Pathophysiology 7 (27) 153 163

www.elsevier.com/locate/pathophys

Review
Oxidative stress and apoptosis
Krishnaswamy Kannan a, Sushil K. Jain b,*
a
Department of Medicine, Centre of Excellence for Arthritis and Rheumatology, Lousiana State Uni6ersity Health Sciences Center, Shre6eport,
LA 71130 -3932, USA
b
Department of Pediatrics, Lousiana State Uni6ersity Health Sciences Center, 1501 Kings Highway, Shre6eport, LA71130 -3932, USA

Received 9 March 2000; accepted 15 May 2000

Abstract

Apoptosis or programmed cell death, is essential for the normal functioning and survival of most multi-cellular organisms. The
morphological and biochemical characteristics of apoptosis, however, are highly conserved during the evolution. It is currently
believed that apoptosis can be divided into at least three functionally distinct phases, i.e. induction, effector and execution phase.
Recent studies have demonstrated that reactive oxygen species (ROS) and the resulting oxidative stress play a pivotal role in
apoptosis. Antioxidants and thiol reductants, such as N-acetylcysteine, and overexpression of manganese superoxide (MnSOD)
can block or delay apoptosis. Bcl-2, an endogenously produced protein, has been shown to prevent cells from dying of apoptosis
apparently by an antioxidative mechanism. Taken together ROS, and the resulting cellular redox change, can be part of signal
transduction pathway during apoptosis. It is now established that mitochondria play a prominent role in apoptosis. During
mitochondrial dysfunction, several essential players of apoptosis, including pro-caspases, cytochrome C, apoptosis-inducing factor
(AIF), and apoptotic protease-activating factor-1 (APAF-1) are released into the cytosol. The multimeric complex formation of
cytochrome C, APAF-1 and caspase 9 activates downstream caspases leading to apoptotic cell death. All the three functional
phases of apoptosis are under the influence of regulatory controls. Thus, increasing evidences provide support that oxidative stress
and apoptosis are closely linked physiological phenomena and are implicated in pathophysiology of some of the chronic diseases

including AIDS, autoimmunity, cancer, diabetes mellitus, Alzheimers and Parkinsons and ischemia of heart and brain. 2000
Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Reactive oxygen species; Apoptosis-inducing factor; Apoptotic protease-activating factor-1

1. Introduction Alzheimers, and Parkinsons are the best examples of

Apoptosis, or programmed cell death (PCD), is a
naturally occurring cell death process, essential for the
normal development and homeostasis of all multicellu-
lar organisms [1]. This process is also important for
removing damaged, infected, or potentially neoplastic
cells. However, both too little and too much apoptotic
cell death can lead to adverse biological consequences
[24]. Rheumatoid arthritis and cancer are the best ex-
amples for too little apoptosis. Ischemic heart disease,
AIDS and neurodegenerative diseases such as
* Corresponding author. Tel.: +1-318-6756086; fax: + 1-318-
6756059.
E-mail address: sjain@lsuhsc.edu (S.K. Jain).
too much apoptosis [26]. The commercial availability
of an ever-growing list of reagents, an explosive number
of research publications, and a number of scientific
meetings devoted to apoptosis signifies the relative im-
portance of this emerging field [6].
Apoptosis is a cell death process that is clearly dis-
tinct from necrosis. The integrity of the cell membrane
is severely compromised or damaged in necrosis, lead-
ing to cell swelling and cell lysis. Often, necrosis occurs
in a group of cells, or in tissue at a particular locus. In
contrast, apoptosis occurs at the single cell level. Dur-
ing this process, an individual cell undergoes an active
process of cell death, set in motion by a genetic pro-
gram and culminating in DNA fragmentation and the
formation of membrane-packaged bits called apoptotic

0928-4680/00/$ - see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 9 2 8 - 4 6 8 0 ( 0 0 ) 0 0 0 5 3 - 5
154 K. Kannan, S.K. Jain / Pathophysiology 7 (2000) 153163

bodies. The plasma membrane of early apoptotic cells highly conserved during the evolution of species. These
and the apoptotic bodies maintain their integrity, pre- observations raise the intriguing possibility of the co-
venting leakage of cellular material and thereby an existence of multiple signaling pathways that converge
inflammatory response and local tissue damage. Bio- upstream of a common mechanism of events, predis-
chemical changes on the outer surface of plasma mem- posing the cell to apoptosis [15]. It is currently be-
brane are recognized by tissue macrophages and several lieved that one such convergent scheme involves the
other neighboring cell types. Apoptotic cells are effi- active participation of mitochondria [912].
