Reactive Oxygen Species in Living Systems
Reactive Oxygen Species in Living Systems -- A Thorough Explanation of Free Radicals
Reactive Oxygen Species in Living Systems -- A Thorough Explanation of Free Radicals
Source: Biochemistry, and Role in Human Disease.
by Halliwell, Barry
from "American Journal of Medicine",
Sept 30, 1991 v91 n3C p14S(9)
Subjects:
Free radicals (Chemistry) Physiological aspects
Antioxidants Physiological aspects
Diseases Causes and theories of causation
Reference #: A12136862
Author's Abstract
COPYRIGHT Cahners Publishing Company 1991
Reactive oxygen species are constantly formed in
the human body
and removed by
antioxidant defenses. An antioxidant is a
substance that, when present at low
concentrations compared to
that of an oxidizable substrate, significantly delays
or prevents
oxidation of that substrate. Antioxidants
can act by scavenging
biologically
important reactive
oxygen species
([O.sub.2].,[H.sub.2][O.sub.2], .OH, HOCl, ferryl,
peroxyl, and
alkoxyl), by preventing their
formation, or by repairing the
damage that they
do. One problem with scavenging-type
antioxidants is that secondary radicals
derived from them can
often themselves do biologic
damage. These various principles
will be illustrated by considering several thiol
compounds.
Full Text
COPYRIGHT Cahners Publishing Company 1991
It is difficult these days to open a medical journal
and not find
some paper on the role of "reactive
oxygen species" or "free
radicals" in human disease.
These species have been implicated in over
50 diseases[1]. This
large number suggests that radicals are not
something esoteric,
but that they participate as a fundamental
component of tissue
injury in most, if not all, human diseases.
What are "free radicals" and
"reactive oxygen species"? Do they
cause disease?
Are they produced in increased amounts as a result of
disease and
then contribute to further tissue
injury? Are they merely an
epiphenomenon of no relevance
to clinical medicine? This
introductory article attempts to answer such questions.
Electrons in atoms occupy
regions of space known as orbitals.
Each orbital can hold a maximum
of two electrons, spinning in
opposite directions. A free radical can be defined as
any species
capable of independent existence
that contains one or more
unpaired electrons, an unpaired electron being
one that is alone
in an orbital. Most
biologic molecules are nonradicals,
containing only paired electrons.
An electron occupying an orbital by
itself has two possible
directions of spin. Indeed, the technique
of measuring electron
spin resonance detects radicals by
measuring the energy changes
that occur as unpaired electrons ~relax'
following alignment in
response to a magnetic field[2].
Since electrons are more stable
when paired together in orbitals,
radicals generally are more
reactive than nonradicals, although
there is a considerable
variation in their reactivity.
Radicals can react with other molecules in
a number of ways[3].
If two radicals meet, they can combine
their unpaired electrons
(symbolized by.) and join to form a covalent bond (a
shared pair
of electrons). The hydrogen atom, with one
unpaired electron, is
a radical and two atoms of hydrogen
easily combine to form the
diatomic hydrogen molecule:
H. + H. [arrow right] [H.sub.2]
(1)
Radicals react with nonradicals in several ways.
A radical may
donate its unpaired electron
to a non-radical (a reducing
radical) or it might take an electron
from another molecule in
order to form a pair (an oxidizing radical).
A radical may also join onto a
nonradical. Whichever of these
three types of reaction occurs, the nonradical species
becomes a
radical. A feature of the
reactions of free radicals with
nonradicals is that they tend
to proceed as chain reactions,
where one radical begets another.
For many years, chemists have been
interested in free radical
reactions. Many plastics, such
as polythene, arise by free
radical chain polymerization[4].
Combustion is a free radical
reaction. The drying and aging of
paint also involves free radical reactions.
Curators of museums
have studied the role of free radical damage in the
age-dependent
deterioration of paintings and
other items[5]. Metabolism of
toxins in the human body
can produce radicals. For example,
carbon tetrachloride ([CCl.sub.4])
is metabolized in the
endoplasmic reticulum of the
liver to produce the damaging
trichloromethyl radical,
[CCl.sub.3.][3].
Chemists and biologists have examined in detail
the role of free
radical reactions in the
damage done to living cells by
high-energy radiation. When tissues are exposed
to, for example,
gamma radiation, most of the energy taken
up is absorbed by the
cell water, largely because there is more water
there than any
other molecule. The radiation causes one of the oxygen
- hydrogen
covalent bonds in water to split,
leaving a single electron on
hydrogen and one on oxygen, thus creating two radicals:
[Mathematical Expression Omitted]
H. is a hydrogen radical
(or hydrogen atom), and .OH is a
hydroxyl radical.
The latter is the most reactive radical known
to chemistry. It
can attack and damage almost every molecule found in
living cells
at a diffusion-controlled rate, i.e.,
.OH reacts as soon as it
comes into contact with another molecule in solution.
Since it is
so reactive, .OH generated in vivo
does not persist for even a
microsecond and rapidly combines with molecules in
its immediate
vicinity.
