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Opinion

Free radicals and antioxidants quo


vadis?
Barry Halliwell
Department of Biochemistry, National University of Singapore, 8 Medical Drive, Singapore 117597

The field of free radicals and antioxidants, or redox


biology, is fundamental to aerobic life. Aerobes constantly make reactive species, but modulate their
actions by synthesizing antioxidants. This balance
allows some reactive species to perform useful functions
while minimizing oxidative damage. In general, dietary
antioxidants are ineffective at modulating the redox
balance in humans. This helps to explain why, although
oxidative damage contributes to the development and
pathology of several human diseases, dietary antioxidant supplements have limited efficacy in disease prevention. Cell culture as usually performed imposes
oxidative stress upon cells, which can lead to artefactual
data in studies of the roles of reactive species and the
actions of added antioxidants in cultured cells. This brief
commentary highlights in broad terms the current status
of the redox biology field and the major challenges it
faces in the next decade.

Introduction
The field of free radicals and antioxidants (for definitions of
terms see Glossary) is often thought of in terms of the value
(or lack of it) of dietary antioxidant supplements in keeping us healthy. However, scientists now realize that the
field is far more than that the complex interplay between
free radicals, other reactive species (RS) (such as H2O2
and peroxynitrite), and antioxidants has been (and still is)
a major driver in the evolution and survival of humans, in
the way cells communicate and respond to danger, and in
age-related diseases of humans and other animals [14].
The purpose of this article, based on a lecture given at
the sixteenth World Congress of Basic and Clinical Pharmacology held in Copenhagen from 1723 July 2010, is to
step back a little from the multitudes of recent publications
in the field and take stock of fundamental principles. What
follows is largely a personal view of where the field of RS/
antioxidants or redox biology is now, and where it might
be heading. I begin by articulating certain key principles.
Humans have a balanced system of RS and antioxidants
that allows some RS to perform useful functions while
minimizing oxidative damage
RS are formed in aerobes both by accidents of chemistry
(e.g. the autoxidation of unstable biomolecules such as
dopamine) and deliberately, for example by activated neutrophils or nitric oxide synthases (nitric oxide, NO, is a

free radical). Mitochondria produce superoxide (O2) radicals; these are known to be potentially damaging in view of
the striking deleterious phenotype of knockout animals
lacking its scavenger, the mitochondrial manganese-containing superoxide dismutase [5]). Is mitochondrial O2
production due to simple accidental leakage of electrons to
O2, or is it part of an intracellular redox-signalling mechanism, as some have argued [6,7]? I tend towards the former
view: there is always an intrinsic potential for electron
leakage when electron transport chain components with
sufficiently-negative redox potentials to reduce O2 directly
to O2 are functioning in the presence of O2 [1]. Strategies
to minimize mitochondrial O2 production include the use
of low intra-mitochondrial pO2 levels, the arrangement of
electron carriers into complexes to facilitate electrons following the correct path towards cytochrome oxidase instead of prematurely escaping to O2, and uncoupling
proteins [1,7].
In terms of human survival, probably the most important source of RS is the immune system [1,4]. When
humans gathered together, initially in nomadic groups
and later congregated into growing populations within
(usually dirty) cities, infectious disease was a major threat.
It has been stated that the black death (bubonic plague)
killed one third of the population of Europe but why not
the other two thirds? Some avoided exposure, but in others
their immune system dealt with it they recovered or
never became sick, a powerful selection for survival of
Glossarya
Antioxidant: a molecule that protects a biological target against
oxidative damage.
Free radical: any species containing one or more unpaired electrons (electrons singly occupying an atomic or molecular orbital).
Oxidative damage: the damage to cells and tissues caused by
reactive oxygen species.
Oxidative stress: a serious imbalance between the generation of
reactive oxygen species and antioxidant protection in favour of the
former, causing excessive oxidative damage.
Reactive oxygen species: the collective term for oxygen radicals
(including hydroxyl, OH, or superoxide, O2S) and some other
nonradical derivatives of oxygen, such as hydrogen peroxide
(H2O2), that can easily generate free radicals and/or cause oxidative
damage. The superscript dot is the symbol used to denote a free
radical. There are also reactive nitrogen, chlorine, iron, copper, and
sulphur species; this article therefore uses the general term reactive species (RS) to encompass these also. Note that each such
species has its own particular types of chemical reactivity.
a

Definitions simplified from [1].

