Free radicals and antioxidants – quovadis?Barry Halliwell

Department of Biochemistry, National University of Singapore, 8 Medical Drive, Singapore 117597

Opinion

Glossarya

Antioxidant: a molecule that protects a biological target against

oxidative damage.

Free radical: any species containing one or more unpaired elec-

trons (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, O2�S) 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 ‘reac-

tive species’ (RS) to encompass these also. Note that each such

species has its own particular types of chemical reactivity.

The field of free radicals and antioxidants, or ‘redoxbiology’, is fundamental to aerobic life. Aerobes con-stantly make reactive species, but modulate theiractions by synthesizing antioxidants. This balanceallows some reactive species to perform useful functionswhile minimizing oxidative damage. In general, dietaryantioxidants are ineffective at modulating the ‘redoxbalance’ in humans. This helps to explain why, althoughoxidative damage contributes to the development andpathology of several human diseases, dietary ‘antioxi-dant’ supplements have limited efficacy in disease pre-vention. Cell culture as usually performed imposesoxidative stress upon cells, which can lead to artefactualdata in studies of the roles of reactive species and theactions of added antioxidants in cultured cells. This briefcommentary highlights in broad terms the current statusof the redox biology field and the major challenges itfaces in the next decade.

IntroductionThe field of free radicals and antioxidants (for definitions ofterms see Glossary) is often thought of in terms of the value(or lack of it) of dietary ‘antioxidant’ supplements in keep-ing us healthy. However, scientists now realize that thefield is far more than that – the complex interplay betweenfree 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, inthe way cells communicate and respond to danger, and inage-related diseases of humans and other animals [1–4].

The purpose of this article, based on a lecture given atthe sixteenth World Congress of Basic and Clinical Phar-macology held in Copenhagen from 17–23 July 2010, is tostep back a little from themultitudes of recent publicationsin the field and take stock of fundamental principles. Whatfollows is largely a personal view of where the field of RS/antioxidants or ‘redox biology’ is now, and where it mightbe 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 asdopamine) and deliberately, for example by activated neu-trophils or nitric oxide synthases (nitric oxide, NO�, is a

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.

free radical). Mitochondria produce superoxide (O2��) radi-

cals; these are known to be potentially damaging in view ofthe striking deleterious phenotype of knockout animalslacking its scavenger, the mitochondrial manganese-con-taining superoxide dismutase [5]). Is mitochondrial O2

��

production due to simple accidental leakage of electrons toO2, or is it part of an intracellular redox-signalling mecha-nism, as some have argued [6,7]? I tend towards the formerview: there is always an intrinsic potential for electronleakage when electron transport chain components withsufficiently-negative redox potentials to reduce O2 directlyto O2

�� are functioning in the presence of O2 [1]. Strategiesto minimize mitochondrial O2

�� production include the useof low intra-mitochondrial pO2 levels, the arrangement ofelectron carriers into complexes to facilitate electrons fol-lowing the ‘correct’ path towards cytochrome oxidase in-stead of prematurely escaping to O2, and uncouplingproteins [1,7].

In terms of human survival, probably the most impor-tant source of RS is the immune system [1,4]. Whenhumans gathered together, initially in nomadic groupsand later congregated into growing populations within(usually dirty) cities, infectious disease was amajor threat.It has been stated that the ‘black death’ (bubonic plague)killed one third of the population of Europe – but why notthe other two thirds? Some avoided exposure, but in otherstheir immune system dealt with it – they recovered ornever became sick, a powerful selection for survival of

a Definitions simplified from [1].

12.002 Trends in Pharmacological Sciences, March 2011, Vol. 32, No. 3 125

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 levelsa

aOne 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’.

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 relevantgenes, breeding children with similarly powerful immunesystems.