Reactions of .OH with
biologic molecules, must of which are
nonradicals, set off chain reactions[1]. Reactions of
.OH include
its ability to interact with the purine and
pyrimidine bases of
DNA, leading to radicals that have a number of
possible chemical
fates[6]. .OH can also abstract hydrogen atoms from
many biologic
molecules, including thiols:
R - SH + .OH [arrow right] RS. +
[H.sub.2.O]
(3)
The resulting sulfur
radicals (thiyl radicals) have many
interesting chemical properties. They can combine
with oxygen to
generate oxysulfur radicals, such as
[RSO.sub.2.] and RSO., a
number of which damage biologic molecules[7-9].
For example, sulfur-containing
radicals derived from the drug
penicillamine are able to attack and damage certain
proteins[10].
When discussing the use of
thiol compounds as free radical
scavengers, it is essential to
ask what may happen to the
resulting sulfur radicals in biologic systems[11].
Perhaps the best-characterized biologic
damage caused by .OH is
its ability to stimulate the free radical chain
reaction known as
lipid peroxidation. This occurs when the
.OH is generated close
to membranes and attacks the fatty
acid side chains of the
membrane phospholipids. It preferentially attacks
polyunsaturated
fatty acid side chains,
such as arachidonic acid. The .OH
abstracts an atom of hydrogen from one of the carbon
atoms in the
side chain and combines with it to form water:
CH - + .OH [arrow right] - C +
[H.sub.2.O]
(4)
Reaction (4) removes the .OH, but leaves behind a
carbon-centered
radical ( - C - ) in
the membrane. Carbon-centered radicals
formed from polyunsaturated
fatty acid side chains usually
undergo molecular rearrangement
to give conjugated diene
structures, which can have
various fates. Thus, if two such
radicals collided in the membrane,
cross-linking of fatty acid
side chains could occur as the two electrons
joined to form a
covalent bond. Reaction
with membrane proteins is also a
possibility. However, under
physiologic conditions, the most
likely fate of carbon-centered
radicals is to combine with
oxygen, creating yet
another radical, the peroxyl radical
(sometimes abbreviated to the peroxy radical):
[Mathematical Expression Omitted]
Peroxyl radicals are reactive enough
to attack adjacent fatty
acid side chains, abstracting hydrogen:
[Mathematical Expression Omitted]
Another carbon-centered radical is generated, and
so the chain
reaction [equations (5) and (6)] continues. One .OH can
result in
the conversion of many hundred fatty acid side
chains into lipid
hydroperoxides. Accumulation of
lipid hydroperoxides in a
membrane disrupts its function and can
cause it to collapse.
Lipid hydroperoxides can also
decompose to yield a range of
highly cytotoxic products, among the most unpleasant of
which are
aldehydes[12]. A great deal of
attention in the literature has
been focused on malonaldehyde (malondialdehyde), but
this is much
less noxious than such
products as 4-hydroxynonenal[12,13].
Peroxyl radicals and cytotoxic aldehydes can
also cause severe
damage to membrane
proteins, inactivating receptors and
membrane-bound enzymes[14].
Biochemists (apart from
those with a special interest in
"background" free radical generation in vivo,
due to exposure to
ionizing radiation) became
interested in radicals only in the
1970s. This interest followed
from the discovery in 1968 of
superoxide dismutase (SOD), and
enzyme specific for a free
radical substrate[15]. SOD removes superoxide
radical, a species
that is formed by adding
an extra electron onto the oxygen
molecule:
[Mathematical Expression Omitted]
SOD removes O2 by catalyzing a dismutation
reaction, involving
oxidation of the O2 to
oxygen and reduction of another O2 to
hydrogen peroxide:
[Mathematical Expression Omitted]
In the absence of SOD, reaction (8) occurs
nonezymically but at a
rate approximately four orders of magnitude less at pH
7.4.
The discovery of SOD led to the realization that
[O.sup.-.sub.2]
is formed in vivo in living organisms, and SOD
removes it. Some
of the O2 formed in vivo arises
from a chemical accident. For
example, when mitochondria are functioning, some of the
electrons
passing through the respiratory
chain leak from the electron
carriers and pass directly onto oxygen, reducing it to
O2[15,16].
Many molecules oxidize on
contact with oxygen, e.g., and
epinephrine solution left on the bench "goes
off" and eventually
forms a pink product. The
first stage in this oxidation is
transfer of an electron from
the epinephrine to [O.sub.2],
forming O2. Such oxidations
undoubtedly proceed in vivo as
well[1]. For example, several sugars, including
glucose, interact
with proteins to produce oxygen radicals.
It has been suggested
that decades of exposure of
body tissues to elevated blood
glucose can result in
diabetic patients suffering "oxidative
stress" that
may contribute to the
side effects of
hyperglycemia[17]. Glycation of proteins involves not
only direct
reaction with the sugar but also free radical
reactions[17].
Thiols can also be oxidized in the presence of oxygen,
generating
sulfur-containing radicals as well as O2 and .OH. Thiol
oxidation
is favored by alkaline pH
values and by the presence of
transition metal ions, especially copper ions[18].
Thus, mixtures
of copper ions and thiols
can be cytotoxic, as shown for
cysteine[19]. Iron ions can also promote free
radical generation
from thiols under certain
circumstances[20]. Attempts to use
thiols as anti-oxidants in systems containing iron or
copper ions
may even result in stimulation of oxidative damage.
