Medical Biology 62:71-77, 1984

Oxygen Radicals: A Commonsense Look at Their Nature and Medical
Importance

B. Halliwell

>From the Department of Biochemistry, University of London King's
College, London, U.K.

Introduction

"Oxygen radicals" are now popular subjects for research papers;
several hundred are published each year. Many of these pass
rapidly into oblivion, joining the great mass of unread
scientific literature that clogs library shelves and dilutes
important research findings to an increasingly great extent. The
basic chemistry of oxygen-derived species was established years
ago by radiation chemists (1,6), but "superoxide" is still
endowed with miraculous properties by the uninitiated.
Demonstration that the action of a disease or toxin in vivo
produces increased lipid peroxidation (a currently-popular
scientific activity) means nothing more than the fact that its
action produces increased lipid peroxidation: it does not
automatically follow that the lipid peroxidation causes the
damaging effects of the drug or disease.

The purpose of this paper is to explain:
i) what oxygen radicals are
ii) the evidence that oxygen radicals are important in vivo
iii) what needs to be done to establish a role for oxygen
radicals and lipid peroxidation in human disease.


What are the oxygen radicals and how are they produced?

Electrons within atoms and molecules occupy regions of space
known as "orbitals". Each orbital can hold a maximum of two
electrons. A single electron alone in an orbital is said to be
"unpaired" and a radical is defined as any species that contains
one or more unpaired electrons. Such a definition embraces the
atom of hydrogen (one unpaired electron) and the ions of such
transition metals as iron, copper and manganese (cf. Holmberg,
this volume).

The diatomic oxygen molecule, O2, has two unpaired electrons and
thus qualifies as a radical. Most of the oxygen taken up by
human cells is reduced to water by the action of the cytochrome
oxidase complex in mitochondria. This requires the addition of
four electrons to each oxygen molecule,

O2 + 4H+ + 4e- ---> 2H2O (1)

For chemical reasons (reviewed in ref. 21 and 28), O2 likes to
receive its electrons one at a time, producing a series of
partially reduced intermediates

O2 add le- O2- add le- H2O2 add le-
---> ---> --->
2H
(two unpaired superoxide hydrogen peroxide
electrons) (one unpaired (no unpaired electron)
electron)

OH + OH-

hydroxyl radical hydroxyl ion (2)
(one unpaired electron) (no unpaired electron)

|
| |
| add le- H+ | add H+
H2O2 H2O2

Cytochrome oxidase keeps the partially reduced intermediates on
the pathway to water tightly bound to its active site (21); they
do not escape into free solution.


Superoxide

Superoxide ion is the one-electron reduction product of oxygen.
Dissolved in organic solvents, it is an extremely reactive
species, e.g. it can displace chlorine from such unreactive
chlorinated hydrocarbons as carbon tetrachloride (CCl4) (40). In
aqueous solution O2- is poorly reactive, acting as a reducing
agent (e.g. it will reduce cytochrome c or nitro-blue
tetrazolium) and slowly undergoing the dismutation reaction, in
which one molecule of superoxide reduces another one to form
hydrogen peroxide (H2O2 ). The dismutation reaction occurs in
stages; O2- must first combine with a proton to yield the
hydroperoxyl radical, HO2,

O2- + H+ ---> HO2 (3)
HO2 + O2 + H+ ---> H2O2 + O2 (4)
--------------------------------------------------------
overall O2- + O2- + 2H+ ---> H2O2 + O2 (5)

At physiological pH the low concentration of H+ ions slows the
rate of dismutation.

Despite the low reactivity of O2- in aqueous solution, systems
producing it do a great deal of damage in vitro (e.g. they
fragment DNA and polysaccharides, kill bacteria and animal cells
in culture) and in vivo (e.g. when O2- generating systems are
injected into the footpads of rats inflammation is produced,
their instillation into the lungs of rats and rabbits produces
oedema and cell death, and infusion of them into vascular beds
produces endothelial cell damage and extensive leakage from the
blood vessels) (21,26,28). Depending on the circumstances,
damage caused by O2- generating systems might be attributed to

(i) O2- itself, e.g. exposure of tissue fluids to O2- causes
formation of a factor chemotactic for neutrophils that
brings more of them into the area and hence can potentiate
inflammation
(ii) HO2 radical, which is more reactive than O2- (6).
Formation of HO2 is favoured at pH values lower than
"physiological", but the phagocytic vacuole operates at an
acid pH and the pericellular pH of macrophages has been
reported to be 6 or less (15)
(iii) H2O2 (see below)
(iv) hydroxyl radical (see below)
(v) singlet oxygen. Singlet O2 is an especially reactive form
of oxygen capable of rapidly oxidising many molecules,
including membrane lipids. Its formation in O2--
generating systems has often been proposed but clear-cut
evidence for a damaging role of singlet O2 in such systems
has not been obtained. One of the problems is that the
"scavengers" of singlet O2 frequently used react with
other radical species as well (for reviews see ref. 26 and
28).


