditis elegans death
gene, ced-3 (Xue et al., 1996). Mammalian CPP32, caspase3, is the closest known homolog to Ced-3. Activation of
CPP32 and other caspases results in cleavage of poly(ADPribose) polymerase, histone HI, phospholipase A2, topoisomerase I, and other proteins, events that are critical in
committing cells to death by the apoptotic pathway (Kumar
and Harvey, 1995). A potential role for mitochondria in
controlling this process arose with the discovery that Bcl-2,
a protein homologous to the cell death protective gene ced9 in C. elegans, is located in mitochondria (Hockenbery et
al, 1990; Monaghan et al., 1992) and is associated with
protection against apoptosis (Vaux et al., 1988; Zhong et
al., 1993).
Wang and co-workers recently found that activation of
CPP32 requires a 15-kDa protein that they identified as cytochrome c (cyt c) (Liu et al., 1996). They showed that cyt c
is released from mitochondria during staurosporine-induced
apoptosis in HL60 cells. This result is surprising because
cyt c is an electron carrier normally associated with the outer
surface of the mitochondrial inner membrane. Recent studies
show that cyt c release can be blocked by Bcl-2 and that
the release of cyt c precedes loss of the mitochondrial Ai/>
(Yang et al., 1997). The release of cyt c is not blocked by
protease inhibitors, but nuclear changes induced by the cyt
c-dependent activation of CPP32 are inhibited by protease
inhibitors (Liu et al., 1996). Thus, these results suggest that
cyt c release is proximal to the activation of CPP32 and that
this release can precede activation of the MPT (Fig. 8).
Independent studies of Fas-mediated apoptosis in Jurkat
Toxicant
Staurosporin
Fas activation
CPP32 activation
m
Apoptosis
/
/
X
Necrosis
r
FIG. 8. Mitochondria] release of cytochrome c in activation of
apoptosis. Mitochondrial toxicants can potentially stimulate cytochrome c
release as a specific signaling event, which initiates apoptosis by activating
CPP32 (solid lines), or activate the mitochondrial permeability transition,
which can cause necrosis and possibly apoptosis (broken lines).
cells (Krippner et al., 1996) revealed an early loss of cyt c
activity but no loss in cytochrome oxidase activity in mitochondria. They found that loss of cyt c activity in mitochondria was associated with a substantial decrease in maximal
respiration rate and also occurred with only a small decline
in mitochondria] A "I*. Together widi the above findings (Liu
et al, 1996; Yang et al, 1997) these results suggest that
mitochondrial cyt c release may also be an early step in the
activation of apoptosis. Compounds that affect mitochondrial function could therefore induce apoptosis by stimulating cyt c release (Fig. 8).
Other relevant findings concerning a possible role of mitochondria in activating apoptosis come from Kroemer and
co-workers, who found that activation of the MPT in isolated
mitochondria was sufficient to activate nuclear changes similar to those of apoptosis (Zamzami et al, 1995). Their results
showed that any of a variety of agents that induced the
MPT with loss of the mitochondrial A * and high-amplitude
swelling of mitochondria caused these nuclear changes. This
process was blocked by agents that block the MPT and by
overexpression of Bcl-2 but was not blocked by protease
inhibitors. They further showed that the MPT was associated
with release of a 50-kDa protein (Susin et al, 1996) which
could activate nuclear condensation without the need for
cytosol. Thus, their results suggest an alternative mechanism
whereby mitochondria could activate apoptosis (Fig. 8).
The findings that mitochondria can activate apoptosis add
to the exciting developments pointing to a central role of
mitochondria in mechanisms of toxicity. Detailed understanding of the molecular signaling of apoptosis will further
allow distinction between necrotic and apoptotic mechanisms and provide the basis for more insightful studies of
the role of mitochondria in toxicity mechanisms. With this
information, the dramatic advances in knowledge as presented in this symposium are certain to continue.
34
WALLACE ET AL.
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