Free radicals, antioxidants, and human disease curiosity, cause, or consequence?
Citation: The Lancet, Sept 10, 1994 v344 n8924 p721(4)
Subjects: Free radicals (Chemistry) Health aspects
Antioxidants Physiological aspects
Oxidation-reduction reaction Physiological aspects
Reference #: A15748015
scientific evidence may support the important role of free radicals in
the development of some diseases. Free radicals are molecules or atoms
that have at least one unpaired electron which usually increases the
chemical reactivity of the molecule. Environmental radiation and
physiological processes in the body cause free radicals to form. Free
radicals can react with other molecules to cause cell damage or DNA
mutation. Molecules called antioxidants protect against free radical
damage. When antioxidants are ineffective, enzymes produced by the body
work to repair free radical damage. Higher levels of free radicals tend
to cause increased cellular damage. This effect is called oxidative
stress. Oxidative stress may contribute to cardiovascular disease and
cancer. Chemical compounds found in some foods may decrease the
accumulated effects of oxidative stress, thus helping to prevent
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"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.sup.*], 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.sup.*] 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.sup.*]. This fearsomely-reactive
radical, 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.
The body makes another
oxygen radical (ie, the unpaired electron is located on oxygen),
superoxide (O.sub.2.sup.* -]). 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. 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, 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.sup.*]), which is made by vascular
endothelium as a relaxing factor, and also by phagocytes and in the
brain. 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:
[O.sub.2.sup.* -] + [NO.sup.*]
[right arrow] [ONOO.sup.-] (peroxynitrite)
At physiological pH,
peroxynitrite damages proteins directly, and decomposes into toxic
products that include nitrogen dioxide gas ([NO.sub.2.sup.*], hydroxyl
radical, and nitronium ion ([NO.sub.2.sup.+). Hence at least some of
the toxicity of excess nitric 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,
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
We have evolved antioxidant
defences to protect against free radicals. Superoxide dismutases (SOD)
convert superoxide to hydrogen peroxide ([H.sub.2.][O.sub.2])
[20.sub.2.sup.* -] +
[2H.sup.+] [right arrow] [H.sup.2] [O.sup.2] + [O.sub.2]
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%. Catalases remove hydrogen peroxide,
are found in peroxisomes in most tissues, and probably serve to remove
peroxide generated by peroxisomal oxidase enzymes. Glutathione
peroxidases are major enzymes that remove hydrogen peroxide generated by
SOD in cytosol and mitochondria, by oxidising the tripeptide
glutathione (GSH) into its oxidised form (GSSG):
2GSH + [H.sub.2] [O.sub.2]
[right arrow] GSSG + [2H.sub.2.O]
GSH also has important roles
in xenobiotic metabolism and leukotriene synthesis and is found at
millimolar concentrations in all human cells. 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. 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 radical, 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 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, remove oxidised fatty acids from
membranes, and repair free-radical damage to DNA (some of the
oxidised bases removed are excreted in urine). 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. Caeruloplasmin is a safe transport form of copper and it
assists loading of iron onto transferrin. Haemopexin and haptoglobin
bind free heme and heme proteins to minimise their ability to catalyse
free-radical damage. 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.
Some antioxidant defences
are located both intracellularly and extracellularly. [alpha]-tocopherol
(TH) occurs in membranes and lipoproteins. It blocks the chain reaction
of lipid peroxidation by scavenging intermediate peroxyl radicals
The tocopherol radical ([T.sup.*])
is much less reactive in attacking adjacent fatty-acid side-chains and
can be converted back to [alpha]-tocopherol by vitamin C. Severe
deficiency of [alpha]-tocopherol causes neurodegeneration. 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. 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, where it may
help to scavenge inhaled toxins (eg, [NO.sub.2.sup.*], ozone) as well as
free radicals produced by activated lung phagocytes.
GSH, urate, [alpha]-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 repaired.[11.12]
Free-radical-damaged proteins are degraded. End-products of lipid
peroxidation (eg, the isoprostanes) and of free-radical attack on
urate are present in vivo.
Because antioxidant defenses
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. If mild oxidative stress occurs,
tissues often respond by making extra antioxidant defenses. 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
One way of imposing
oxidative stress is by the action of certain toxins, namely those that
produce free radicals or deplete antioxidant defenses.[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
([NO.sub.2.sup.*]), the brown toxic gas in polluted air. This compound
is a good initiator of lipid peroxidation:
lipid-H + [NO.sub.2.sup.*]
[right arrow] [lipid.sup.*] + [HNO.sub.2]
Second, the toxin is
metabolised to a free radical - eg, carbon tetrachloride is converted to
a free radical by hepatic cytochrome P-450:
This radical reacts with
oxygen to give a peroxyl radical:
[O.sub.2] [right arrow] [Ccl.sub.3] [O.sub.2.sup.*]
which is a good initiator of
lipid peroxidation. In addition, several drugs can be metabolised to
generate toxic free-radical products (eg, phenylbutazone, nitrofurantoin,
penicillamine[28,29]). 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. Alloxan, a
diabetogenic agent, works through a mechanism similar to that of
paraquat. In addition, although doxorubicin's anti-cancer effect
probably involves interference with DNA-unwinding during replication,
its major side-effects (especially cardiotoxicity) may involve excess of
superoxide and hydroxyl radical production. 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 defenses 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 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
Oxidative stress In disease
When a new mediator of
tissue injury is first described (eg, prostaglandins, leukotrienes,
interleukin-6, interleukin-8, tumor necrosis factor alpha, excess
nitric oxide) scientists rush to measure it in a host of human diseases
and imply diat 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. 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,
isoprostanes, free-radical-damaged aminoacids on proteins) and
improved methods for "trapping" free radicals are changing the
In our 1984 article in this
journal Gutteridge and I emphasized 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 cause
of disease have been reviewed. What is 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
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.
