seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 12, 2001: pp.

449457
doi:10.1006/scdb.2001.0282, available online at http://www.idealibrary.com on

Reactive oxygen species and mitochondrial diseases

Ilias G. Kirkinezosa and Carlos T. Moraesa,b,
produce ROS at a rate higher than their scavenging
capacity, resulting in the incomplete metabolism of
approximately 13% of the consumed oxygen. 4,5 The
byproducts of incomplete oxygen metabolism are
.
superoxide (O
2 ), hydrogen peroxide (H2 O2 ),
and hydroxyl radical (OH). The formation
of superoxide occurs via the transfer of a free
electron to molecular oxygen. This reaction occurs
at specific sites of the electron transport chain
(ETC), which resides in the inner mitochondrial
membrane [Figure 1(a)]. ETC complexes I (NADH
dehydrogenase) and III (ubisemiquinone) produce
most of the superoxide, 4,6,7 which is then scavenged
by the mitochondrial enzyme manganese superoxide
dismutase (MnSOD) to produce H2 O2 . Since
mitochondria do not contain catalase, their only
defense against the potentially toxic properties
of H2 O2 is the enzyme glutathione peroxidase
(GSPx). GSPx requires reduced glutathione (GSH)
as a coenzyme and converts H2 O2 to water, thus
completely detoxifying ROS. However, in the
presence of reduced transition metals, H2 O2 can
produce the highly reactive OH, which can cause
extensive damage to DNA, proteins, and lipids
[Figure 1(a)]. 8
Two other important radical species are nitric
oxide (NO) and peroxynitrite (ONOO ). Recent
evidence has demonstrated that mitochondria
possess their own nitric oxide synthase (mtNOS) 9
and can produce endogenous NO and ONOO . 10,11
Although the main source of ONOO is through
the reaction of NO with superoxide [Figure 1(a)],
there are also several other ways to produce ONOO
in a cell. 1216 Mitochondrial NO decays mainly
via ONOO formation, ubiquinol oxidation, and
reversible binding to cytochrome c oxidase. 17

A variety of diseases have been associated with excessive reactive oxygen species (ROS), which are produced mostly in the
mitochondria as byproducts of normal cell respiration. The
interrelationship between ROS and mitochondria suggests
shared pathogenic mechanisms in mitochondrial and ROSrelated diseases. Defects in oxidative phosphorylation can
increase ROS production, whereas ROS-mediated damage
to biomolecules can have direct effects on the components of
the electron transport system. Here, we review the molecular
mechanisms of ROS production and damage, as well as the
existing evidence of mitochondrial ROS involvement in human diseases.
Key words: mitochondria / reactive oxygen species /
mtDNA diseases / neurodegeneration / aging
c 2001 Academic Press

Molecular mechanisms of mitochondrial ROS

production
Mitochondria are unique organelles, as they are
the main site of oxygen metabolism, accounting for
approximately 8590% of the oxygen consumed
by the cell. 1,2 Incomplete processing of oxygen
and/or release of free electrons results in the
production of oxygen radicals. Mitochondria
constantly metabolize oxygen thereby producing
reactive oxygen species (ROS) as a byproduct.
These organelles have their own ROS scavenging
mechanisms that are required for cell survival. 3
It has been shown, however, that mitochondria

From the Departments of a Cell Biology and Anatomy and b Neurology,

University of Miami School of Medicine, Miami, Florida 33136.
*Corresponding author. University of Miami School of Medicine, 1095 NW
14th Terrace, Miami, Florida 33136.
E-mail: cmoraes@med.miami.edu
c
2001
Academic Press
10849521 / 01 / 060449+ 09 / $35.00 / 0

ROS-mediated damage
Under normal conditions, the effects of ROS are
counteracted by a variety of antioxidants, by both
enzymatic and nonenzymatic mechanisms. Oxidative
449

I. G. Kirkinezos and C. T. Moraes

(a)

(b)

Figure 1. Generators and targets of reactive oxygen species (ROS) in mitochondria.