ciently cleared in vivo by antigen processing cells within It is now well established that mitochondria is the
2 4 h [1,3,5]. main site of the generation of oxygen radicals, such as,
Apoptosis can be triggered by numerous factors in- superoxide anion, hydroxyl radical, singlet oxygen and
cluding receptor-mediated signals, withdrawal of hydrogen peroxide [7,13]. It is estimated that 1 4% of
growth factors, anti-tumor drugs and, under certain oxygen reacting with the respiratory chain leads to the
conditions, damage to DNA (1 7). A partial list of formation of superoxide radicals (O2). Other sources
some of these inducers is included in Table 1. Each of of reactive oxygen species include radiation, cytotoxic
these stimuli has its own specific pathway that leads to chemicals and drugs. The formation and the sources of
activation of apoptotic process, however, all appear to free radicals have been the subject of many reviews
converge at a highly conserved sequence of events. The [9,1417].
caspases are central component of this apoptotic pro- Excess oxidative stress kills cells either by necrosis or
gram [8]. While the initial signal for apoptotic program- by apoptosis [9,10]. In many models of apoptosis, alter-
ming may vary, the morphological and biochemical ations in the redox status of the cell to a more oxidizing
characteristics of PCD are uniformly similar, and are environment occurs prior to activation of the final
phase of caspase activation [1015]. This argument is
further supported by the ability of various anti-oxidants
Table 1 such as N-acetylcysteine (NAC) to block apoptosis in a
A partial list of inducers of oxidative stress and or apoptosis similar way that caspase inhibitors do [18]. The anti-ox-
idant properties of Bcl-2, a potent inhibitor of apopto-
Inducers of oxidative stress
Reactive oxygen Superoxide radical, hydroxyl sis, further supports this view [1922]. Under normal
species radial, hydrogen peroxide conditions, aerobic cells are endowed with extensive
Metals Iron, cadmium, mercuric anti-oxidant defense mechanisms to counteract the
chloride damaging effects of ROS [7,13,14]. When pro-oxidants
Pathophysiologic Hyperglycemia, ischemic heart
overwhelm anti-oxidant defense mechanisms, oxidative
condition disease, Alzheimers and
Parkinsons stress occurs. Interestingly, apoptosis may serve as a
fail-safe device to prevent cells from running amok and
Inducers of apoptosis that in most cases, involve severe oxidative
stress
proliferating uncontrollably in the face of a persistent
Pro-oxidants H2O2, diamide, etoposide and oxidative stress [23]. This review presents an overview
semiquinones of the relationship between oxidative stress and apopto-
Ionizing radiation Gamma UV radiation sis and its relevance to human health.
Protein synthesis inhibitor Cycloheximide
Apoptotic stimuli Fas (CD95), TNFa, ceramide,
glutamate, growth factor
withdrawl (IL-2, IL-3, nerve 2. Mediators of apoptosis
growth factor and serum
starvation) Most of the mediators of apoptosis can be broadly
Physiologic stimuli Glucocorticosteroids, calcium, placed in one of five categories based on the primary
TNF-a, glutamate
Pathophysiologic Serum starvation, IL-2 and perturbation within the cell, i.e. (1) the cell surface, (2)
conditions IL-3 withdrawal, the cytosol, (3) the cytoskeleton, (4) the mitochondrion,
hyperglycemia, ischemia and (5) and the nucleus. At the cell surface, some of the
reperfusion well-characterized death receptors are Fas (CD95), tu-
Pro-apoptotic genes p53, Bax, c-myc
mor necrosis factor receptor-1 (TNF-Rl), CAR1, DR3,
Organic solvents and Benzene and their metabolites
metabolites and 2,5-hexanedione
DR4, and DR5 [2429]. The ligands that activate these
Pesticides DDT, endosulfan, dieldrin, death receptors are structurally related molecules that
and 2,3,7,8 belong to the tumor necrosis factor (TNF) gene super-
tetrachrolorodibenzo-p-dioxin family [1,30]. Interaction of death receptors with lig-
Drugs Actinomycin D, cisplatin, ands may lead to the initiation of a death signal,
cycloheximide, taxol,
camptothecin and
depending upon the presence of intracellular death
staurosporine domains and its physical association with the adapter
protein called Fas-associated death domain (FADD)
 K. Kannan, S.K. Jain / Pathophysiology 7 (2000) 153163
[28,29]. Additionally, FADD also contains another
death effector domain that communicates with caspase
8, one of the key enzymes in the death machinery. This,
in turn, activates downstream caspases, thus commit-
ting the cell to apoptosis. Further examples of ligands
that act at the cell surface include the CD40/CD40
ligand on T and B cells, and the glutamate/glutamate
receptor in the nervous system [28,31 33].