Superoxide and Phagocyte Action
Some of the O2 production in vivo may be accidental
but much is
functional.
Activated phagocytic cells generate
O2 as shown for monocytes,
neutrophils, eosinophils, and
macrophages of all types[21].
Radical production is important in
allowing phagocytes to kill
some of the bacterial strains that
they engulf. This can be
illustrated by examining
patients with chronic granulomatous
disease, a series
of inborn conditions in
which the
membrane-bound reduced
nictotinamide adenine dinucleotide
phosphate (NADPH) oxidase system in phagocytes that
makes the O2
fails to work[21]. Such patients have phagocytes
that engulf and
process bacteria normally, but several bacterial
strains are not
killed and are released in viable form when the
phagocytes die.
Thus, patients suffer sever, persistent, and
multiple infections
with such organisms as
Staphylococcus aureus. Another killing
mechanism used by neutrophils (but not by
macrophages) is the
enzyme myeloperoxidase[22]. It uses [H.sub.2.O.sub.2]
produced by
dismutation of O2 to oxidize chloride ions into
hypochlorous acid
(HOCl), a powerful anti-bacterial agent:
[H.sub.2.O.sub.2] + [Cl.sup.- arrow right] HOCl + [OH.sup.-]
Thiol groups are easily oxidized by
HOCl. Hence, low molecular
mass thiol compounds such as glutathione (GSH),
N-acetylcysteine,
and mercaptopropionylglycine are very
effective at protecting,
for example, proteins against oxidative damage by
HOCl[23.24].
Superoxice formed in vivo, whether functionally
or accidentally,
is disposed of by SOD
[equation (8)]. Recent studies using
genetic engineering techniques
to manipulate SOD levels of
organisms, or to delete the genes encoding
SOD, provide further
evidence of the importance of SOD[25]. It is
interesting to note
that no complete inborn deficiencies of SOD have been
reported in
humans, perhaps because they would be lethal mutations.
Reactive Oxygen Species
SOD removes O2
by converting it into hydrogen peroxide
([H.sub.2.O.sub.2]) and [O.sub.2] [equation (8)].
[H.sub.2.sub.2]
itself can be quite toxic to cells.
For example, incubation of cells with
[H.sub.2.O.sub.2] causes
deoxyribonucleic acid (DNA) damage,
membrane disruption, and
release of [Ca.sup.2+]
ions within the cells, leading to
activation of [Ca.sup.2+]-dependent proteases and
nucleases[26].
At least some of this damage may be mediated
by a reaction of
[H.sub.2.O.sub.2] with O2 in the presence of iron or
copper ions,
to form highly reactive radicals, one of which .OH.
This reaction
proceeds in a number of
stages, but the overall process is
summarized by
[Mathematical Expression Omitted]
Thus, removal of
[H.sub.2.O.sub.2], as well as of O2, is
biologically advantageous[27].
SOD therefore works in conjunction with two enzymes.
catalase and
glutathione peroxidase[27], that
remove [H.sub.2.O.sub.2] in
human cells. The study of inborn
errors of metabolism suggests
that glutathione peroxidase (GSH-Px) is the more
important of the
two in removing [H.sub.2.O.sub.2], probably because it
is located
in the same subcellular compartments (cytosol
and mitochondria)
as SOD. GSH-Px has the distinction of being the only
human enzyme
known requiring the
element selenium for its activity; a
selecnocysteine residue (side chain -SeH
instead of -SH, as in
normal cysteine) is present at its active site.
However, it is
unlikely that the sole function of selenium
in humans is to act
as a cofactor for GSH-Px[28]. GSH-Px removes
[H.sub.2.O.sub.2] by
suisng it to oxidize reduced
glutathione (GSH) into oxidized
glutathione (GSSG):
2GSH + [H.sub.2.O.sub.2 arrow right] GSSG + [2H.sub.2O
[H.sub.2.O.sub.2] has no unpaired electrons and does
not qualify
as a radical.
Hence, the term reactive oxygen
species has been introduced to
describe collectively not only O2
and .OH (radicals) but also
[H.sub.2.O.sub.2] (nonradical). Hypochlorous acid
(HOCl)produced
by myeloperoxidase is also
a nonradical, having no unpaired
electrons. [H.sub.2.O.sub.2.],
.OH, and HOCl are sometimes
collectively called "oxidants."
This is valid description of
[H.sub.2.O.sub.2], .OH, and HOCl,
which are oxidizing agents.
However, O2 has both oxidizing
and reducing properties. The
latter property is used
in a popular assay for O2, the
SOD-inhibitable reduction of
cytochrome c, often applied to
measure [O.sup.-.sbu.2.] production by phagocytes:
cyt c (Fe.sup.3+] + [O.sup.-.sub.2 [arrow right]
O.sub.2] + cyt c
(Fe.sup.2+
Many transition metals have
variable oxidation numbers, e.g.,
iron has
[Fe.sup.2+] and [Fe.sup.3+] ions and copper
has [Cu.sup.+] and
[Cu.sup.2+]
ions. Changing between oxidation
states involves accepting and
donating single electrons, e.g.,
[Mathematical Expression Omitted]
Transition metal ions are
remarkably good promoters of free
radical reactions[29]. Polymer scientists and food
chemists have
been aware of this for years[4,30], and
biochemist are learning
it too [1,17-20,26,31-34]. It has already been
noted that copper
ions promote oxidation of thiols:
R-SH + [Cu.sub.2+ arrow right R-S.] + [Cu.sup.+] + [H.sup.+]
and that [Fe.sup.2+] ions reduce [H.sub.2.O.sub.2] to
give * OH
[equation (10)].