(a) A schematic diagram of the generation of the main mitochondrial reactive oxygen species and their targets. Solid
) indicate
arrows with solid arrowheads ( I) indicate generation of molecules. Solid arrows with pointed arrowheads (
diffusion of molecules. Hatched arrows indicate damaging effects.
(b) An illustration of the vicious cycle hypothesis. MtDNA damage leads to defective ETC complexes, which consequently
increase ROS production, potentially leading to further mtDNA damage.

450

Reactive oxygen species and mitochondrial diseases

NO are cytotoxic, although the exact mechanism

associated with this effect is still unclear. It may be
involved in inflammatory, neurodegenerative, and
cardiovascular pathological processes. 20 NO can
regulate aerobic respiration by reversible inhibition
of cytochrome c oxidase. Interestingly, because
of the short half-life and diffusibility of NO, any
cell that produces excess NO will inhibit its own
respiration and respiration of surrounding cells,
which may contribute to the cytotoxic effects of
NO. For example, in co-cultures of astrocytic and
neuronal cells, astrocytic-derived NO causes both
reversible and irreversible damage to the neuronal
electron transport chain. 22
Peroxynitrite is a highly damaging agent with a
vast repertoire of targets and detrimental cellular
effects. ONOO modifies proteins by nitrating
tyrosine residues, forming dityrosine, and oxidizing
tryptophan and cysteine. 23 The main mitochondrial
targets of peroxynitrite are complexes I, II, IV and V,
aconitase, creatine kinase, superoxide dismutase,
mitochondrial membranes, and mtDNA. Damage of
these molecules may induce mitochondrial swelling,
depolarization, calcium release, and permeability
transition. 24 Uptake of calcium by mitochondria
induces mtNOS which increases ONOO levels in
the organelle, leading to release of cytochrome c,
increase of lipid peroxidation 25 and to a subsequent
release of calcium in the cytosol, 26 a process
which has been suggested to be a feedback loop
preventing mitochondrial calcium overload. Another
important finding is that ONOO inhibits MnSOD
enzymatic activity by nitration and oxidation of
critical tyrosine residues. 27 Curiously, ONOO
seems to preferentially damage certain proteins in
a cell-type specific manner. The molecule affects
the electron transport chain of neurons, but not
astrocytes, 28 however, it preferentially depletes
glutathione (GSH) in astrocytes. 29

stress is considered to be the result of an imbalance

of two opposing and antagonistic forces, ROS
and antioxidants, in which the effects of ROS are
more potent than the compensatory capacity of
antioxidants. In the case of mitochondrial-derived
ROS, superoxide is the first radical produced. It
is a highly reactive species and does not diffuse
easily throughout the cell. Because the main site
.
of O
2 production is the inner mitochondrial
membrane, the mitochondrial DNA (mtDNA) has
been hypothesized to be a major target for ROS
damage [Figure 1(b)]. 18 A circumstantial correlation
between ROS and mtDNA damage was shown by
measuring the levels of mtDNA rearrangements in
heart and skeletal muscle tissue of mice. The level
of mtDNA rearrangements were higher in the heart,
which coincides with the fact that heart has lower
antioxidant defenses than skeletal muscle. 18 H2 O2 is
the next key player in mitochondrially derived ROS,
as it is the product of superoxide detoxification by
MnSOD. H2 O2 is not a free radical by definition
because it lacks free electrons. Nevertheless, its role
in ROS-mediated damage is extremely significant
by virtue of chemical versatility and diffusibility.
H2 O2 is a substrate in many physiological and
abnormal chemical reactions both intracellularly and
extracellularly. Due to its small size and relatively
benign reactivity, compared to the rest of the ROS,
H2 O2 can diffuse freely across several cell radii;
therefore, it is able to mediate toxic effects far from
the site of ROS production. Although by itself H2 O2
is not a major source of oxidative damage, it can react
with free transition metals via the Fenton reaction
(Fe2+ + H2 O2 Fe3+ + OH + OH), producing
the extremely reactive hydroxyl radical. OH has
a very short half-life and reacts with virtually any
molecules in close proximity [Figure 1(a)]. There is
no known scavenger for OH, but OH toxicity can
be avoided by minimizing the levels of H2 O2 and
most importantly the availability of free transition
metals (e.g. Fe2+ , Cu+ ).
Nitric oxide has also been implicated in ROSmediated damage. NO has a dual personality,
which is both beneficial, 19 and detrimental. 20
In some instances, different intracellular levels
of NO can mediate diverse effects. For example,
physiological levels of NO inhibit the opening of
the mitochondrial permeability transition pore
(PTP), whereas high NO concentrations promote
PTP opening. 21 In this review, however, we will only
discuss the harmful effects of NO. High levels of