How is this relevant to redox biology? RS play anessential role in the immune system. First, they help tokill some infecting organisms. It is essential to mountvigorous RS production and ‘hit microorganisms hardand fast’, because many such pathogens respond to lowlevels of RS with rapid increases in antioxidant defencesystems that render them resistant to much higher RSlevels [1,8]. Second, RS help to restrain the immune system(especially T-lymphocytes) from over-activation andprolonging inflammation once the threat has been elimi-nated [9–11]. Persistent or chronic inflammation is a majordeterminant of disease later in the human lifespan, and RSplay key roles in the origin and pathology of several age-related diseases, particularly cancer and neurodegenera-tive disorders [1,12].

There is, I feel, less evidence that RS (apart of coursefrom the well-established case of nitric oxide) play a majorrole in signalling in the healthy human body; many claimsfor ‘redox signalling’ (e.g. by H2O2) are based on experi-ments with cell cultures and tissue explants (about whichmore later). Nevertheless, evidence that RS play at leastsome role is steadily accumulating, and the mechanismsthat might allow them to be generated at selected sites,persist for a short time, and then be removed, are slowlybeing elucidated [13]. Evidence that RS are truly impor-tant physiological (as opposed to pathological) signallingmolecules is presently stronger in plants than in animals(reviewed in [2]). However, RS do seem to be involved inadaptation to ischaemia and to exercise, for example [14–[()TD$FIG]

PeroxiredoxinsGlutathione/glutathi

peroxidase system

Arachidonic acidmetabolism

Phagocytes Other sources

Iron c(eg.la

Mitochondrialrespiration

Nitric oxide synthase

Xanthine oxidase

RS- reactive species

SOD- superoxide dismutase

O2.-/H2O2/NO

./other RS

Figure 1. The approximate balance of antioxidants and reactive species in vivo. Adapted

continually even in healthy aerobes, and repair (e.g. DNA repair) and replacement (e.g. of d

the evidence for the effects of carotenoids or polyphenols as anti-or pro-oxidants in vivo i

126

17]. Therefore I list the question of the physiological im-portance of redox signalling as one of the challenges facingthe RS/antioxidant field in the coming years (Table 1).

The human antioxidant defence network is complex andinterlocking; it functions to minimize levels of RS whileallowing useful roles to continue (Figure 1). Two recentexamples: some O2

�� in muscle appears to be useful, butexcess causes harm [14,17,19]; stem cells need some RS tofunction properly, but too many RS can impair function[20]. The properties of the peroxiredoxin proteins, whichscavenge H2O2 but can be inactivated by it, could beespecially important in allowing localized and transientH2O2-dependent signal transduction [13,21]. In humans,the greatmajority of ‘total antioxidant capacity’ of cells andtissues is contributed by endogenously-synthesized anti-oxidants such as reduced glutathione (GSH), peroxiredox-ins and superoxide dismutase; whereas diet-derivedantioxidants are far less important, a point upon whichI elaborate below. It follows that, instead of using dietary‘antioxidants’, a more effective way of strengthening over-all antioxidant defence in humans might be mild pro-oxidants, increasing the generation of RS and electrophiles

one

upregulation

upregulation

SOD, Catalase

helatorsctoferrin)

Blood componentseg.Albumin,Caeruloplasmin,Haemopexin,HaptoglobinTransferrin

Antioxidants

Diet-derivedpro-oxidants(eg. polyphenols?)

Diet-derivedAntioxidants(eg. polyphenols?)Vitamin E, Vitamin C,Carotenoids?

TRENDS in Pharmacological Sciences

from [18] with permission from Elsevier. Some degree of oxidative damage occurs

amaged lipids and proteins) mechanisms are therefore essential. The ‘?’ signifies that

s not compelling, but some effects could be exerted within the gastrointestinal tract.

Table 3. If antioxidants rarely change oxidative damage levelsin humans, what does?

� Obesity (humans)

� Hyperglycaemia (humans)

� High plasma LDL–cholesterol (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)aThis could depend on how well glucose and lipids have been normalized in the

diabetic cohorts studied.bDespite the propensity of PUFAs to oxidize in vitro, growing evidence suggests that

they reduce oxidative damage in vivo [38].Abbreviations: LDL, low-density lipopro-

tein; PUFAs, polyunsaturated fatty acids.