A major shift in the focus of research on apoptosis
from the nucleus to the cytoplasm has occurred in
recent years. A substantial fraction of the pro-apoptotic
members is localized in the cytosol or cytoskeleton
[12,13,30]. Following a death signal, the pro-apoptotic
members undergo a conformational change that en-
ables them to target and integrate various subcellular
systems including mitochondrial outer membrane [34].
Another major player in cell death by apoptosis is a
class of enzymes called caspases, which are cysteine-
dependent enzymes and are sensitive to the redox status
of the cell [8,30,35,36]. These cysteine proteases are
synthesized as precursors that have little, if any, cata-
lytic activity. The precursor is usually converted to the
active protease by proteolytic processing. One role of
caspases is to inactivate proteins that are vital to cell
survival. These enzymes cut off the cell cell contact to
the surrounding cells, disorganize the cytoskeleton, shut
down DNA replication and repair, interrupt splicing,
destroy DNA, disrupt the nuclear structure, induce
biochemical changes on the cell surface for easy recog-
nition by phagocytes, and finally disintegrate the cell to
vesicular bodies [35,37 42]. At present, 13 different
caspases have been identified and more are being added
to the list [37]. Initiation of apoptotic events, however,
depends on the ability of the signaling complexes to
generate an active protease [8].
Poly (ADP-ribose) polymerase (PARP) is a cellular
substrate for caspase 3 and 7. Caspase 3 has also been
demonstrated to cleave all of the three following sub-
strates, DNA dependent protein kinase (DNA-PKcs,
the 70 kDa protein component of the U1-ribonucle-
oprotein (U1-70 kDa), and PARP, at very similar sites,
defining the DXXD motif as the key determinant for
cleavage specificity. Specific inactivation of these sub-
strates initiate an irreversible stress on the organization
of nuclear DNA structure and the regulation of gene
expression [45,46,51].
Under normal conditions, mitochondria possess an
efficient biochemical defense mechanism to neutralize
the effect mediated by ROS. This system is composed
of GSH, glutathione peroxidase, glutathione reductase,
superoxide dismutase, NADP dehydrogenase
(NADPH), Vitamin E and C [13,18,43 46]. For exam-
ple, superoxide radicals are scavenged by superoxide
dismutase, leading to the production of hydrogen per-
oxide, which again is detoxified by glutathione perox-
idase or catalase. Oxidative stress can occur under
155
conditions when oxygen radicals production is greater
than the detoxification capacity of the cell [47,48].
Several lines of experimental evidence recognize the
mitochondrial dysfunction as one of the important
mediators of apoptosis [915,23,4956].
3. Oxidative stress and its significance in biology
ROS has been shown to play both beneficial as well
as deleterious roles. At very low concentration, it may
act as a second messenger in some of the signal trans-
duction pathways [57]. However, when produced in
excess, it can cause oxidative damage to many vital
components of the cell. There exists a dynamic relation-
ship between ROS production and antioxidant capacity
of the given cell system. Some oxidation processes such
as cysteine oxidation play a role in a dynamic regula-
tory process within the cell. Such a variation may cause
a drastic modulation of the oxidized or reduced ratio of
signaling proteins, such as, transcription factors. One
mechanism through which these effectors may elicit
oxidative stress is the small G-protein Ras. Indeed,
Ras is suspected to activate a cascade of kinases via
ROS production [58]. Similarly, transcriptional factors
such as NF-kB, p53 and AP-1 have been shown to be
modulated by oxygen species (reviewed in [13]). Sub-
lethal ROS production, therefore, can interfere with
signal transduction pathways. ROS, in particular H2O2,
are indeed second messengers for various physiological
stimuli, such as, angiotensin inflammatory cytokines
and growth factors or transforming factors [59,60].