Transition Metals and Lipid Peroxidation
Transition metal ions are involved in lipid
peroxidation in two
ways. They can participate
in first-chain initiation, which
involves attack by any species capable of abstracting
a hydrogen
atom. .OH, which has this property, is
produced by the reaction
of O2 and [H.sub.2.O.sub.2] with
iron ion catalysis [equation
(10)]. It is also be produced by reaction
of [H.sub.2.O.sub.2]
with copper ions,
probably in addition to
oxidizing
copper(III)-oxygen complexes[26,31].
Several iron ion-oxygen
complexes,
such as perferryl,
ferryl, or
[Fe.sup.2+]/[Fe.sup.3+]/[O.sup.2]
complexes, have also been
claimed to initiate peroxidation[32], although
their ability to
do so is uncertain[33,34].
Transition metal
ions also affect lipid peroxidation by
decomposing peroxides.
Commercial fatty acids are heavily
contaminated with peroxides[34].
Cell disruption to isolate
membrane fractions increases rates of
nonenzymic free radical
reactions and
activates enzymes (cyclooxygenases and
lipooxygenases) that
produce peroxides (Figure 1). When
transition metal ions are
added to lipid systems already
containing peroxides, their
main action is to decompose these
peroxides into peroxyl and alkoxyl (lipid-O
*) radicals that in
turn abstract hydrogen and perpetuate the chain
reaction of lipid
peroxidation[34]. This may be
represented by the following
simplified equations,
in which lipid * symbolises
a
carbon-centered radical
[Mathematical Expression Omitted]
lipid O. + lipid-H [arrow right] lipid-OH +
lipid.
(18)
lipid-OO. + lipid-H [arrow right] lipid-OOH + lipid.
(19)
lipid. + [O.sub.2] [arrow right] lipid-OO.
(20)
Reducing agents, such as ascorbic acid or [O.sup.2-*],
accelerate
these metal ion-dependent
peroxidation reactions because
[Cu.sup.+] and [Fe.sup.2+] ions
seem to react with peroxides
faster than do [Cu.sup.2+] and [Fe.sup.3+]
respectively. The end
products of these complex metal ion-catalyzed
breakdowns of lipid
hydroperoxides include
the cytotoxic aldehydes mentioned
previously (malonaldehyde,
4-hydroxynonenal), as well as
hydrocarbon gases such as
ethane and pentane[1]. Some thiyl
compounds can also reduce metal ions and
accelerate peroxidation
of lipids, e.g., cysteine[35]. It has
been suggested that some
thiyl radicals (RS.]
initiate peroxidation by abstracting
hydrogen atoms from lipids
[36]. Different thiols behave
differently in peroxidizing lipid systems,
presumably depending
on their metal ion-reducing ability and the
reactivity of their
thiyl radicals.
Organisms use superoxide dismutases, catalase,
and glutathione
peroxidase as protection against
generation of reactive oxygen
species. Organisms also keep as
many iron and copper ions as
possible safely bound in storage or
transport proteins[37-39].
There is three times as much transferrin iron-binding
capacity in
plasma as iron needing to
be transported, so that there are
essentially no free iron ions in the plasma[38].
Iron ions bound
to transferrin cannot stimulate lipid
peroxidation or formation
of free .OH radicals. The same is
true of copper ions bound to
the plasma proteins ceruloplasmin or albumin[37-40].
The value of
this sequestration is shown by
an inspection of the pathology
suffered by patients with iron-overload
disease, in whom iron
ion-citrate chelates circulate in the
blood[40]. These patients
can suffer liver damage,
diabetes, joint inflammation, and
hepatoma, among other problems[41]. Metal ion
sequestration is an
important antioxidant defense. For
example, recent papers have
referred to ascorbic acid as a
major antioxidant in plasma.
However, ascorbate can only exert
antioxidant properties in the
absence of transition metal ions[11].
Tocopherol
As well as the primary defenses (scavenger
enzymes and metal-ion
sequestration), secondary defenses are also
present. The cell
membranes and plasma lipoproteins contain
[alpha]-tocopherol, a
lipidsoluble molecule
that functions as a chain-breaking
antioxidant. Attached
to the hydrophobic structure of
[alpha]-tocopherol is an -OH group whose hydrogen atom
is easily
removed. Hence, peroxyl and
alkoxyl radicals generated during
lipid peroxidation preferentially combine
with the antioxidant,
e.g.,
[Mathematical Expression Omitted]
instead of with an adjacent fatty acid side chain. This
therefore
terminates the chain reaction, whence
the term chain-breaking
antioxidant. It also converts the
[alpha]-tocopherol into a new
radical, tocopherol-O., which is
poorly reactive and unable to
attack adjacent fatty acid side chains, consequently
stopping the
chain reaction. Evidence
exists[43,44] that the tocopherol
radical can migrate to
the membrane surface and reconvert to
[alpha]-tocopherol by reaction with
ascorbic acid (vitamin C).