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

that then trigger increases in the levels of our own anti-oxidants, such as GSH [22,23].

Because RS are not completely removed in vivo(Figure 1), it follows that the ability to repair (e.g. DNArepair) or replace (e.g. lipid turnover, degradation of ab-normal proteins by the proteasome and by lysosomes)oxidatively-damaged molecules is essential. Failures insome or all of these repair systems could contribute moreto ageing and age-related disease than changes in antiox-idants or RS production, although the magnitude of thecontribution remains unclear [24–26].

Human antioxidant capacity resists modulation by

dietary antioxidants

In recent years, better (although still not perfect) biomark-ers of oxidative damage in human tissues and body fluidshave been developed (Table 2). Note that most are mea-sured in blood or urine, and they could easily fail to detectchanges in oxidative damage in small groups of cells orindividual organs. For example, rises in oxidative damagelevels in the brain are probably not usually reflected in theblood (discussed in [27]). Nevertheless, studies using bio-markers reveal that giving antioxidant supplements tohealthy or diseased humans rarely causes much changein systemic levels of oxidative damage [28–34]. The failureof most human intervention trials with antioxidants tomodify disease outcome [1,35,36] is therefore not surpris-ing. Even if oxidative damage does play a role in thedisease, ‘antioxidants’ will be ineffective if they fail to alterthe levels of oxidative damage. If RS are involved in theadaptation to tissue injury (as discussed earlier and sum-marized in Figure 2), then high doses of antioxidants couldsometimes 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 enzymesc

To be avoided because the results are often misleading

� Simple TBA assays

� Total antioxidant capacity determination

� Many commercial kit methods for biomarkersaMass spectrometric methodology only.bMore research is needed on the origins of 8OHdG and whether their relative

contributions of different sources vary with age or disease.cProbably underestimates oxidative DNA damage [61] and needs careful calibration

[61–63]. Table adapted from [28] with permission.

because otherwise they might sometimes have causedharm rather than good.

If antioxidants generally fail to modulate oxidativedamage in humans, what does? Table 3 attempts to sum-marize our limited knowledge to date. One important pointis that laboratory rats andmice appear to bemore sensitiveto dietary antioxidant levels than are humans, and dietaryantioxidant supplementation is therefore more likely toreduce oxidative damage in rodents than in humans[1,39,40]. Indeed, the antioxidant content of animal feedcould account for several discrepancies between resultsreported from different laboratories [39]. It follows thatrodent models of human diseases in which oxidative dam-age appears to be important in the pathology (such asstroke, atherosclerosis, amyotrophic lateral sclerosis anddementia) are more likely to be responsive to administeredantioxidants than are humans. This should be borne inmind when evaluating the potential uses of ‘therapeuticantioxidants’ 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 baldnessto failed pregnancy (perhaps the opposite is true for thelatter [36]). Their role in many diseases is likely to beperipheral, but in some it is fundamental (Figure 2). Can-cer is probably an example of the latter: RS can causecancer, contribute to its progression, and sometimes evencure it [42]. Neurodegenerative disease is another exam-ple: marked oxidative damage is present in affected brainregions in both Parkinson and Alzheimer diseases; there issome evidence that this damage precedes neurodegenera-tion and studies on animal models suggest that decreasingoxidative damage can preserve neuronal function [1,27,43].

Developing better antioxidants?Developing better antioxidants is a major challenge for theredox biology field (Table 1). Many antioxidants are avail-able (Table 4) but most have failed dismally in clinicalstudies even when there is strong evidence that oxidativedamage plays a role in the disease pathology (and theevidence is often not robust). Probable reasons for thisfailure have been summarized [1,41,44]. Promising anti-oxidants under evaluation include low molecular mass

127

[()TD$FIG]

Tissue injury

Oxidative damage

Late stage in tissueinjury, accompanying

cell death

Induction ofantioxidant andother defence/repair systems (e.g. heat shock

proteins,proteasome,autophagy,

haem oxygenase)

Early stage in tissueinjury

Neutralization ofoxidative stress.