What is the evidence that O2- is formed in vivo in human cells?

Any electron transport chain operating in the presence of O2
"leaks" some of the electrons, passing them directly onto O2.
Since O2 prefers to take electrons one at a time, O2- is
produced. Such O2- production can be demonstrated in vitro using
mitochondria and microsomes from a range of animal tissues. The
rate at which O2- is produced rises as the concentration of O2 in
the system is raised (e.g. see ref. 20). A number of compounds
slowly become oxidised on exposure to O2 and O2- is generated;
these include adrenalin, tetrahydrofolate, reduced FMN and
oxyhaemoglobin (21,24).

Since human cells contain mitochondria, endoplasmic reticulum,
oxidisable compounds and oxygen, it is likely that O2- is formed
within them in vivo. Backing up this evidence, for those who do
not like extrapolating from in vitro experiments, is the fact
that human cells contain high levels of superoxide dismutase
(SOD) activity (45). This enzyme, for which O2- is the specific
substrate (35), is known to be a very important anti-oxidant in
bacteria and small mammals (26) and its presence in human cells
is good evidence that O2- is formed in vivo. During the
maturation of erythrocytes most enzymes are lost, but SOD
remains. It is not a great stretch of the imagination to
associate this with the ability of oxyhaemoglobin to release O2-
radical and methaemoglobin.

Another source of O2- in vivo is the respiratory burst of
phagocytic cells such as neutrophils, monocytes, eosinophils and
macrophages (3, 16, 25). The amount of O2- produced might
sometimes be controlled by the O2 tension of body fluids (14).
Host defence against invading bacteria is dependent on the
circulating neutrophils, which respond to contact with particles
they recognise as foreign by producing a "burst" of O2 radical.
The particle is engulfed (the piece of membrane surrounding it
being the segment that produces O2- on contact; cf. Segal, this
volume), and other vesicles then fuse with the phagocytic
vesicle. This exposes the engulfed particle to other anti-
bacterial mechanisms, including cationic proteins, lysosomal
enzymes and myeloperoxidase (3, 16, 25).

Which of these processes is the most important in bacterial
killing? Human and other animal neutrophils can kill some
strains of bacteria under anaerobic conditions, when O2- cannot
form. Obviously, the other mechanisms are important here. Many
other bacterial strains are not killed in the absence of O2,
however, even though engulfment and vesicle fusion proceed
normally. In chronic granulomatous disease (CGD), an inborn
error of metabolism, the respiratory burst does not occur but
other aspects of phagocytic action proceed normally. CGD was
first described in humans because it is accompanied by severe and
recurrent infections affecting lymph nodes, skin, lungs and liver
(43). The symptoms of CGD provide direct evidence for the
production of O2- by human phagocytic cells in vivo and for its
role in bacterial killing.

It follows therefore that if neutrophils become activated in the
wrong place, or to excessive extents (as in the autoimmune
diseases, 25) then the oxygen radicals they release could do a
lot of damage. It must be remembered, however, that phagocytic
cells also produce hydrolytic enzymes (elastase, neutral
proteases etc.), chemotactic factors, prostaglandins,
leukotrienes and other chemicals, so that damage by activated
phagocytes could be due to any one of these factors or to any
combination of them. It cannot be attributed a priori to oxygen
radicals.


Hydrogen Peroxide

O2- generating systems produce H2O2 by the dismutation reaction
(eqn. 5) and a number of oxidase enzymes produce H2O2 directly,
examples being glycollate oxidase and amino acid oxidases. SOD
enzymes remove O2- by greatly accelerating the dismutation
reaction, so if we accept that O2- is formed in vivo in humans
then we must accept that H2O2 vapour is present in expired human
breath (48), a likely source being H2O2 released from alveolar
macrophages (3, 25) although a contribution from peroxide-
producing oral bacteria (10) cannot be ruled out.