Both vitamin C and
[alpha]-tocopherol seem to minimize the
consequences of lipid
peroxidation in lipoproteins and in
membranes, should this process begin. Some thiol
compounds, such
as GSH, might also be involved in regenerating
[alpha]-tocopherol
from its radical in vivo[44].
The terms "[alpha]-tocopherol" and
"vitamin E" are often used
synonymously, which is not strictly correct. Vitamin E
is defined
nutritionally as a factor needed in the
diet of pregnant female
rats to prevent resorption of the fetus[45]
and compounds other
than
[alpha]-tocopherol (e.g.,
[beta-, gamma-, and
delta-tocopherols) have some effect
in this assay. However,
[alpha]-tocopherol is the most effective, and it
seems to be the
most important lipid-soluble chainbreaking antioxidant
in vivo in
humans[46]. The content of
[alpha]-tocopherol in circulating
low-density lipoproteins helps to determine their
resistance to
lipid peroxidation and thus
may affect the development of
atherosclerosis, a disease
in which lipid peroxidation is
involved[47]. Low plasma levels
of f [alpha]-tocopherol and
vitamin C correlate with an increased
incidence of myocardial
infarction and of some forms of cancer[47].
Other Antioxidants and Repair Systems
Some other compounds may also function as
antioxidants in vivo,
such as uric acid, ubiquinol, and bilirubin
(reviewed in[11].).
Antioxidant defenses are not quite perfect. Cells
contain systems
that can repair DNA after
attack by radicals[48], degrade
proteins damages by
radicals[49], and metabolize lipid
hydroperoxides[1].
What Is an Antioxidant?
"Antioxidant" can be define in
various ways. Often, the term is
implicitly restricted to chainbreaking antioxidant
inhibitors of
lipid peroxidation, such as
vitamin E. However, the author
prefers a broader definition - an
antioxidant is any substance
that, when present at low concentrations compared
with those of
an oxidizable substrate,
significantly delays or prevents
oxidation of that substrate[1]. The term
"oxidizable substrate"
includes almost everything found
in living cells, including
proteins, lipids, carbohydrates, and DNA.
Antioxidants act in many different ways
(Table I). In proposing
antioxidants for use in human
disease, it is important to note
the following: (a) the precise
role played in the disease
pathology by reactive oxygen
species; and (b) the molecular
targets of oxidative damage that need protecting. Thus,
oxidative
stress can damage a multiplicity of targets
in living cells and
the initial damage to one target can
then affect others[26].
Figure 2 attempts to illustrate some of the
complex interacting
mechanisms by which express production of reactive
oxygen species
can produce cell damage. If, for
example, the primary event in
damage to DNA, then an inhibitor of
lipid peroxidation might
offer little or no protection.
Questions to Ask When Evaluating the Proposed Role of
an
"Antioxidant" In Vivo
1. What biomolecule is
the compound supposed to protect? An
inhibitor of lipid peroxidation is unlikely
to be useful if the
oxidative damage is mediated by an attack on proteins
or DNA.
2. Is the compound present in vivo at or near that
biomolecule at
sufficient concentration? For example, many
compounds have been
suggested to act as .OH scavengers in vivo.
In order to compete
with biologic molecules for .OH, a
scavenger must be present in
at least millimolar concentrations in
vivo. Most drugs never
achieve this sort of concentration.
3. How does it protect: by scavenging reactive oxygen
species, by
preventing their formation, or by repairing damage?
4. For naturally
occurring antioxidants, is antioxidant
protection the primary biologic
role of the molecule or a
secondary one? For example, SOD
has probably evolved as an
antioxidant enzyme. By contrast, transferrin has
probably evolved
as an iron transport protein, although the
binding of iron ions
to transferrin prevents them from accelerating radical
reactions,
giving this protein an important secondary role
in extracellular
antioxidant defense.
5. If the antioxidant acts by
scavenging a reactive oxygen
species, can the
antioxidant-derived radicals themselves do
biologic damage?
6. Can the antioxidant cause damage in biologic systems
different
from those in which it exerts protection?
Free Radicals and Human Disease: Causation or
Consequence?
Does increased formation of
free radicals and other reactive
oxygen species cause
any human disease? Radiation-induced
carcinogenesis be initiated by free radical damage[48].
The signs
produced by chronic dietary
deficiencies of selenium (Keshan
disease) or of vitamin E (neurologic
disorders seen in patients
with inborn errors in the mechanism of intestinal fat
absorption)
could also be mediated by reactive oxygen
species[28,50]. In the
premature infant, exposure of
the incompletely vascularized
retina to elevated
concentrations of oxygen can lead to
retinopathy of prematurity, which in
its most severe forms can
result in blindness. Several
controlled clinical trials have
documented the efficiency of [alpha]-
tocopherol in minimizing
the retinopathy[51], suggesting a role for
lipid per-oxidation.
For most human diseases, increased
formation of reactive oxygen
species is secondary to the primary disease process.