Mitochondrial dysfunction and ROS in human

pathology
There has been a great deal of research on the
role of ROS in the pathogenesis of a number of
human diseases. A multitude of theories exist,
attempting to explain the mechanisms of ROSmediated damage and its effects in the progression
of diseases. In many diseases, primary mitochondrial
involvement is profound and evident. In others,
the mitochondrial participation is only suspected
451

I. G. Kirkinezos and C. T. Moraes

rate of accumulation of mtDNA deletions has not

been observed in patients with mitochondrial
disorders. Using a specific PCR approach to
detect the so-called mtDNA common deletion
and long PCR to detect a plethora of mtDNA
rearrangements, Tengan et al. could not find an
increase in mtDNA deletions in muscle of patients
with pathogenic mtDNA point mutations. 32 This
observation argues against the vicious cycle theory,
as one would expect that the ETC defect caused
by the mtDNA point mutations should lead to an
increase in ROS, and consequently an increase in
mtDNA rearrangements. Therefore, the vicious
cycle hypothesis [Figure 1(b)] continues to be an
attractive but unproven hypothesis.
Several studies have demonstrated that mtDNA
mutations associated with human disease lead to
ETC complex dysfunction, increased production of
ROS, and oxidative damage, as is the situation in
MELAS, where hydroxyl radical damage to mtDNA
can be accelerated by a specific mitochondrial
genotype associated with the disease. 33 In MELAS
and MERRF patients, the intracellular levels of
hydrogen peroxide and oxidative damage to DNA
and lipids were increased in skin fibroblasts. 34 In
whole blood cells of patients with MELAS-related
mitochondriopathy and patients with LHON, ROSassociated telomere shortening was observed. 35
Finally in a mouse model of sarcopenia, mtDNA
deletion mutations in muscle fibers co-localized
with increased levels of oxidative damage to nucleic
acids. 36 Furthermore, ROS-mediated damage
selectively to mtDNA has been reported in other
diseases, like Down syndrome, 37 acute pancreatitis, 38
multiple sclerosis, 39 end-stage renal disease, 40 and
left ventricular remodeling and failure following
myocardial infarction. 41
As discussed above, most mitochondrial ROS
are formed at complexes I and III. Many clinical
syndromes have been associated with isolated
complex I deficiencies have been described,
including fatal infantile lactic acidosis, adultonset exercise intolerance, focal dystonia, LHON,
cardiomyopathy
with
cataracts,
hepatopathy
with tubulopathy, Leighs disease, cataracts and
developmental delay, and lacticacademia in the
neonatal period followed by mild symptoms. 4247 It
is unclear at this point if some of these symptoms
are exacerbated by increased ROS production, even
though complex I impairment leads to increased ROS
production in submitochondrial particles 6 and cell
systems. 46,48 In studies of human xenomitochondrial