There is a paradox surrounding the physiological and
patho-physiological roles played by reactive oxygen
species such as the superoxide radical. It is universally
accepted that the production of superoxide radical by
activated neutrophils and other phagocytes is an essen-
tial component of immunological defense mechanisms
against bacteria [61].
Oxidative stress occurs when redox homeostasis
within the cell is altered. It has been suggested that the
global shutdown of mitochondrial function under con-
ditions of oxidative stress could contribute to apoptosis
because of the dramatic decrease in cellular energy
supply. Injury to cells occurs only when the ROS
overwhelm the biochemical defenses of the cell [43,62
65]. Reactive oxygen species, (in particular, hydroxy
radicals) can react with all biological macromolecules,
i.e. lipids, proteins, nucleic acids and carbohydrates.
The initial reaction generates a second radical, which
reacts with a second macromolecule and so on in a
continuing chain reaction. Among the more susceptible
targets are polyunsaturated fatty acids [48,6365].
The polyunsaturated fatty acyl side chains, because
of their susceptibility to oxidative damage in membrane
phopholipids, pose a constant threat to cellular in-
156 K. Kannan, S.K. Jain / Pathophysiology 7 (2000) 153163
Table 2
Diseases that are influenced by oxidative stress or apoptosis
Neurodegenerative diseases (Alzheimers, Parkinsons)
Autoimmune disease (e.g. rheumatoid arthritis)
Human immunodeficiency virus (AIDS)
Diabetes mellitus
Cancers of lung, colon, breast and others
Alcohol induced liver disease
Hepatitis-C induced liver disease
Ischemic reperfusion damage-heart, liver
tegrity and function [62 72]. The lipid radicals gener-
ated during the early encounter with an oxidant add
molecular oxygen to produce lipid dioxyl radical. This
pro-oxidant abstracts, an allylic hydrogen from another
unsaturated side chain, producing a lipid hydroperoxide
(LOOH) and thus propagating the chain [62 64]. Iron,
a transition metal, is also well known for its crucial role
in the initiation of new lipid-radical chain reactions
[65].
Lipid peroxidation is an important biological conse-
quence of oxidative cellular damage and aging [6673].
A partial list of conditions or diseases that are likely to
involve oxidative stress is given in Table 2. This in-
cludes several neurodegenerative diseases and AIDS.
Other sources of ROS include radiation (e.g. UV), toxic
chemicals (e.g. paraquat and endosulfan) and
chemotherapeutic agents (e.g. adriamycin and
bleomycin) [13,74,75]. The classical view of ROS as
villains that indiscriminately destroy biological macro-
molecules has undergone a shift, in which, positive
physiological roles are considered as well. In summary,
oxidative stress can have positive responses, such as,
proliferation or activation, as well as negative re-
sponses, such as, lipid peroxidation, DNA damage, cell
growth inhibition or cell death.
4. Redox imbalance and apoptosis
Oxidative stress has been implicated in the pathogen-
esis of several disease processes including, ischemia/
reperfusion injury, Alzheimers, Parkinsons and
diabetes mellitus. In cancer, ROS has been implicated
in damage to DNA resulting in altered gene expression.
Consequently, changes in cell cycle-related protein ex-
pression, activation of proto-oncogenes, and the inacti-
vation of some tumor suppressor genes have been
reported [7678]. Yet another modulation that deserves
mention here is the increased expression of Bcl-2
protein that favors prolonged cell survival. Recently, a
therapeutic approach led to the yet incomplete investi-
gation of antisense Bcl-2 oligonucleotides in the treat-
ment of B-cell lymphomas [79].
In colon cancer, it is believed that a mutated p53
gene allows pre-cancerous cells to accumulate tumor-
promoting genetic changes that favor the origin of
tumor cells of malignant type [78]. Interestingly, current
chemotherapeutic agents such as anthracycline-deriva-
tives, which are frequently used as chemotherapeutics in
the treatment of numerous types of cancers, target
some of these apoptotic pathways. For example, adri-
amycin is known to chelate iron and generate ROS that
result in apoptosis of cancer cells [13,80]. As similar to
cancer, oxidative stress has also been implicated in
AIDS disease [81]. In particular, the bystander effect
in uninfected lymphocytes, in HIV infection, seems to
involve increased apoptosis. This increased cell death
has been linked to the effects of free extracellular gp120
and Tat protein that may have a major impact on the
progression of AIDS [81,82].