For example, activated neutrophils produce
[O.sub.2-], and HOCl
in order to kill bacteria. If a large number of
phagocytes become
activated in a localized area, they
can produce tissue damage.
The synovial fluid in the
swollen knee joints of rheumatoid
patients swarm with activated neutrophils. There is
evidence that
reactive oxygen species and
other products derived from
neutrophils contribute to the
joint injury. Whether this is a
major or a minor contribution
to joint damage remains to be
established[52]. In some forms of
adult respiratory distress
syndrome (ARDS), lung damage seems to be mediated by an
influx of
neutrophils into the lung, where they become activated
to produce
prostaglandins, leukotrienes,
proteolytic enzymes such as
elastase, and reactive oxygen species[53].
Among other effects,
reactive oxygen species inactivate
proteins (such as [alpha
1]-antiproteinase) within the
lung that normally inhibit the
action of elastase and prevent
it from attacking lung elastic
fibers. The precise contribution of
oxidative damage to lung
injury in ARDS is unknown, but deserves
investigation in view of
the high mortality rate.
In both ARDS and in rheumatoid arthritis, increased
generation of
reactive oxygen species is secondary to the
processes that cause
neutrophil infiltration, but they then may
make an additional
detrimental contribution to tissue injury.
There are several examples
in which injury, by a nonradical
mechanism, leads to increased free radical
reactions. Mechanical
(e.g., crushing) or chemical injury to tissues can
cause cells to
rupture and release their contents,
including transition metal
ions (Figure 1), into the
surrounding area. Administration of
cytotoxic drugs to patients with acute myeloid
leukemia has been
shown to create a temporary "iron-overload"
state, probably due
to extensive drug-induced lysis
of the leukemic cells. This
increased iron availability could contribute to
the side effects
of cytotoxic chemotherapy[54].
Perhaps the greatest interest in this area lies
in the sequelae
of traumatic or ischemic injury to the
brain. Some Areas of the
human brain are rich in
iron. Cerebrospinal fluid has no
significant ironbinding
capacity, since its content of
transferrin is low. It has been proposed[55] that
injury to the
brain by mechanical means
(trauma) or by oxygen deprivation
(stroke) can result in release of iron ions into
the surrounding
area. These ions facilitate further damage
to the surrounding
areas by accelerating free radical
reactions. This proposal has
been given some support from animal
studies, using antioxidants
such as chelating agents that bind iron
ions and prevent from
catalyzing radical
reactions. Promising results have been
obtained with amino-steroid-based
antioxidants. Thus, one such
"lazaroid," U74006F, has been observed to
decrease the effects of
reperfusion injury upon the
brain of cats[56] to decrease
post-traumatic spinal cord
degeneration in cats[57] and to
minimize neurologic damage after head injury in
mice[58].
Free Radicals in Human Disease: A Triviality?
Tissue destruction and degeneration
can result in increased
oxidative damage, by such
processes as metal-ion release,
phagocyte activation, lipoxygenase activation,
and disruption of
mitochondrial electron transport chains, so that
more electrons
"escape" to oxygen to form [O.sub.2-] .
(Figure 1).
If follows that almost any disease is likely to be
accompanied by
increased formation of reactive
oxygen species. It is not
therefore surprising that the
list of diseases in which their
formation has been implicated is long and
is growing longer[1].
For atherosclerosis[43,59], rheumatoid arthritis[52],
some forms
of ARDS, reoxygeneration injury[60,61], and traumatic
or ischemic
damage to the central
nervous system, there is reasonable
evidence to suggest
that free radical reactions make a
significant detrimental contribution to the
pathologic process.
As previously stressed[62], it is equally
likely that in some
(perhaps most) diseases, the
increased ROS formation is an
epiphenomenon, making no
significant contribution to the
progression of the disease. Each proposal
must be subject to
stringent examination, because
the likely clinical value of
"antioxidant therapy" will depend on
how well the exact role of
reactive oxygen species is known.
[1.] Halliwell B, Gutteridge, JMC. Free
radicals in biology and
medicine. Oxford: Clarendon Press, 1989.
[2.] Cammack R. Electron spin
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plants, Vol. 13. New York: Academic Press, 1987-229-57.
[3.] Slater TF. Free radical mechanisms
in tissue injury. Biochem
J 1984; 222: 1-15.
[4.] Scott G. Potential toxicological
problems associated with
antioxidants in plastic and
rubberconsumable. Free Radic Res
Commun 1988; 5: 141-7.
[5.] Daniels V.Oxidative damage and the
preservation of organic
artefacts. Free Radic Res Commun 1988; 5: 213-20.
[6.] Aruoma Ol, Halliwell B,
Dizdaroglu M. Iron ion-dependent
modification of bases in DNA by the superoxide
radical-generating
system hypoxanthine/xanthine oxidase. J
Biol Chem 1989; 264:
13024-8.
[7.] Asmus KD, Sulphur-centered free radicals. In:
Slater TF, ed.
Radioprotectors and anticarcinogens.
London: Academic Press,
1983: 23-42.