Corresponding author: Halliwell, B. (bchbh@nus.edu.sg)


0165-6147/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2010.12.002 Trends in Pharmacological Sciences, March 2011, Vol. 32, No. 3

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Opinion

Trends in Pharmacological Sciences March 2011, Vol. 32, No. 3

people with robust and well-coordinated immune systems.


Perhaps then the survivors passed on some of the relevant
genes, breeding children with similarly powerful immune
systems.
How is this relevant to redox biology? RS play an
essential role in the immune system. First, they help to
kill some infecting organisms. It is essential to mount
vigorous RS production and hit microorganisms hard
and fast, because many such pathogens respond to low
levels of RS with rapid increases in antioxidant defence
systems that render them resistant to much higher RS
levels [1,8]. Second, RS help to restrain the immune system
(especially T-lymphocytes) from over-activation and
prolonging inflammation once the threat has been eliminated [911]. Persistent or chronic inflammation is a major
determinant of disease later in the human lifespan, and RS
play key roles in the origin and pathology of several agerelated diseases, particularly cancer and neurodegenerative disorders [1,12].
There is, I feel, less evidence that RS (apart of course
from the well-established case of nitric oxide) play a major
role in signalling in the healthy human body; many claims
for redox signalling (e.g. by H2O2) are based on experiments with cell cultures and tissue explants (about which
more later). Nevertheless, evidence that RS play at least
some role is steadily accumulating, and the mechanisms
that might allow them to be generated at selected sites,
persist for a short time, and then be removed, are slowly
being elucidated [13]. Evidence that RS are truly important physiological (as opposed to pathological) signalling
molecules is presently stronger in plants than in animals
(reviewed in [2]). However, RS do seem to be involved in
adaptation
to ischaemia and to exercise, for example [14
[()TD$FIG]

Arachidonic acid
metabolism

Table 1. Major challenges in the RS field


 Establishing the true role of RS in ageing
 Developing effective antioxidants for human use, particularly in
the brain (treating neurodegeneration, especially dementia)
 Using our knowledge of redox biology to alter the ageing process
 Understanding the physiological importance and mechanism
of redox signalling at the cellular and organismal levels a
a
One exciting possibility (suggested by a reviewer) is that disruption of ROS
signalling during specific periods of development could cause long-lasting changes
in physiology. We must therefore be cautious in assuming that ROS are deleterious
in pregnancy, newborns or during child development, and ensure that children and
mothers are not overloaded with nutritional antioxidants.

17]. Therefore I list the question of the physiological importance of redox signalling as one of the challenges facing
the RS/antioxidant field in the coming years (Table 1).
The human antioxidant defence network is complex and
interlocking; it functions to minimize levels of RS while
allowing useful roles to continue (Figure 1). Two recent
examples: some O2 in muscle appears to be useful, but
excess causes harm [14,17,19]; stem cells need some RS to
function properly, but too many RS can impair function
[20]. The properties of the peroxiredoxin proteins, which
scavenge H2O2 but can be inactivated by it, could be
especially important in allowing localized and transient
H2O2-dependent signal transduction [13,21]. In humans,
the great majority of total antioxidant capacity of cells and
tissues is contributed by endogenously-synthesized antioxidants such as reduced glutathione (GSH), peroxiredoxins and superoxide dismutase; whereas diet-derived
antioxidants are far less important, a point upon which
I elaborate below. It follows that, instead of using dietary
antioxidants, a more effective way of strengthening overall antioxidant defence in humans might be mild prooxidants, increasing the generation of RS and electrophiles
Diet-derived
pro-oxidants
(eg. polyphenols?)

upregulation
Peroxiredoxins
Glutathione/glutathione
peroxidase system

upregulation
Phagocytes

.-

Other sources

O2 /H2O2/NO /other RS

SOD, Catalase

Mitochondrial
respiration
Nitric oxide synthase
Xanthine oxidase

RS- reactive species

Diet-derived
Antioxidants
(eg. polyphenols?)
Vitamin E, Vitamin C,
Carotenoids?
Blood components
eg.
Albumin,
Caeruloplasmin,
Haemopexin,
Haptoglobin
Transferrin

Antioxidants

Iron chelators
(eg.lactoferrin)

SOD- superoxide dismutase


TRENDS in Pharmacological Sciences

Figure 1. The approximate balance of antioxidants and reactive species in vivo. Adapted from [18] with permission from Elsevier. Some degree of oxidative damage occurs
continually even in healthy aerobes, and repair (e.g. DNA repair) and replacement (e.g. of damaged lipids and proteins) mechanisms are therefore essential. The ? signifies that
the evidence for the effects of carotenoids or polyphenols as anti-or pro-oxidants in vivo is not compelling, but some effects could be exerted within the gastrointestinal tract.