Sometimessuperprotection of

the tissue

Necrotic death of some cells spreadsdamage 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

No adverse contribution of“oxidative damage” to disease

pathology. RS can sometimes bebeneficial.

(e.g. in ischemic preconditioningor 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 Examples

Naturally occurring 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

Synthetic 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.

Agents already in

clinical use that

might have some

antioxidant activity

in vivo (but were

not developed as

antioxidants)

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

aCompounds 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.bSeveral 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.

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

128

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

catalytic scavengers of RS [45,46] and targeted antioxi-dants (for example to mitochondria) [44,47,48]. However, Ihave doubts that these redox-active agents are alwaysantioxidants in vivo; indeed they can sometimes be mildpro-oxidants [46,49], and could thereby bemore effective inreducing oxidative damage (Figure 1).

Flavonoids and other polyphenols, widely touted asbeneficial to health, can act as both antioxidants andpro-oxidants in vitro, but there is little or no evidence thatthey act as either in vivo, except possibly in the stomach,small intestine and colon [1,29,50,51], although they couldbe beneficial in other ways. It is possible that if sufficientflavonoids could be introduced into the brain they couldoxidize gently and thereby upregulate endogenous antiox-idants, and this could help to treat neurodegeneration(Figure 1).

Time to abandon cell culture?Cell culture studies are used extensively in research anddrug development, but a substantial proportion of cell linesare not the cells they are supposed to be [52]. To quote theauthors of [52] ‘thousands of misleading studies and poten-tially erroneous papers have been published’. The scale ofthe problem is under-appreciated by most scientists [52].Equally under-appreciated is the fact that the effects of‘‘antioxidants’’ such as ascorbate, lycopene, epigallocatechingallate and several other polyphenols on cells in culture areoften artifacts – reflecting the reactions of these compoundswith components of cell culture media and/or their rapiddecomposition into other bioactive agents in cell culture.Several groups have highlighted and tabulated these pro-blems (summarized in [18,53]) but artefactual reports de-scribing the effects of flavonoids and other ‘antioxidants’ oncells in culture continuetoproliferate faster thanHeLacells,and (sadly) continue to pass peer-review. Time to call a halt.

Antioxidants and ageingRS are intimately involved in ageing and age-relateddisease [54,55]. RS probably do not cause ageing (theycould perhaps account for some features of ageing, suchas skin wrinkling), nor will dietary antioxidants stop age-ing [24–26,56,57]. Perhaps mild pro-oxidants might help,by raising endogenous antioxidant defences [57–59]. IfROS 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 wrongtype of RS). As the world population ages, we need to solvethis issue urgently; this is at the top of my list for personalresearch in the next few years (Table 1).

Concluding remarksThe biology of ROS and antioxidants is not an esoteric fieldof study: these species are involved in all aspects of aerobiclife. One cannot live without them, nor would one wish to,but ultimately they no doubt contribute to individual mor-tality. Learning how to stop the latter while preserving theuseful functions of RS should be a major research priority.

References1 Halliwell, B. and Gutteridge, J.M.C. (2007) Free Radicals in Biology

and Medicine (4th edn), Oxford University Press

2 Halliwell, B. (2006) Reactive species and antioxidants. Redox biology isa fundamental theme of aerobic life. Plant Physiol. 141, 312–322

3 Janssen-Heininger, M.W. et al. (2008) Redox-based regulation of signaltransduction: principles, pitfalls and promises. Free Radic. Biol. Med.45, 1–17

4 Lambeth, J.D. (2007) Nox enzymes, ROS, and chronic disease: anexample of antagonistic pleiotropy. Free Radic. Biol. Med. 43, 332–347

5 Huang, T.T. et al. (2001) Genetic modification of prenatal lethality anddilated cardiomyopathy in Mn superoxide dismutase mutant mice.Free Radic. Biol. Med. 31, 1101–1110