Although several risk factors can trigger the develop-
ment of insulin-dependent diabetes (IDDM), several
studies suggest a role for ROS in beta-cell death and
disease progression [83,84]. In a pancreatic beta-cell
line, stable transfectants of aldose reductase gene in-
duced apoptosis by causing a redox imbalance [85].
Taken together, these observations show that oxidative
stress can negatively modulate the expression of genes
that control carbohydrate metabolism by repressing the
insulin signaling pathway. Accumulating evidence indi-
cates that increased antioxidant defense systems and
ingestion of antioxidants reduce the susceptibility to
IDDM in animal models or in human study [84]
[86,87]. In vitro studies have demonstrated inhibition of
apoptosis by the antisense nucleotide to the p65NF-kB
[88]. Whether the intervention with antisense therapy
can prevent apoptosis and the progression of the dis-
ease, it needs further investigation. Gene therapy, there-
fore, remains a big challenge in translating some of the
recent advances made in molecular biology to patient
care.
Ischemia and reperfusion injury are other manifesta-
tions of ROS-mediated injury during surgery [89]. Is-
chemic injury occurs when the blood supply to an area
of tissue is cut off under physiological conditions, or
during surgery when blood vessels are cross-clamped,
and in organs for transplant. Oxygen deprivation dur-
ing this period leads to necrotic lesions depending on
duration. On the other hand, restoration of blood
supply leads to reperfusion injury, which is due to
sudden increase of ROS in the target tissue or organ.
Under the in vivo conditions, a complex interplay
between endothelial cells, neutrophils and other cells of
immune system occurs after anoxia-reoxygenation.
Some of the consequences of this interaction are the
oxidative cell damage including lipid peroxidation, acti-
vation of inflammatory cytokines, followed by neu-
trophil and macrophage attack on the reperfused tissue.
A second consequence being accelerated apoptotic cell
death in these tissues resulting in various clinical com-
plications [90]. Oxidative stress can also repress the
 K. Kannan, S.K. Jain / Pathophysiology 7 (2000) 153163
activity of T-lymphocytes, and thus alter the immune
response. Depletion of intracellular GSH pool elicits
oxidative stress by raising the intracellular redox poten-
tial, and causes a marked inhibition of the T cell-medi-
ated immune response. In particular, IL-2 expression,
an important cytokine, is decreased by oxidative stress.
This could contribute to the alteration of protective
immune response as in patients suffering from rheuma-
toid arthritis and HIV-infected people [13,91].
5. Genes, oxidative stress and apoptosis
Gene expression, in general, is modulated by both
physiological and environmental stimuli. It appears that
oxidative stress acts as a pleiotropic modulator in both
these pathways. Indeed, ROS have been described as
second messengers for several growth factors and cy-
tokines. It has been shown that the transcription factors
such as NF-kB and AP-1, which are stimulated by
ROS, could mediate such inductions [13]. Most of the
literature has historically focused on gene induction and
less on gene repression by ROS. Under conditions of
mild oxidative stress, cell cycle-related genes are re-
pressed to increase the lengthening of G1-phase. A cell
cycle arrest is required in order to assess the amount of
macromolecule alterations, and, if necessary, to enter
the apoptotic pathway instead of carrying on the cellu-
lar division process. This delay in cell cycle is impor-
tant, since a base alteration could be converted into an
irreversible mutation if a mismatch escaped the repair
systems before replication. Hence, there is a necessity
not to activate S-phase too quickly when a cellular
stress occurs.
As compared with genomic DNA, mitochondrial
genome appears to be more sensitive to oxidative dam-
age. The mitochondrion possesses its own genome and
produces its own RNAs that are necessary to its func-
tion. Crawford et al., in 1998 [92] have shown that
mitochondrial RNAs undergo specific degradation
upon oxidative stress. Similarly, a direct relationship
between glutathione oxidation and mtDNA damage in
apoptosis has been suggested by Esteve and co-workers
[93]. Mitochondrial DNA is also heavily damaged by
ROS at the bases, as indicated by the high steady-state
level of 8-hydroxydeoxyguanosine, i.e. the presence of
which causes mispairing and point mutations [94].
These mutations can be detected by long-extension
PCR, a method for detecting a variety of mutations of
mitochondrial genome [95]. In mammalian cells, UVB
radiation has been reported to repress mitochondrial
function by strongly inhibiting its transcription [96].