126

Opinion
that then trigger increases in the levels of our own antioxidants, such as GSH [22,23].
Because RS are not completely removed in vivo
(Figure 1), it follows that the ability to repair (e.g. DNA
repair) or replace (e.g. lipid turnover, degradation of abnormal proteins by the proteasome and by lysosomes)
oxidatively-damaged molecules is essential. Failures in
some or all of these repair systems could contribute more
to ageing and age-related disease than changes in antioxidants or RS production, although the magnitude of the
contribution remains unclear [2426].
Human antioxidant capacity resists modulation by
dietary antioxidants
In recent years, better (although still not perfect) biomarkers of oxidative damage in human tissues and body fluids
have been developed (Table 2). Note that most are measured in blood or urine, and they could easily fail to detect
changes in oxidative damage in small groups of cells or
individual organs. For example, rises in oxidative damage
levels in the brain are probably not usually reflected in the
blood (discussed in [27]). Nevertheless, studies using biomarkers reveal that giving antioxidant supplements to
healthy or diseased humans rarely causes much change
in systemic levels of oxidative damage [2834]. The failure
of most human intervention trials with antioxidants to
modify disease outcome [1,35,36] is therefore not surprising. Even if oxidative damage does play a role in the
disease, antioxidants will be ineffective if they fail to alter
the levels of oxidative damage. If RS are involved in the
adaptation to tissue injury (as discussed earlier and summarized in Figure 2), then high doses of antioxidants could
sometimes be deleterious (legend to Table 1 and [35,36]).
We are perhaps fortunate that diet-derived antioxidants
do not markedly decrease oxidative damage in humans
Table 2. Biomarkers (a personal view)
Generally robust (for human use)
 F2-isoprostanes and other isoprostanes in tissues and body fluids
(lipid peroxidation) a
 8-Hydroxy-20 -deoxyguanosine (8OHdG) in urine (damage to DNA
and the DNA precursor pool) b
Usable with cautious interpretation: for example in controlling
for possible effects of absorption of the products from the diet.
 Cholesterol oxidation products (COPs)/fatty acid hydroperoxides/
hydroxides (lipid peroxidation)
 Peroxide assays (lipid peroxidation)
 8-Hydroxyguanosine (8OHG) in urine
 DNA base oxidation in DNA isolated from cells (with care taken
to minimize artefactual oxidation)
 Direct chemical determination of malondialdehyde, other
aldehydes (e.g. 4-hydroxynonenal), and their adducts
with proteins and nucleic acids
 HPLC-based thiobarbituric acid (TBA) tests
 Comet assay with cleavage enzymes c
To be avoided because the results are often misleading
 Simple TBA assays
 Total antioxidant capacity determination
 Many commercial kit methods for biomarkers
a

Mass spectrometric methodology only.


More research is needed on the origins of 8OHdG and whether their relative
contributions of different sources vary with age or disease.
c
Probably underestimates oxidative DNA damage [61] and needs careful calibration
[6163]. Table adapted from [28] with permission.
b

Trends in Pharmacological Sciences March 2011, Vol. 32, No. 3

Table 3. If antioxidants rarely change oxidative damage levels


in humans, what does?









