Fre
e ra di cals and antiox idans
Freeradicals, antioxidants, and human disease: curiosity, cause,
or consequence?
"Free radicals are implicated in Parkinson’s disease";
"taking vitamin E capsules will prevent heart attacks"; "our
anti-ageing cream contains free radical scavengers."
Comments such as these, found in the expert and lay press,
show that free radicals and antioxidants have become "hot
news".
What is a free radical? Atoms contain a nucleus, and
electrons move around the nucleus, usually in pairs. A free
radical is any atom or molecule that contains one or more
unpaired electrons. The unpaired electrons alter the
chemical reactivity of an atom or molecule, usually making
it more reactive than the corresponding non-radical.
However, the actual chemical reactivity of radicals varies
enormously. The hydrogen radical (H*, the same as a
hydrogen atom), which contains 1 proton and 1 electron
(therefore unpaired), is the simplest free radical. Free-
radical chain reactions are often initiated by removal of H*
from other molecules (eg, during lipid peroxidation, figure
1). A superscripted dot is used to denote free radicals.
What radicals are made In the human body?
We are exposed to electromagnetic radiation from the
environment, both natural (eg, radon and cosmic radiation)
and from man-made sources. Low-wavelength
electromagnetic radiation (eg, gamma rays) can split water
in the body to generate hydroxyl radical, OH*. This
fearsomely-reactive radical,l once generated, attacks
whatever it is next to. Its lifetime in vivo is vanishingly
small because hydroxyl radical reacts at its site of formation,
usually leaving behind a legacy in the form of propagating
free-radical chain reactions.1
The body makes another oxygen radical (ie, the unpaired
electron is located on oxygen), superoxide (0;-).
Superoxide is made by adding one electron to the oxygen
molecule. It is generally poorly reactive.1,2 Some
superoxide is made by "accidents of chemistry", in that
many molecules in the body react directly with oxygen to
make superoxide. Examples include the catecholamines,
tetrahydrofolates, and some constituents of mitochondrial
and other electron-transport chains.1,2 Such superoxide
generation is unavoidable. In addition, some superoxide is
made deliberately., For instance, activated phagocytes
(neutrophils, monocytes, macrophages, eosinophils)
generate large amounts of superoxide as part of the
mechanism by which foreign organisms are killed.3 During
chronic inflammations, this normal protective mechanism
may become damaging (see later article in this series by
Grisham).
About 1-3% of the oxygen we breathe in is used to make
superoxide. Since human beings consume a lot of oxygen,
Neurodegenerative Disease Research Group, King’s College,
London SW3 6LX, UK (Prof B Halliwell DSc)
we may produce over 2 kg of superoxide in the body every
year; people with chronic infections may make much more.
Another physiological free radical is nitric oxide (NO*),
which is made by vascular endothelium as a relaxing factor,
and also by phagocytes and in the brain.4 Nitric oxide has
many useful physiological functions, but excess nitric oxide
can be toxic.4,5 Neither superoxide nor nitric oxide is highly
reactive chemically, but under certain circumstances they
can generate more toxic products. 1,5
How do radicals react?
Radical plus radical
If two free radicals meet, they can join their unpaired
electrons and make a covalent bond (a shared pair of
electrons). Thus superoxide and nitric oxide combine:
Oi- + NO.ONOO- (peroxynitrite)
At physiological pH, peroxynitrite damages proteins
directly, and decomposes into toxic products that include
nitrogen dioxide gas (N02•), hydroxyl radical, and
nitronium ion (N02 +). Hence at least somes of the toxicity
of excessnitric oxide may involve its interaction with
superoxide. In addition, superoxide can react with iron and
copper ions,1,2 eventually to made hydroxyl radical.
Radical plus non-radical
Most molecules in the body are not radicals. Hence any
reactive free radical generated is likely to react with a
non-radical. When a free radical reacts with a non-radical, a
free-radical chain reaction results and new radicals are
formed. Figure 1 shows two important reactions of this
type. Attack of reactive radicals on membranes or
lipoproteins starts lipid peroxidation,l which is particularly
implicated in the development of atherosclerosis (see later
article by Witztum). If hydroxyl radicals are generated
close to DNA, they can attack the purine and pyrimidine
bases and cause mutations. For example, guanine is
converted into 8-hydroxyguanine and other products.6
Antioxidant defences -
We have evolved antioxidant defences to protect against
free radicals. Superoxide dismutases (SOD) convert
superoxide to hydrogen peroxide (H202):
20i- + 2H+→ H202 + O2
These enzymes are found in mitochondria and cytosol.