Following the treatment of hamster fibroblasts with
H2O2, these authors observed that the 16S rRNAs, a
major component of mitochondrial ribosomes, were
specifically degraded, whereas cytosolic mRNAs were
157
not. This resulted in a dramatic shutdown of mitochon-
drial protein biosynthesis. Transcription factors such as
NF-kB, p53 and AP-1 are also sensitive to redox
changes in mammalian cells, primarily through redox
regulation of their DNA binding regions [13,84,88,97].
Cell death in the nematode Caenorhabditis elegans is
undoubtedly the best system for the study the genetics
of programmed cell death. During development, a pro-
cess resembling apoptosis deletes 131 cells out of 1090
cells of this nematode. A total of 14 suicide genes that
are important for development have been identified so
far, of which three genes Ced3, Ced4 and Ced9
have received most attention. The Ced9 gene negatively
regulates the Ced3 and Ced4 genes [1,35,9799]. The
homologues of Ced9 in humans have been identified as
Bcl-2, which protect mammalian cells. Caspase 3
(YAMA/CPP32), a proteolytic enzyme in humans is
believed to be an equivalent of Ced3 in nematode. The
homologue to Ced4 in human has been identified to be
APAF-1 (apoptotic protease-activating factor-1) [35].
Other genes of importance are c-fos, c-jun as well as
c-myc, p53, clusterin, RP-2 and RP-8 [100,101]. How-
ever, the complex mechanisms controlling gene expres-
sion that determines cell survival and cell death are still
unresolved.
6. Bcl-2 family members and apoptosis
Bcl-2 is the first mammalian regulator gene that was
identified to have anti-apoptotic potential in a variety
of cell systems. At least 15 Bcl-2 family members have
been identified in mammalian cells [107]. All members
possess at least one of four conserved motifs known as
Bcl-2 homology domains (BH1BH4). Generally
speaking, family members that act as inhibitors of cell
death harbor at least three domains (BH1, BH2, and
BH3), which are important for proteinprotein interac-
tion and the suppression of apoptosis, whereas BH3
serves as the minimal death domain in the prop-apop-
totic members studied so far [11]. Some of the family
members, such as Bax, Bak, and Bok, work in a
manner opposite to that of Bcl-2, though they resemble
Bcl-2 closely. Pro- and anti-apoptotic family members
of Bcl-2 can homodimerize or heterodimerize and seem-
ingly can neutralize one anothers function, suggesting
that their relative concentration in the given cell may
act as a rheostat for the suicide program. Bcl-2 is
localized on the cytoplasmic face of the mitochondrial
outer membrane, endoplasmic reticulum and nuclear
envelope. It is currently believed that this strategic
localization allows it to register and counterbalance the
oxidative damage done to these compartments and
affect their behavior [19,108,109]. This pro-survival
protein also seems to maintain the membrane integrity
of mitochondria, by directly or indirectly preventing the
158 K. Kannan, S.K. Jain / Pathophysiology 7 (2000) 153163
release of cytochrome C, which, along with dATP and
APAF-1, facilitates the activation of pro-caspase 9 to
its active form of caspase 9 [11,108,109]. Bcl-2 protects
cells against diverse cytotoxic insults, for example,
gamma radiation, cytokine withdrawal, hypoxia, ROS,
dexamethasone, staurosporine, and cytotoxic drugs. All
pro-survival Bcl-2-like genes are potentially oncogenic,
whereas pro-apoptotic family members may act as tu-
mor suppressors. Clarifying how Bcl-2 family members
govern apoptosis under various conditions might
provide clues for the development of novel therapies in
clinical settings.
7. Oxidative stress and mitochondria
The mitochondria is sensitive to changes in the redox
state of the cell. Several studies have shown that the
global shutdown of mitochondrial function under con-
ditions of oxidative stress could contribute to apoptosis
[9 13]. Maintenance of mitochondrial membrane in-
tegrity is a dynamic process. Under severe oxidative
stress, the mitochondrial permeability transition (PT)
occurs. PT involves a sudden increase of the inner
mitochondrial membrane permeability to solutes
greater than 1500 Da (protons, calcium, GSH etc.). It is
currently believed that PT functions as a voltage sensor,
a thiol sensor, a sensor of oxidation reduction equi-
librium of adenine nucleotide pool, and as a sensor of
divalent cations. As a consequence, defective PT pore
opening to larger molecules causes uncoupling of the
respiratory chain resulting in hypergeneration of ROS,
cessation of ATP synthesis, matrix Ca++ outflow and
depletion of reduced glutathione and other reductants.