Obesity (humans)
Hyperglycaemia (humans)
High plasma LDLcholesterol (humans)
High-cholesterol diet (rabbits and rats, humans probably not)
Zinc intake (rabbits, some other animals, human data mixed)
Body iron levels (rabbits, rats, mice, possibly humans)
Certain foods (humans, e.g. dark soy sauce, tomato)
Diabetes (in some studies, not others)a, but probably not
the metabolic syndrome [37]
 Intake of PUFAsb (docosahexaenoic acid, possibly
eicosapentaenoic acid)

a
This could depend on how well glucose and lipids have been normalized in the
diabetic cohorts studied.
b
Despite the propensity of PUFAs to oxidize in vitro, growing evidence suggests that
they reduce oxidative damage in vivo [38].Abbreviations: LDL, low-density lipoprotein; PUFAs, polyunsaturated fatty acids.

because otherwise they might sometimes have caused


harm rather than good.
If antioxidants generally fail to modulate oxidative
damage in humans, what does? Table 3 attempts to summarize our limited knowledge to date. One important point
is that laboratory rats and mice appear to be more sensitive
to dietary antioxidant levels than are humans, and dietary
antioxidant supplementation is therefore more likely to
reduce oxidative damage in rodents than in humans
[1,39,40]. Indeed, the antioxidant content of animal feed
could account for several discrepancies between results
reported from different laboratories [39]. It follows that
rodent models of human diseases in which oxidative damage appears to be important in the pathology (such as
stroke, atherosclerosis, amyotrophic lateral sclerosis and
dementia) are more likely to be responsive to administered
antioxidants than are humans. This should be borne in
mind when evaluating the potential uses of therapeutic
antioxidants in humans; such trials could well fail [41].
Oxidative damage contributes to the development and
pathology of several human disease
RS have been suggested to cause everything from baldness
to failed pregnancy (perhaps the opposite is true for the
latter [36]). Their role in many diseases is likely to be
peripheral, but in some it is fundamental (Figure 2). Cancer is probably an example of the latter: RS can cause
cancer, contribute to its progression, and sometimes even
cure it [42]. Neurodegenerative disease is another example: marked oxidative damage is present in affected brain
regions in both Parkinson and Alzheimer diseases; there is
some evidence that this damage precedes neurodegeneration and studies on animal models suggest that decreasing
oxidative damage can preserve neuronal function [1,27,43].
Developing better antioxidants?
Developing better antioxidants is a major challenge for the
redox biology field (Table 1). Many antioxidants are available (Table 4) but most have failed dismally in clinical
studies even when there is strong evidence that oxidative
damage plays a role in the disease pathology (and the
evidence is often not robust). Probable reasons for this
failure have been summarized [1,41,44]. Promising antioxidants under evaluation include low molecular mass
127

()TD$FIG][ Opinion

Trends in Pharmacological Sciences March 2011, Vol. 32, No. 3

Tissue injury

Oxidative damage

Induction of
antioxidant and
other defence/
repair systems
(e.g. heat shock
proteins,
proteasome,
autophagy,
haem oxygenase)

Early stage in tissue


injury
Late stage in tissue
injury, accompanying
cell death

Necrotic death of some cells spreads


damage to others
e.g. by Fe/Cu/haem protein release,
and triggers inflammation, causing
more RS formation.
Release of peroxides from apoptosing
cells may sometimes affect
surrounding cells

Neutralization of
oxidative stress.
Sometimes
superprotection of
the tissue

No adverse contribution of
oxidative damage to disease
pathology. RS can sometimes be
beneficial.
(e.g. in ischemic preconditioning
or adapation to exercise).
Antioxidant interventions fail to
give benefit and could
conceivably be deleterious.

Aggravates disease.
Potential therapeutic
benefit from antioxidant
intervention, provided
that the antioxidants are
effective in decreasing
oxidative damage to the
important biomolecular
targets.

TRENDS in Pharmacological Sciences

Figure 2. The significance of oxidative stress in human disease. Adapted from [1] with permission from Oxford University Press.

Table 4. Some antioxidants available for therapeutic use


Category of compound
Naturally occurring

Synthetic

Agents already in
clinical use that
might have some
antioxidant activity
in vivo (but were
not developed as
antioxidants)
a