Patients with the familial dominant form of amyotrophic
lateral sclerosis have a gene defect that decreases activity of
cytosolic SOD by about 40%.7 Catalases remove hydrogen
peroxide, are found in peroxisomes in most tissues, and
probably serve to remove peroxide generated by
peroxisomal oxidase enzymes.8 Glutathione peroxidases
are major enzymes that remove hydrogen peroxide
generated by SOD in cytosol and mitochondria,8 by
721

(A) Lipid peroxidation
Reactive radical (such as N02 OH*, or CCl302•) abstracts
atom of hydrogen from polyunsaturated fatty-acid side-chain
in membrane or lipoprotein. This leaves unpaired electron on
carbon (hydrogen atom has only one electron, so its removal
must leave spare electron):
Carbon radical reacts with oxygen:
Resulting peroxyl radical attacks adjacent fatty-acid side-
chain to generate new carbon radical
And chain reaction continues:
Overall, attack of one reactive free radical can oxidise
multiple fatty-acid side-chains to lipid peroxides, damaging
membrane proteins, making the membrane leaky, and
eventually causing complete membrane breakdown.
(8) Attack of hydroxyl radical on guanine in DNA"
Figure 1: Radicals beget radicals: some biologically Important
consequences
oxidising the tripeptide glutathione (GSH) into its oxidised
form (GSSG):
GSH also has important roles in xenobiotic metabolism and
leukotriene synthesis and is found at millimolar
concentrations in all human cells.8 The glutathione
peroxidase that removes hydrogen peroxide contains
selenium (essential for catalytic function) at its active site, as
does a similar enzyme that can remove lipid hydroperoxides
from membranes.9 Inborn defects in enzymes of
glutathione metabolism can have severe clinical
consequences. Since iron (and copper) ions are powerful
promoters of free-radical damage, accelerating lipid
peroxidation and causing formation of hydroxyl radica1,1
we have evolved a complex system of transport and storage
proteins to ensure that these essential metals are rarely
allowed to be "free". Ferritin is the usual storage-form of
722
iron. Iron within ferritin will not stimulate free-radical
reactions.
Since antioxidant defences are not completely effective,
repair enzymes exist that destroy free-radical-damaged
proteins,’O remove oxidised fatty acids from membranes,9
and repair free-radical damage to DNAII (some of the
oxidised bases removed are excreted in urine12). All these
defences are largely intracellular.
Other antioxidant defences are largely extracellular.
They include the plasma iron-transport-protein transferrin
and the similar iron-binding-protein lactoferrin found in
many body secretions (eg, tears, nasal lining fluid); iron
bound to these proteins cannot catalyse free-radical
damage.1 Caeruloplasmin is a safe transport form of copper
and it assists loading of iron onto transferrin. 13 Haemopexin
and haptoglobin bind free heme and heme proteins to
minimise their ability to catalyse free-radical damage.14
Albumin, which has one sulphydryl group per molecule
and is present at about 0-5 mmol/L in plasma, can scavenge
several radicals and binds copper ions.14
Some antioxidant defences are located both
intracellularly and extracellularly. a-tocopherol (TH)
occurs in membranes and lipoproteins. It blocks the chain
reaction of lipid peroxidation by scavenging intermediate
peroxyl radicals (figure 1):
The tocopherol radical (T*) is much less reactive in
attacking adjacent fatty-acid side-chains and can be
converted back to a-tocopherol by vitamin C. Severe
deficiency of a-tocopherol causes neurodegeneration.1s
Urate is present at about 0-5 mmol/L in body fluids and is
the end-product of purine metabolism. It scavenges several
free radicals.16 Reduced glutathione occurs in millimolar
concentrations in human cells but in only trace amounts in
plasma and most other body fluids, except in fluids lining
the lower part of the respiratory tract,17 where it may help to
scavenge inhaled toxins (eg, N02, ozone) as well as free
radicals produced by activated lung phagocytes.
GSH, urate, a-tocopherol, and ascorbate remove free
radicals by reacting directly with them non-catalytically.
Some "free-radical scavengers" come from the diet (table).
A diet rich in fruits, nuts, grains, and vegetables seems to be
protective against several human diseases. This effect may
be due to the antioxidants they contain, to the many other
compounds present, or both.
Despite all these antioxidants, some free radicals still
escape to do damage. Thus DNA undergoes constant
"oxidative damage", and has to be repairedy,12 Free-
radical-damaged proteins are degraded.10 End-products of
lipid peroxidation (eg, the isoprostanes25) and of free-,
radical attack on urate16 are present in vivo.
Oxidative stress
Because antioxidant defences are not completely efficient,
increased free-radical formation in the body is likely to
increase damage. The term "oxidative stress" is often used
to refer to this effect .16 If mild oxidative stress occurs,
tissues often respond by making extra antioxidant defences.