[8.] Sevilla MD, Yan M, Becker D, Gillich
S. ESR investigations
of the reactions of
radiation-produced thiyl and DNA peroxyl
radicals: formation of sulfoxyl radicals. Free Radic
Res Commun
1989; 6: 21-4.
[9.] Monig J, Asmus KD,Forni LG,
Wilson RL. On the reaction of
molecular oxegen with thiyl radicals:
a re-examination. Int J
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[10.] Aruoma Ol, Halliwell B,
Butler J, Hoey BM. Apparent
inactivation
of [alpha.sub.1-]
antipoteinase by
sulphur-containing radicals derived from
penicillamine. Biochem
Pharmacol 1989; 38: 4353-7.
[11.] Halliwell B. How to characterize a
biologic antioxidant.
Free Radic Res Commun 1990; 9: 569-71.
[12.] Esterbauer H, Zollner
H, Schaur RJ. Hydroxyalkenals:
cytotoxic products of lipid peroxidation.
ISI Atlas Sci Biochem
1988; 1: 311-5.
[13.] Curzio
M. Interaction between
neutrophils and
4-hydroxylkenals and consequences on neutrophil
mobility. Free
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[14.] Dean RT, Thomas
SM, Garner A. Free-radical-mediated
fragmentation of monoamine oxidase in the mitochondrial
membrane.
Role of lipid radicals. Biochem J 1986; 240: 489-94.
[15.] Fridovich 1. Superoxide dismutases.
Adv Enzymol 1974; 41:
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[16.] Fridovich I. Superoxide radical: an
endogenous toxicant.
Annu Rev Pharmacol Toxicol 1983; 23: 239-57.
[17.] Wolff SP,Dean
RT. Glucose autoxidation and protein
modification. The potential role of "Autoxidative
glycosylation"
in diabetes. Biochem J 1987; 245: 243-50.
[18.] Albro PW, Corbett JT, Schoeder JL.
General of hydrogen
peroxide by incidental
metal ion-catalyzed autoxidation of
glutathione. J Inorg Biochem 1986; 27: 191-203.
[19.] Vina J, Saez GT, Wiggins D, Roberts AFC, Hems R,
Krebs, HA.
The effect of cysteine oxidation on isolated
hepatocytes. Biochem
J 1983; 212: 39-44.
[20.] Rowley DA, Halliwell B.
Superoxide-dependent formation of
hydroxyl radicals in the presence of thiol compounds.
FEBS Letts
1982; 138: 33-6.
[21.] Curnutte JT, Babior Chronic granulomatous
disease. Adv Hum
Genet 1987; 16: 229-45.
[22.] Weiss SS. Tissue destruction by
neutrophils. N Engl J Med
1989; 320: 365-76.
[23.] Aruoma Ol, Halliwell B, Hoey BM, Butler J.
The antioxidant
action of N-acetylcysteine. Free Radic Biol Med 1989;
6: 593-7.
[24.] Puppo A, Cecchini
R, Aruoma Ol, Bolli R,Halliwell B.
Scavenging of hypochlorous acid and of myoglobin-derived
species
by the cardioprotective agent
mercaptopropionylglycine. Free
Radic Res Commun 1990; 10: 371-81.
[25.] Farr SB, D'Ari R, Touati D. Oxygen-dependent
mutagenesis in
Escherichia coli lacking superoxide dismutase. Proc
Natl Acad Sci
USA 1986; 83: 8268-72.
[26.] Halliwell B, Aruoma Ol.
DNA damage by oxygen-derived
species. Its mechanism and measurement in mammalian
systems. FEBS
Lett 1991; 281: 9-19.
[27.] Change B, Sies H,Boveris
A. Hydroperoxide metabolism in
mammalian organs. Physiol Rev 1979; 59: 527-605.
[28.] Levander OA. A global
view of human selenium nutrition.
Annu Rev Nutr 1987; 7: 227-50.
[29.] Hill HAO, Oxygen, oxidases and the
essential trace metals
Philos Trans R Soc Lond Ser B 1981; 294: 294-28.
[30.] Grootveld M, Jain R. Recent advances in the
development of
a diagnostic test for
irradiated foodstuffs. Free Radic Res
Commun 1989; 6:271-92.
[31.] Aruoma Ol, Jalliwell B,Gajewski E,
Dizdaroglu M. Copper
ion-dependent damage to the
bases in DNA in the presence of
hydrogen peroxide. Biochemistry J 1991; 273: 601-04.
[32.] Minotti G, Aust SD. The role of iron
in the initiation of
lipid peroxidation. Chem Phys Lipids 1987; 44: 191-208.
[33.] Aruoma Ol, Halliwell B, Laughton MJ, Quilan JG,
Gutteridge
JMC. The mechanism of initiation of lipid
peroxidation. Evidence
against a requirement for an iron(ll)-iron(lll)
complex. Biochem
J 1989; 258: 617-20.
[34.] Gutteridge JMC. Lipid
peroxidation: some problems and
concepts. In: Halliwell B, ed. Oxygen radicals and
tissue injury.
Kansas: Allen Press, 1988; 9-19.
[35.] Haenen GRMM, Vermeulen NPE, Timmerman H,
Bast A, Effect of
thiols on lipid peroxidation in rat liver
microsomes. Chem Biol
Interact 1989; 71: 201-12.