Examples
SOD (CuZnSOD, MnSOD, EC-SOD; recombinant or purified), superoxide reductase, tocopherols, tocotrienols,
coenzyme Q, lipoic acid, vitamin C, adenosine, transferrin, lactoferrin, cysteine, GSH, histidine-containing
dipeptides, pyridoxamine, carotenoids, flavonoids, other plant phenolics, desferrioxamine, other natural
iron chelators, melatonin, coelenterazine, antibiotics
Thiols (e.g. mercaptopropionylglycine, N-acetylcysteine), synthetic metal-ion chelators (e.g. ICRF-187, Exjade,
hydroxypyridones), fullerenes, xanthine oxidase inhibitors, inhibitors of O2 generation by phagocyte or by
other NADPH oxidases, lipid-soluble chain-breaking antioxidants, inhibitors of phagocyte adhesion, GSH
precursors, SOD/catalase mimetics, derivatives of vitamins E or C, coelenterazine derivatives, modified
antibiotics, mitochondrially-targeted antioxidants, NXY-059, PBN/other spin traps.
Penicillaminea, bucillaminea, aminosalicyatesa (alone or as components of sulphasalazine), apomorphinea,
selegiline, flupirtine, omeprazole, 4-hydroxytamoxifena, ACE inhibitors (e.g. quinapril, ramipril, captopril),
or angiotensin II receptor antagonists (e.g. losartan), ketoconazole, probucol, propofol, some b-blockers
(e.g. carvedilol, metoprolol)a, cimetidine, some Ca2+ channel blockersb, phenylbutazonea, nitecaponea,
entecaponea, idebenone, troglitazonea, tacrolimus

Compounds which react with RS to form products with potential to cause damage. When an RS reacts with a putative antioxidant the possible biological effects of the
oxidation products must always be considered.
b
Several b-blockers/Ca2+ blockers inhibit peroxidation in vitro; but it is uncertain if they do so in vivo at the therapeutic levels normally achieved. Table adapted from [1] with
permission from Oxford University Press.Abbreviations: ACE, angiotensin converting enzyme; EC, extracellular; PBN, phenyl N-tert-butylnitrone; SOD, superoxide
dismutase.

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Opinion
catalytic scavengers of RS [45,46] and targeted antioxidants (for example to mitochondria) [44,47,48]. However, I
have doubts that these redox-active agents are always
antioxidants in vivo; indeed they can sometimes be mild
pro-oxidants [46,49], and could thereby be more effective in
reducing oxidative damage (Figure 1).
Flavonoids and other polyphenols, widely touted as
beneficial to health, can act as both antioxidants and
pro-oxidants in vitro, but there is little or no evidence that
they act as either in vivo, except possibly in the stomach,
small intestine and colon [1,29,50,51], although they could
be beneficial in other ways. It is possible that if sufficient
flavonoids could be introduced into the brain they could
oxidize gently and thereby upregulate endogenous antioxidants, and this could help to treat neurodegeneration
(Figure 1).
Time to abandon cell culture?
Cell culture studies are used extensively in research and
drug development, but a substantial proportion of cell lines
are not the cells they are supposed to be [52]. To quote the
authors of [52] thousands of misleading studies and potentially erroneous papers have been published. The scale of
the problem is under-appreciated by most scientists [52].
Equally under-appreciated is the fact that the effects of
antioxidants such as ascorbate, lycopene, epigallocatechin
gallate and several other polyphenols on cells in culture are
often artifacts reflecting the reactions of these compounds
with components of cell culture media and/or their rapid
decomposition into other bioactive agents in cell culture.
Several groups have highlighted and tabulated these problems (summarized in [18,53]) but artefactual reports describing the effects of flavonoids and other antioxidants on
cells in culture continue to proliferate faster than HeLa cells,
and (sadly) continue to pass peer-review. Time to call a halt.
Antioxidants and ageing
RS are intimately involved in ageing and age-related
disease [54,55]. RS probably do not cause ageing (they
could perhaps account for some features of ageing, such
as skin wrinkling), nor will dietary antioxidants stop ageing [2426,56,57]. Perhaps mild pro-oxidants might help,
by raising endogenous antioxidant defences [5759]. If
ROS really are key components of physiological signalling,
perhaps the general signalling failure with ageing [60]
could even be due to insufficient RS (or perhaps the wrong
type of RS). As the world population ages, we need to solve
this issue urgently; this is at the top of my list for personal
research in the next few years (Table 1).
Concluding remarks
The biology of ROS and antioxidants is not an esoteric field
of study: these species are involved in all aspects of aerobic
life. One cannot live without them, nor would one wish to,
but ultimately they no doubt contribute to individual mortality. Learning how to stop the latter while preserving the
useful functions of RS should be a major research priority.
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