That H2O2 is formed in vivo in humans is further supported by the
presence of enzymes specific for its removal, such as catalase
and glutathione peroxidase. The latter enzyme requires selenium
for its activity (13; cf. Diplock, this volume). H2O2 is
probably more damaging than is O2- in in vitro experiments in
aqueous solution, but many cells seem to tolerate its presence
and bacteria often produce H2O2 (e.g. ref. 10). On the other
hand, the toxicity of O2- generating systems to several animal
cells in culture has been attributed to formation of H2O2 (e.g.
ref. 44). Why this should be so is discussed in the next
section.


Hydroxyl radical

Hydroxyl radical is produced when water is exposed to high-energy
ionising radiation and hence its properties have been well
documented by radiation chemists (6, 49). Unlike the hydroxyl
ion, the hydroxyl radical is fearsomely reactive, combining with
most molecules found in vivo at near diffusion-controlled rates.
Hence any OH produced in vivo will react at or close to its site
of formation. The extent of the damage done would therefore
depend on what the site of formation was (e.g. production of OH
close to DNA could lead to strand breakage whereas production
close to an enzyme molecule already present in excess in the
cell, such as lactate dehydrogenase, might have no biological
consequences).

Hydroxyl radical is produced whenever H2O2 comes into contact
with copper (I) ions (Cu+) or iron (II) ions (Fe2+). Dr.
Gutteridge has reviewed in this volume the substantial evidence
that metal complexes capable of causing hydroxyl radical
formation are present in vivo in human cells (also see ref. 28).
Particularly important in vivo are complexes of iron salts with
phosphate esters such as ATP and GTP (17, 19) or with DNA (18).
Organisms take great care to ensure that as much iron or copper
as possible is bound to transport proteins or functional proteins
such as transferrin, caeruloplasmin or haemoglobin. Metals bound
to these proteins are inactive or only weakly active in
catalysing OH production (28, 50).

Since both H2O2 and metal complexes are present in vivo in
humans, it is logical to assume that OH radicals can form.
Direct evidence for this is difficult to obtain. Many methods
exist for demonstrating the existence of OH in vitro (see ref. 24
and 28 for reviews) but in vivo any OH formed is likely to react
so close to its site of formation that the use of these methods
is impractical, although some new techniques (such as the ability
of OH to convert dimethylsulphoxide into methane (36) or its
ability to hydroxylate aromatic rings in characteristic ways (37)
show promise for in vivo use. One can also attempt to infer the
formation of OH radical in vivo by observing the damage done (as
in rheumatoid arthritis, see below). In vitro, phagocytic cells
have been shown to produce OH radical (11-13) and the killing of
bacteria can sometimes be prevented by reagents that react with
this species (3, 16, 25).

It was mentioned in the previous section that the killing of
animal cells in culture by O2- generating systems can sometimes
be attributed to H2O2. It could, of course, be achieved by H2O2
itself; some enzymes are known to be inactivated by H2O2 although
the best examples come from plant rather than animal systems
(11). There is another possibility, however, H2O2 generated
externally crosses cell membranes easily and could penetrate
inside the cell and cause OH to be formed. Externally added
scavengers of OH would not prevent this since they could not
reach the correct place. By contrast, O2- crosses cell membranes
only slowly (42) unless there is a specific channel for it (the
only known example of this being the erythrocyte membrane, which
has an "anion channel" through which O2- can move(3). Hydroxyl
radical will never cross a membrane: it will react with whatever
membrane component if meets first.


What is lipid peroxidation and is it of medical importance?

Lipid peroxidation has been broadly defined by A. L. Tappel in
the USA as "oxidative deterioration of polyunsaturated fatty
acids", i.e. fatty acids that contain more than two carbon-carbon
double bonds. Oxygen-dependent deterioration, leading to
rancidity, has been long recognised as a problem in the storage
of fats and oils and is even more relevant today with the
popularity of "polyunsaturated" food products. Some of the best
studies on peroxidation chemistry have been carried out by food
chemists.