or suggested. For the scope of this paper we will

consider all diseases potentially affected by ROS
as having a mitochondrial component. The main
concept connecting mitochondrial diseases and ROS
is that increases in ROS production and decreases in
ATP production can cause oxidative phosphorylation
deficiency, leading to mitochondrial disease. 30 Also,
a primary defect in oxidative phosphorylation may
increase superoxide production, thereby increasing
oxidative stress.
Mitochondrial diseases
In the last 15 years, several clinical syndromes were
associated with mtDNA mutations, the most common
being NARP (neurogenic muscle weakness, ataxia
and retinitis pigmentosa), MELAS (mitochondrial
encephalomyopathy lactic acidosis, and strokelike episodes), MERRF (myoclonic epilepsy and
ragged-red fibers), LHON (Leber hereditary optic
neuropathy), and KSS (Kearns-Sayre syndrome,
ophthalmoplegia, ataxia, retinitis pigmentosa,
cardiac conduction defect and elevated cerebrospinal
fluid protein) (see article by DiMauro in this issue).
MtDNA encodes few proteins which are involved
in the electron transport chain, the main source
of ROS in cells. This special situation highlights
the theory of the vicious cycle, a theory attractive
within the realm of degenerative processes. In this
cycle, an inherited or random primary mitochondrial
mutation initially induces a respiratory defect, that
increases the leakage of ROS from the electron
transport chain. Subsequently, ROS may trigger
accumulation of secondary mtDNA mutations
exacerbating mitochondrial respiratory defects and
consequently increasing production of ROS and lipid
peroxides from mitochondria.
It has been demonstrated that after exposing
cells to oxidative stress, mtDNA damage is more
extensive and persists longer than damage in nuclear
DNA (nDNA). 31 Several reasons may contribute
to this selective vulnerability: (i) mtDNA lacks
histones which are protective against free radical
damage, (ii) mtDNA lacks an adequate repair
system, rendering it unable to cope with extensive
damage, especially strand breaks, (iii) mtDNA has
very few non-coding sequences, therefore increasing
the likelihood of a DNA alteration to affect a
gene, and (iv) mtDNA is located near the inner
mitochondrial membrane, a major site of oxygen
radical production. 2 However, a correlation between
defective oxidative phosphorylation and increased
452

Reactive oxygen species and mitochondrial diseases

These changes can lead to neuronal death, mainly

through excitotoxic pathways, involving oxidation of
macromolecules and apoptosis.
More than a decade ago, the first strong evidence
of mitochondrial involvement in the pathogenesis
of a neurodegenerative disease came to light
when complex I deficiency was identified in
substantia nigra 56 and platelet mitochondria
of Parkinson disease patients. 57 Subsequently,
enzymatic deficiencies in the electron transport chain
were identified in additional neurodegenerative
diseases: complex IV deficiency in AD and ALS, 5860
and complex II and III in HD and FA. 61 The
common denominator of mitochondrial dysfunction
in these diseases led to hypotheses related to
mtDNA involvement. In HD and FA the genetic
defects appear to be in nuclear genes that encode
non-respiratory proteins (huntingtin for HD,
and frataxin for FA). This fact suggests that the
observed respiratory deficiencies are secondary to
the pathogenic initiating factors. For PD and AD
the picture is unclear. Some reports showed that
a proportion of patients, have evidence of mtDNA
abnormalities as a key player in the progress of
disease. 62 Additional evidence of an association
between ROS and mitochondrial dysfunction
comes from the observation that mutations in the
SOD1 gene can cause ALS. 63 Also, mitochondrial
abnormalities are seen early in the development
of the disease. 60 However, it is still unclear if
the mutated SOD1 alters ROS production, or
even whether the mitochondrial abnormalities are
secondary to a cell death stimulus.
Oxidative damage is also a common finding in all
neurodegenerative diseases. Evidence of oxidative
damage in PD, 48,62,6467 AD, 6870 ALS, 7174 HD, 7577
and FA 78,79 strengthened the link between abnormal
mitochondrial function, increased ROS production,
and neurodegeneration. Recent interesting evidence
suggest that motor neurons are particularly
susceptible to damage from endogenously produced
ONOO , 80,81 and protein bound nitrotyrosine
has been observed in motor neurons from human
ALS patients and tissue of a mouse model harboring a
transgene coding for a mutated form of SOD1 found
in some patients (G93A-SOD1). 82 An interesting
hypothesis is that the high polyunsaturate level of
brain mitochondrial phospholipids might predispose
them to peroxidation, hence brain mitochondria may
be more susceptible to neurodegenerative disorders
that progress via mechanisms of mitochondrial
oxidative damage. 83