[36.] Schoneich C, Asmus KD, Dillinger
U,Bruchhausen F. Thiyl
radical attack on polyunsaturated fatty
acids: a possible route
to lipid peroxidation. Biochem
Biophys Res Commun 1989; 161:
113-20.
[37.] Halliwell B.
Albumin, an important extracellular
antioxidant? Biochem Pharmacol 1988; 37: 569-71.
[38.] Halliwell B, Gutteridge JMC. Oxygen free
radicals and iron
in relation to biology and medicine: some
problems and concepts.
Arch Biochem Biophys 1986; 246: 501-14.
[39.] Aruoma Ol,
Haliwell B. Superoxide-dependent and
ascorbate-dependent formation of hydroxyl radicals
from hydrogen
peroxide in the presence of iron. Are lactoferrin and
transferrin
promoters of hydroxyl-radical
generation? Biochem J 1987; 241:
273-8.
[40.] Grootveld M, Bell JD, Halliwell
B, Aruoma Ol, Bomford A,
Sadler PJ. Non-transferrin-bound iron in
plasma or serum from
patients with idiopathic
hemochromatosis. Characterization by
high performance liquid
chromatography and nuclear magnetic
resonance spectroscopy. J Biol Chem 1989; 264: 4417-22.
[41.] McLaren GD, Muir
WA, Kellermeyer RW. Iron overload
disorders: natural history, pathogenesis, diagnosis
and therapy.
CRC Crit Rev Clin Lab Sci 1983; 19: 205-66.
[42.] McCay PB. Vitamin E:
interactions with free radicals and
ascorbate. Annu Rev Nutr 1985; 5; 323-40.
[43.] Esterbauer H, Striegl G, Puhl
H, Rotheneder M.Continuous
monitoring of in vitro
oxidation of human low density
lipoprotein. Free Radic Commun 1989; 6: 67-75.
[44.] Wefers H,
Sies H.The protection by ascorbate and
glutathione against microsomal lipid peroxidation is
dependent on
vitamin E. Eur J Biochem 1988; 174: 353-7.
[45.] Diplock AT, ed. Fat-soluble
vitamins, their biochemistry
and applications. London: Heinemann, 1985: 154-224.
[46.] Ingold KU, Burton GW, Foster DO, et al.
A new vitamin E
analogue more active than
[alpha]-tocopherol in the curative
myopathy biossay. FEBS Lett 1986; 205: 117-20.
[47.] Gey KF, Brubacher
GB, Stahelin HB. Plasma levels of
antioxidant vitamins in
relation to ischemic heart disease
cancer. Am J Clin Nutr 1987; 45: 1368-77.
[48.] Breimer LH. lonizing radiation-induced
mutagenesis. Br J
Cancer 1988; 57: 6-18. [49.] Marcillat O. Zhang Y, Lin
SW, Davies
KJA, Mitochondria contain aproteolytic system which can
recognize
and degrade oxidatively-denatured proteins.
Biochem J 1988; 254:
677-83.
[50.] Muller DPR, Lloyd JK, Wolff OH. Vitamin E
and neurological
function. Lancet 1983; 1: 225-7.
[51.] Kretzer FL, Mehta RS, Johnson AT,
Hunter DG, Brown ES,
Hittner HM. Vitamin E protects against retinopathy of
prematurity
through action on spindle cells. Nature (Lond) 1984;
309: 793-5.
[52.] Halliwell B, Hoult JRS, Blake
DR. Oxidants, inflammation
and the anti-inflammatory drugs. FASEB J 1989; 2:
2867-73.
[53.] Baldwin SR, Simon RH, Grum CM, Ketai
LH, Boxer LA, Devall
LJ. Oxidant activity in expired breath of
patients with adult
respiratory distress syndrome. Lancet 1986; 1: 11-4.
[54.] Halliwell B,
Aruoma Ol, Mufti G, Bomford
A.
Bleomycin-detectable iron in serum from leukaemic
patients before
and after chemotherapy. Therpeutic
implications for treatment
with oxidant-generating drugs. FEBS Lett 1988; 241:
202-4.
[55.] Halliwell B, Gutteridge
JMC. Oxygen radicals and the
nervous system. Trends Neurosci 1985; 8: 22-6.
[56.] Hall ED Yonkers PA. Attenuation of
postischemic cerebral
hypoperfusion by the 21-aminosteroid
U74006F. Stroke 1988; 19;
340-4.
[57.] Hall ED. Effect
of the 21-aminosteroid U74006F on
post-traumatic spinal cord ischemia in cats.
J Neurosurg 1988;
68: 462-5.
[58.] Hall ED, Yonkers PA, McCall JM,
Braughler JM. Effects of
the 21-aminosteroid U74006 on experimental head injury
in mice. J
Neurosurg 1988; 68: 456-61.
[59.] Steinberg D, Parthasarathy S, Carew
TE, Khoo JC, Witztum
JL, Beyond cholesterol. Modifications of
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that increase its atherogenicity. N Engl J Med 1989;
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1988; 12: 239-49.
[61.] McCord JM. Oxygen -derived free radicals in
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[62.] Halliwell B, Gutteridge
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1396-8.
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