Initiation of peroxidation in a membrane or polyunsaturated fatty
acid is due to the attack of any species that can "pull off" a
hydrogen atom from one of the - CH2 - groups in the carbon chain.
Hydroxyl radical and possibly HO2 can do this, but H2O2 and O2-
cannot. Hence O2- does not initiate lipid peroxidation. Since a
hydrogen atom has only one electron, removing it leaves behind an
unpaired electron on the carbon. The resulting carbon radical -
CH -, undergoes molecular rearrangement to form a conjugated
diene, which then combines rapidly with O2 to give a

O2
|
peroxy radical, - CH -. Peroxy radicals are capable of
abstracting a hydrogen atom from other fatty acids and so setting
off a chain reaction that can continue until the membrane fatty
acids are completely oxidised to hydroperoxides (eqn. 6)

O2
|
- CH - + - CH2 - --->
peroxy adjacent fatty acid
radical carbon chain

O2H
|
- CH - + - CH - (6)
carbon radical, lipid
forms another hydroperoxide
peroxy radical

Lipid hydroperoxides are stable under physiological conditions
until they come into contact with transition metals such as iron
or copper salts. Cu2+, Fe2+ or Fe3+ salts as well as haem and
haem proteins (e.g. cytochromes, haemoglobin) can interact with
lipid peroxides. These metals or their complexes cause lipid
hydroperoxides to decompose in very complicated ways, producing
radicals that can continue the chain reaction of lipid
peroxidation (as in eqn. 6), as well as cytotoxic aldehydes and
hydrocarbon gases. Most attention is paid in the literature to
malonaldehyde, but this is a very minor endproduct of lipid
peroxidation (for reviews see ref. 4, 26, 32).

Does lipid peroxidation occur normally in vivo in humans? This
question is surprisingly difficult to answer: little evidence for
lipid peroxides or their decomposition products can be found in
healthy human tissues (28). Expired human breath contains
gaseous hydrocarbons that might have originated from
decomposition of lipid hydroperoxides, but they might also have
been produced by bacteria in the gut or even on the skin. Animal
cell membranes contain tocopherol (vitamin E), which is a
powerful inhibitor of lipid peroxidation, and proteins such as
caeruloplasmin and glutathione peroxidase probably help to
protect against this process in vivo (27).

Diseased tissues, or tissues isolated after exposure of animals
to such toxins as ethanol, phenylhydrazine and paraquat often
show evidence of increased peroxidation. Simple in vitro
experiments demonstrate quite clearly that dead or damaged
tissues peroxidise more rapidly than living ones, presumably
because of membrane disruption by enzymes released from
lysosomes, release of metal ions from their storage sites and
failure of antioxidant mechanisms. Thus evidence that a toxin
increases lipid peroxidation in vivo does not prove the sequence
of events


toxin ---> lipid peroxidation ---> damage (7)

but is equally explained by the sequence

toxin ---> cell damage or death ---> lipid peroxidation (8)

Of course, toxins released by dead or dying cells undergoing
peroxidation might cause further damage to healthy cells,
although there is little evidence for this in vivo. Among the
many claims I have seen in the literature for lipid peroxidation
as an agent of the damage induced by a toxin, I have seen clear
evidence for sequence 7 only in the case of the hepatotoxic
effects of carbon tetrachloride (32). Sequence 8 is a much
better explanation of the in vivo effects on membrane lipids of,
for example, paraquat.

An often quoted illustration of the importance of lipid
peroxidation in vivo is the accumulation of "age pigment" in
various human tissues. Chemical analysis of age pigment shows
convincingly that it is an endproduct of oxidative damage to
lipids (41). However, the lipids in question seem to be taken
into lysosomes before they are degraded; they are not "normal
cell lipids". The exposure of lipids to hydrolytic enzymes and
metal ions within lysosomes no doubt facilitates their
peroxidation, and so more peroxidised material accumulates within
cells as lysosomes get older and have engulfed more lipid
material.


The TBA test

The TBA (thiobarbituric acid) test is one of the most widely used
(and abused!) tests for measuring lipid peroxidation. The
simplicity of performing the test (the material under study is
merely heated with acid and TBA and the formation of a pink
colour measured at 523 nm) conceals its essential complexity.

Consider a typical experiment. A lipid system, perhaps with
added metal ions, chelating agents or other reagents, in
incubated in the presence of air. Then TBA plus acid are added
and the mixture heated at 100 degrees Celcius. The air, metals
and other reagents are still present, so as much or even more
oxidative damage to the lipid can be done during the TBA test
itself as happened during the initial incubation.