Following the inner membrane permeability and the
release of matrix solutes, a colloidal osmotic pressure
arises in the mitochondrial matrix due to the high
concentrations of proteins, which are slow to equili-
brate [101103]. In order to correct the osmotic bal-
ance, the diffusion of H2O results in a massive swelling
of the mitochondria [102]. This change in membrane
potential predisposes these cells to oxidative damage by
impairment of endogenous antioxidant defense mecha-
nisms [4956]. Another crucial step in the early changes
in mitochondria is alteration of mitochondrial
transmembrane potential (DCm) [9 13]. A decline in
the DCm alone can induce oxidative stress and cell
death [12]. The measurement of cell respiration, per-
formed with whole cell populations, showed that the
lower DCm correlates, as expected, with an uncoupling
of electron transport from ATP production [104,105].
These events are detected at early stages of the apop-
totic process before most of the cells are irreversibly
committed to death suggesting that mitochondria could
be a primary target during apoptosis. Recently, the
release of cytochrome C from mitochondria has been
shown as another important effector molecule in the
mediation of programmed cell death [12,56,106]. It is
postulated that cytochrome C is somehow able to inter-
act with pre-existing cytoplasmic factor(s), which subse-
quently mediate the cleavage of zymogens and resulting
in the activation of caspase 9.
Permeability transition is also associated with the
release of apoptotic inducing factor (AIF), which has
recently been identified and characterized [11,53,54].
This is a  50 kDa protein located in the inner mito-
chondrial membrane whose functions are inhibited by
Bcl-2. Under in vitro conditions, purified AIF induces
dose and temperature-dependent apoptosis in isolated
nuclei. This is an important finding in that membrane
permeability transition predisposes the mitochondria to
lose protein thiols and other factors including AIF to
the cytosol for the initiation of a biochemical cascade
resulting in programmed cell death [11]. It is not clear
at this time whether the release of AIF and cytochrome
C is interrelated, or occurs as biochemical events of
separate pathways. In both instances, Bcl-2, an anti-
apoptotic protein, prevented the actions of AIF and
cytochrome C release. This partly explains the impor-
tance of the physical location of Bcl-2 on the outer
membrane of the mitochondria, nuclear envelope and
endoplasmic reticulum [53,54,110]. A schematic repre-
sentation of the association between oxidative stress
and apoptosis is presented in Fig. 1.
Calcium is another important mediator of apoptosis
[111113]. Also, a close relationship exists between
calcium status and oxidative stress in many isolated cell
systems, the best example being isolated rat hepatocytes
[114]. When cells are deprived of calcium, mitochondria
become sensitive to this calcium-deficient state and their
calcium regulatory mechanism is disturbed. Calcium
omission results in a marked decline of mitochondrial
transmembrane potential (DCm), leading to oxidative
stress and induction of programmed cell death. Many
of the antioxidants can prevent this collapse, thus indi-
cating that the DCm is critical to the process and is
reversible in the early phases. However, it remains to be
determined what dictates the efflux of mitochondrial
calcium. It must be emphasized here that oxidative
stress induced by the absence of extracellular calcium is
distinct from that generated by ROS, in that it appears
to induce calcium cycling in and out of mitochondrial
matrix, which serves to promote oxidative stress
[15,64,115]. In the human erythrocyte model, pretreat-
ment of erythrocytes with calcium increases their sensi-
tivity to calcium [115]. Thus, it appears that calcium
can promote lipid peroxidation both under conditions
of excess and of deficiency. Calcium homeostasis is,
thus, vital to the function and survival of the cell [116].
 K. Kannan, S.K. Jain / Pathophysiology 7 (2000) 153163 159

8. Mitochondria and apoptosis
While the central role of mitochondria in apoptosis
has been widely accepted, experimental evidence now
points to the existence of yet another pathway indepen-
dent of mitochondria. However, it appears that both
these pathways involve pro-caspase 8 as one of the
important intermediary step. Using the Xenopus cell-
free system as the experimental model, Kuwana et al.,
in 1998 [117] have provided experimental evidence that
suggests that caspase 8 participates in both pathways.