 Halliwell B, Gutteridge JMC. Free radicals in biology and medicine.
2nd ed. Oxford: Clarendon Press, 1989.
 Liochev SI, Fridovich I. The role of [O.sub.2] in the production of
[HO.sup.*]: in vitro and in vivo. Free Radic Biol Med 1994;16:29-33.
 Babior BM, Woodman RC. Chronic granulomatous disease. Semin Hemarol
 Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J
 Beckman JS, Chen J, Ischiropoulos H, Crow JP. Oxidative chemistry of
peroxynitrite. Meth Enzymol 1994;233:229-40.
 Dizdaroglu M. Chemistry of free radical damage to DNA and
nucleoproteins. In: Halliwell B, Aruoma OI, eds. DNA and free radicals.
Chichester: Ellis Harwood, 1993:19-39.
 Robberecht W, Sapp P, Viaene MK, et al. Cu/Zn-superoxide dismutase
activity in familial and sporadic amyotrophic lateral sclerosis. J
 Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian
organs. Physiol Rev 1979;59:527-605.
 Maiorino M, Chu FF, Ursini F, Davies K, Doroshow JH, Esworthy RS.
Phospholipid hydroperoxide glutathione peroxidase is the 18 kDa
selenoprotein expressed in human tumor cell lines. J Biol Chem
 Stadtman ER, Oliver CN. Metal-catalyzed oxidation of proteins:
physiological consequences. J Biol Chem 1991;266:2005-08.
 Breimer L. Repair of DNA damage induced by reactive oxygen species.
Free Radic Res Commun 1991;14:159-71.
 Stillwell WG, Xu HX, Adkins JA, Wishnock JS, Tannenbaum SR.
Analysis of methylated and oxidized purines in urine by capillary gas
chromatography-mass spectrometry. Chem Res Toxicol 1989;2:94-99.
 Gutteridge JMC, Stocks J. Caeruloplasmin: physiological and
pathological perspectives. Crit Rev Clin Lab Sci 1981;14:257-329.
 Halliwell B, Gutteridge JMC. The antioxidants of human
extracellular fluids. Arch Biochem Biophys 1990;280:1-8.
 Muller DP, Goss-Sampson MA. Neurochemical, neurophysiological and
neuropathological studies in vitamin E deficiency. Crit Rev Neurobiol
 Kaur H, Halliwell B. Action of biologically-relevant oxidizing
species upon uric acid: identification of uric acid oxidation products.
Chem Biol Interact 1990;73:235-47.
 Slade R, Crissman K, Norwood J, Hatch G. Comparison of antioxidant
substances in bronchoalveolar lavage cells and fluid from humans, guinea
pigs, and rats. Exp Lung Res 1993;19:469-84.
 Byers T. Vitamin E supplements and coronary heart disease. Nutr Rev
 Meister A. On the antioxidant effects of ascorbic acid and
glutathione. Biochem Pharmacol 1992;44:1905-15.
 Halliwell B, Cross CE, Gutteridge JMC. Free radicals, antioxidants
and human disease: where are we now? J Lab Clin Med 1992;119:598-620.
 Burt MJ, Halliday JW, Powell LW. Iron and coronary heart disease.
BMJ 1993;307:575-76.  Krinsky NI. Effect of carotenoids in cellular
and animal systems. Am J Clin Nutr 1991;53:238S-46S.
 Hertog MGI, Feskens EJM, Hollman PCH, Katan MB, Kromhout D. Dietary
antioxidant flavonoids and risk of coronary heart disease: the Zutphen
Elderly Study. Lancet 1993;342:1007-11.
 Laughton MJ, Evans PJ, Moroney MA, Hoult JRS, Halliwell B.
Inhibition of mammalian 5-lipoxygenase and cyclo-oxygenase by flavonoids
and phenolic dietary additives: relationship to antioxidant activity and
to iron ion-reducing ability.Biochem Pharmacol 1991;42:1673-81.
 Morrow JD, Hill KE, Burk RF, Nammour TM, Badr KF, Roberts LJ II. A
series of prostaglandin [F.sub.2]-like compounds are produced in vivo in
humans by non-cyclooxygenase, free radical catalyzed mcchanism. Proc
Natl Acad Sci USA 1990;87:9383-87.
 Sies H, ed. Oxidative stress II: oxidants and antioxidants. New
York: Academic Press, 1991.
 Sarafian TA, Bredesen DE. Is apoptosis mediated by reactive oxygen
species? Free Radic Res 1994;20:1-6.
 Aust SD, Chignell CF, Bray TM, Kalyanaraman B, Mason RP. Free
radicals in toxicology. Toxicol Appl Pharmacol 1993;120:168-78.
 Hofstra AH, Uetrecht JP. Myeloperoxidase-mediated activation of
xenobiotics by human leukocytes. Toxicology 1993;82:221-42.
 Keizer HG, Pinedo HM, Scbuurhuis GJ, Joenje H. Doxorubicin (adriamycin):
a critical review of free radical-dependent mechanisms of cytotoxicity.
Pharmacol Ther 1990;47:219-31.
 Gutteridge JMC. Free radicals in disease processes: a compilation
of cause and consequence. Free Radic Res Commun 1993;19:141-58. 
Halliwell B, Gutteridge JMC. Lipid peroxidation, oxygen radicals, cell
damage, and antioxidant therapy. Lancet 1984;i:1396-98.