The pink colour is due to the formation of an adduct between TBA
and malonaldehyde (MDA) under acidic conditions. Indeed, the TBA
assay is often calibrated with MDA and the results of
peroxidation assays are often expressed as "amounts of MDA
formed". Some papers in the literature give the mistaken
impression that TBA reacts only with free MDA and so measures the
production, but it was shown as long ago at 1958 in studies with
peroxidising fish oil that 98 % of the MDA that reacts in the TBA
test was not present in the original sample assayed but forms
from lipid peroxides that decomposed during the acid-heating
stage of the TBA assay. More recent studies confirm this and
show that the apparent "TBA reactivity" of say, serum, varies
with the exact concentration of acid, type of acid and period of
heating used in the TBA assay (23). The amount of MDA formed
during the initial incubation of the system as opposed to during
the assay depends on such factors as the iron salt concentration
(4, 23, 32). An apparent "inhibitor" of lipid peroxidation as
detected by the TBA test might actually inhibit the peroxidation
process, but could equally well interfere with decomposition of
the peroxides during the acid-heating stage of the assay.
Similarly, absolute values for the "TBA reactivity" of body
fluids or tissue extracts are meaningless, although changes in
these values may be significant provided that the same assay in
employed in the same way each time.

Of course, many scientists are aware of these problems with the
TBA assay and there are ways around them (2, 41), including the
use of other assay systems in conjunction with the TBA test (4,
27). I have included these cautions to encourage a more critical
attitude to some of the published literature.


Oxygen Radicals and Disease

Free radicals have been suggested to be involved in the pathology
of a number of diseases. In several cases the evidence consists
only of observations of increased lipid peroxidation in diseased
tissues, which is ambiguous (see above). I have chose to look in
detail at two cases where the evidence at first sight is more
convincing, cancer and inflammatory joint disease.

Cancer

Any substance that reacts with DNA is potentially carcinogenic.
Exposure of DNA to O2- generating systems causes extensive strand
breakage and degradation of deoxyribose (9, 39), an effect shown
in vitro to be due to formation of OH. Both bacteria and animal
cells in culture suffer DNA damage on exposure to O2- generating
systems, which can be shown to be mutagenic (46, 47). It is
therefore tempting to attribute the increased risk of development
of cancer in chronically inflamed tissues to generation of oxygen
radicals by phagocytic cells, although there is no direct
evidence for this.

Great excitement was generated by reports that cancer cells in
culture and from some transplantable tumours in animals are
deficient in SOD activity, especially in their mitochondria (for
a review see ref. 34). The relevance of these studies to human
cancer is not at all clear, however, since human tumours biopsied
during surgery show no defects in any SOD activity (31, 45).

Rheumatoid arthritis

I have already speculated on the role of oxygen radicals in the
autoimmune diseases. Rheumatoid arthritis has some of the
features of an autoimmune disease but its exact cause is unknown.
The synovial fluid of the inflamed joint swarms with neutrophils.
Since the fluid contains increased concentrations of products
that activated neutrophils release (including lactoferrin, 5) and
end-products of arachidonic acid metabolism), then at least some
of these neutrophils must be activated and thus producing
superoxide, and hence H2O2 in vivo. Human synovial fluid is poor
in SOD, catalase and glutathione peroxidase activities (8) but
does contain iron complexes capable of catalyzing a reaction
between O2- and H2O2 to form OH (38). There is as yet no direct
proof that OH is formed in vivo, but evidence consistent with its
formation includes the observation that the hyaluronic acid in
synovial fluid is degraded in rheumatoid joints, and the type of
degradation observed can be reproduced by exposing pure
hyaluronic acid in vitro to OH radical (22). TBA-reactive
material is also present in serum and synovial fluid of
rheumatoid patients. There are significant correlations (38)
between the content of TBA-reactive material in synovial fluid,
its content of catalytic iron complexes and both clinical ("knee
score") and laboratory ("white cell count" and "fluid content of
C-reactive protein") assessments of disease activity.

Thus there is certainly evidence for oxygen radicals being
produced in the rheumatoid joint and having some deleterious
effects. The question to be answered in how important are oxygen
radicals in relation to other agents of damage. The pathology of
rheumatoid arthritis is very complex and the number of
potentially damaging agents, including hydrolytic enzymes,
prostaglandins and leukotrienes, is enormous (29). Some
scientist have tried to assess the importance of oxygen radicals
by examining the effects of injecting SOD directly into inflamed
joints (33; see Marklund, this volume), whereas our group,
reasoning that iron complexes are required for O2- dependent
formation of highly reactive OH radical, is examining the effect
of iron-chelating drugs that can prevent OH formation (such as
desferrioxamine, 12) on animal models of acute and chronic
inflammation (7).


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Address: B. Halliwell
Department of Biochemistry, University of London
King's College
Strand, London
UK