cybrids harboring a 40% complex I deficiency and

a drug-induced model of complex I inhibition, 49,50
correlations between the levels of complex I
impairment and cell respiration, cell growth,
ROS production, lipid peroxidation, mitochondrial
membrane potential, and apoptosis were observed.
Interestingly, cell death was quantitatively associated
with ROS production, rather than with complex I
deficiency. 51 This suggests that partial ETC defects
may, in some instances, mediate cellular damage by
ROS-related processes.
It has been shown that complex I impairment
induces MnSOD and/or increases ROS production. 45,46,52 Therefore, a complex I defect may not
be accompanied by increased ROS, if MnSOD is
elevated. However, MnSOD as the only scavenger
might not be sufficient to prevent oxidative damage.
Its action need to be coupled with GSPx/GSH
detoxification of H2 O2 , in order to avoid the
formation of the highly reactive OH, as observed
in cells from a patient with cardiomyopathy with
cataracts. 45
Fewer studies have been described for complex III
deficiencies. Mutations in the apocytochrome
b gene have been described in patients with
myopathies 53 but also with encephalopathies. 54 In
transmitochondrial cybrids containing mtDNA from
a patient with Parkinsonism and MELAS, high levels
of a mtDNA with a mutation in the apocytochrome
b gene were associated with respiratory deficiency
and complex III defect. This deficiency was also
associated with increased hydrogen peroxide
production. 55 Such an increase in the ROS levels
in the central nervous systems could explain the
Parkinsonism in this patient.
Neurodegenerative diseases
Increasingly, research efforts are investigating
the involvement of ROS and mitochondria in
the pathogenesis of common neurodegenerative
diseases: Parkinson disease (PD), Alzheimers
disease (AD), amyotrophic lateral sclerosis (ALS),
Huntingtons disease (HD), and Friedreichs
ataxia (FA). Although an extensive review of research
efforts in this field would be beyond the scope of
this paper, we will briefly discuss the mitochondrial
and ROS involvement in these conditions. Increased
ROS production in neurodegenerative processes
may affect normal mitochondrial parameters like
ATP production, membrane potential, permeability
transition pore activation, and calcium uptake.
453

I. G. Kirkinezos and C. T. Moraes

in this area, the next few years should bring answers

to many of these questions.

Aging
Although it is doubtful that aging is a disease,
mitochondrial ROS involvement in this process
is strongly suggested by experimental data. A
prevalent theory for aging is a variant of the
vicious cycle concept we described previously.
MtDNA is constantly exposed to ROS generated by
the mitochondrial electron transport chain, and
mutations may accumulate exponentially with age.
The simultaneous increase in lipid peroxidation
and oxidation of mitochondrial proteins adds to the
oxidative stress effects, initiating the vicious cycle
of molecular degeneration. This putative vicious
cycle can operate at different rates in various tissues,
leading to differential accumulation of oxidative
damage, which could explain the differences in
functional impairment and deterioration of different
tissues in the aging process.
There is substantial evidence that damage to
mtDNA accumulates with age. Among them, the
most significant is the 10-fold increase in an oxidative
damage marker (8-hydroxy-2-deoxyguanosine) in
mtDNA versus nDNA from human brain. 84 A study
of aging in rhesus monkeys revealed that there
are significant decreases with increasing age in
the activities of complex I and IV, as well as in
mitochondrial ATP generation. 85 Finally, several
mtDNA point mutations also increase with normal
aging. 8689 What is not clear at this point, is whether
such mutations are generated by ROS-mediated
damage.

Acknowledgements
The work from our laboratory is supported by National Institutes of Health Grants GM55766, CA85700
and EY10804 and by the Muscular Dystrophy Association.

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