However, severe oxidative stress can cause cell injury and
death. Free-radical-induced cell death can proceed as
necrosis or apoptosis, and "anti-apoptosis genes" in certain
cells appear to encode free-radical scavengers.27
One way of imposing oxidative stress is by the action of
certain toxins, namely those that produce free radicals or
Table: Antioxidants from the diet
deplete antioxidant defences.1,28 One particular area of
interest is the possibility that the side-effects of several
drugs involve increased oxidative damage.28-30
Examples fall into four main groups. First, the toxin is
itself a free radical-eg, nitrogen dioxide (N02), the brown
toxic gas in polluted air. This compound is a good initiator
of lipid peroxidation:
Second, the toxin is metabolised free radical-eg,
to a
carbon tetrachloride is converted to a free radical by hepatic
cytochrome P-450:
This radical
reacts with oxygen to give a peroxyl radical:
which is a good initiator of lipid peroxidation.28 In addition,
several drugs can be metabolised to generate toxic free-
radical products (eg, phenylbutazone, nitrofurantoin,
penicillamine28,29). Third, the toxin is metabolised to
generate oxygen free-radicals. Thus, paraquat, which
accumulates selectively in the lung, is reduced to a free
radical that re-oxidises to make superoxide and regenerate
paraquat. The overall effect is to catalyse the reduction of
oxygen to superoxide in large excess.1 Alloxan, a
diabetogenic agent, works through a mechanism similar to
that of paraquat.1 In addition, although doxorubicin’s
anti-cancer effect probably involves interference with
Figure 2: Tissue injury and oxidative stress
DNA-unwinding during replication, its major side-effects
(especially cardiotoxicity) may involve excess of superoxide
and hydroxyl radical production.3O Fourth, the toxin
depletes antioxidant defences. Paracetamol’s metabolism
by liver cytochrome P-450 generates a product that reacts
with and removes glutathione. Loss of glutathione causes
secondary oxidative damage, which contributes to hepatic
failure in paracetamol overdose.
Oxidative stress and human disease
prevention
There is growing evidence that the major "killers",
cardiovascular disease and cancer, can be prevented or
delayed to some extent by dietary changes, such as
reduction in fat intake and increased consumption of fruits,
grains, and vegetables. There is also increasing evidence
that free-radical damage is involved in the development of
these diseases (see later articles by Cerutti and Witztum).
We obtain several compounds from a healthy diet that act
(or may act) to diminish oxidative damage in vivo (table).
Because our endogenous antioxidant defences are not
completely effective, it seems reasonable to propose that
dietary antioxidants are particularly important in
diminishing the cumulative effects of oxidative damage
over the long human lifespan, and that they account for
some of the beneficial effects of fruits, grains, and
vegetables. For example, if continuous free-radical damage
723
to DNA, perhaps not always efficiently repaired, is
involved in the development of spontaneous cancers, more
dietary antioxidants might help. An increased dietary
intake of vitamin E seems to decrease death from
myocardial infarction.18
Oxidative stress In disease
When a new mediator of tissue injury is first described
(eg,
prostaglandins, leukotrienes, iriterleukin-6, interleukin-8,
tumour necrosis factor alpha, excess nitric oxide) scientists
rush to measure it in a host of human diseases and imply that
it is important in the pathology. Many years have to pass
before the real importance is established. Free radicals have
followed the same pattern-they have been implicated in
over one-hundred human diseases.31 However, implicated
does not mean "important". 1,20 These evanescent species
are difficult to assay. Fortunately, "fingerprints" of
products of free-radical damage (such as oxidised DNA
bases,6 isoprostanes,25 free-radical-damaged aminoacids on
proteins10) and improved methods for "trapping" free
radicals20 are changing the situation rapidly.
In our 1984 article in this journa132 Gutteridge and I
emphasised that oxidative damage could be just as much a
consequence of tissue injury as a cause of it. Indeed, tissue
injury (however caused) almost certainly leads to oxidative
stress, for many reasons (figure 2). The oxidative stress
could then contribute significantly to worsening the tissue
injury, or it might be irrelevant. Thus demonstration of
increased free-radical damage is not the same as proof that it
is important (any more than is detecting, for example,
increased concentrations of cytokines). The criteria that are
needed to implicate free radicals as important contributors
in the of disease have been reviewed.2O What is
cause
exciting at the moment
is that in neurodegenerative disease,
chronic inflammatory disease, cardiovascular disease, and
cancer (the major scourges of life in "advanced" countries),
evidence is accumulating to show that free-radical damage
is important. Hopefully, this realisation will contribute to
the development of new preventive and therapeutic
strategies.
I am grateful to many scientific colleagues all over the world for their
excellent research and helpful discussions, and to the research councils,
government bodies, industries, and medical charities which have
supported my work.
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