In the absence of mitochondria, activation of a caspase
cascade by caspase 8 produces only a partial apoptotic
phenotype in nuclei added to the Xenopus cell extract.
In contrast, the mitochondria-independent pathway,
which involves the release of cytochrome C from mito-
chondria into cytosol, triggers full nuclear apoptosis.
Moreover, engagement of the mitochondria provides an

Fig. 1. Schematic model of mammalian cell death pathway. Death signal may be delivered at the cell surface by direct ligand-receptor interaction.
This followed by clustering of death receptors and activation of caspase-8. Alternatively, cytotoxic drugs, ionizing radiation may directly activate
caspase-9, otherwise a later event. Mitochondria plays a central role in apoptosis by releasing apoptogenic factors and proteases into the cytosol.
A major checkpoint in this pathway is the ratio between pro-apoptotic (Bax) to anti-apoptotic (Bcl-2) members. Miitochondrial dysfunction
includes PT, change in mitochondrial membrane potential, production of ROS, release of cytochrome C, AIPF-1 and caspases-2, -3, and -9 into
the cytosol, where they form multimeric complexes. This activates downstream caspases and degradation of death substrates in the nucleus, which
ultimately leads to cell death.
160 K. Kannan, S.K. Jain / Pathophysiology 7 (2000) 153163
efficient means of amplifying the apoptotic signal trans-
duced by caspase 8 even under very low concentration.
In the absence of mitochondria, high concentrations of
caspase 8 were required to activate downstream caspase
cascade. Interestingly, caspase 8 can activate the mito-
chondria-dependent pathway even when the Bcl-2
protein is present. This helps explain the failure of Bcl-2
to inhibit CD95-dependent apoptosis consistently [117].
Scaffidi et al., in 1998 [118] have identified two types of
CD95-mediated cell death. Recently, it has been
demonstrated that Apaf-1 / and caspase 9 / T
cells remain sensitive to Fas-induced killing [119,120].
However, Fas-induced apoptosis was markedly reduced
in embryonic fibroblasts with Apaf-1 / phenotype
[121], suggesting Fas (CD95) can activate different
pathways in different cell types. These observations in
T cells indicate that the apoptotic function of mito-
chondria (at least the cytochrome C release part) can be
bypassed in these cells.
Other less defined pathways that lead to apoptosis
are the ones initiated by potent cytotoxic chemicals or
gamma irradiation. These pathways seem to rely on the
apoptotic function of mitochondria, since cells with
Apaf-1 / and caspase-9 / phenotypes are princi-
pally resistant to these death-inducing agents [119
121]. Further research in this area may help us to better
understand some of the missing links.
9. Conclusions
The role of oxidative stress as a mediator of apop-
totic cell death in diverse cell systems is now better
understood. In recent years, mitochondria have gained
considerable importance both as a site for ROS produc-
tion and as a major player in apoptosis. The involve-
ment of mitochondria or ROS in apoptosis is not
without controversy. In some experimental systems,
apoptosis can occur at very low oxygen tension, when
ROS are unlikely to occur. The protooncogene
product, Bcl-2, can inhibit apoptosis both in the pres-
ence and in the absence of reactive oxygen products.
Further research is needed to clarify the underlying
mechanisms.
Recent developments suggest that mtDNA may be a
good indicator of oxidative damage in apoptosis. It is
becoming apparent that the redox status of a cell can
have a complex and multilayered effect on cell survival
and cell death. Future investigations will unravel
whether oxidative stress is a pre-requisite for all apop-
totic events. This would be an attractive mechanism
whereby a panoply of seemingly diverse injuries could
rapidly converge on common apoptotic pathways.
More in-depth studies are needed to elucidate the role
of BH3-domain-only subset of molecules and their in-
terplay with ROS. This could open new sites of drug
targeting. Future studies may also improve our under-
standing whether the oxidation states of mitochondrial
thiols alone function as a prominent apoptotic sensor.
A better understanding of the molecular machinery of
apoptosis in the pathophysiology will undoubtedly
provide novel therapeutic interventions and care for a
large number of patients with chronic diseases.
Acknowledgements
We thank Georgia Morgn First for the critical read-
ing of the manuscript. This work was supported in part
by the funds by Feist Weiller Cancer Center and the
Center of Excellence for Arthritis and Rheumatology of
the Louisiana State University Health Sciences Center
at Shreveport, and a grant-in aid from the American
Heart Association (Southeast).
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