6 Kim, A. et al. (2010) Enhanced expression of mitochondrial superoxidedismutase leads to prolonged in vivo cell cycle progression and up-regulation of mitochondrial thioredoxin. Free Radic. Biol. Med. 48,1501–1512

7 Stowe, D.F. and Camara, A.K.S. (2009) Mitochondrial reactive oxygenspecies production in excitable cells: modulators of mitochondrial andcell function. Antioxid. Redox Signal. 11, 1373–1414

8 Imlay, J.A. (2008) Cellular defenses against superoxide and hydrogenperoxide. Annu. Rev. Biochem. 77, 755–776

9 Hultqvist, M. et al. (2009) The protective role of ROS in autoimmunedisease. Trends Immunol. 30, 201–208

10 Halliwell, B. (2006) Phagocyte-derived reactive species: salvation orsuicide? Trends Biochem. Sci. 31, 509–515

11 Morgenstern, D.E. (1997) Absence of respiratory burst in X-linkedchronic granulomatous disease mice leads to abnormalities in bothhost defense and inflammatory response to Aspergillus fumigatus. J.Exp. Med. 185, 207–218

12 Finch, C.E. (2007) The Biology of Human Longevity: Inflammation,Nutrition, and Aging in the Evolution of Lifespans, Academic Press

13 Woo, H.A. et al. (2010) Inactivation of peroxiredoxin I byphosphorylation allows localized H2O2 accumulation for cellsignaling. Cell 140, 517–528

14 Ristow, M. et al. (2009) Antioxidants prevent health-promoting effectsof physical exercise in humans. Proc. Natl. Acad. Sci. U.S.A. 106, 8665–

867015 Tang, X.L. et al. (2002) Oxidant species trigger late preconditioning

againstmyocardial stunning in conscious rabbits.Am. J. Physiol. HeartCirc. Physiol. 282, H281–H291

16 Pagliaro, P. et al. (2010) Cardioprotective pathways duringreperfusion: focus on redox signaling and other modalities of cellsignaling. Antioxid. Redox Signal. DOI: 10.1089/ars.2010.3245

17 Palomero, J. and Jackson, M.R. (2010) Redox regulation in skeletalmuscle during contractile activity and aging. J. Anim. Sci. 88, 1307–

131318 Halliwell, B. (2008) Are polyphenols antioxidants or pro-oxidants?

What do we learn from cell culture and in vivo studies? Arch.Biochem. Biophys. 476, 107–112

19 Jang, Y.C. et al. (2010) Increased superoxide in vivo accelerates age-associated muscle atrophy through mitochondrial dysfunction andneuromuscular junction degeneration. FASEB J. 24, 1376–1390

20 Juntilla, M.M. et al. (2009) AKT1 and AKT2 maintain hematopoieticstem cell function by regulating reactive oxygen species. Blood 115,4030–4038

21 Barranco-Medina, S. et al. (2009) The oligomeric conformation ofperoxiredoxins links redox state to function. FEBS Lett. 583, 1809–

181622 Kaspar, J.W. et al. (2009) Nrf2:INrf2 (Keap1) signaling in oxidative

stress. Free Radic. Biol. Med. 47, 1304–130923 Leiser, S.F. and Miller, R.A. (2010) Nrf2 signaling, a mechanism for

cellular stress resistance in long-lived mice. Mol. Cell Biol. 30, 871–

87424 Gems, D. and Doonan, R. (2009) Antioxidant defense and aging in

C.elegans: is the oxidative damage theory of aging wrong? Cell Cycle 8,1681–1687

25 Perez, V.I et al. (2009) Is the oxidative stress theory of aging dead?Biochim. Biophys. Acta 1790, 1005–1014

26 Jang, Y.C. and Remmen, V.H. (2009) The mitochondrial theory ofaging: insight from transgenic and knockout mouse models. Exp.Gerontol. 44, 256–260

27 Halliwell, B. (2006) Oxidative stress and neurodegeneration; where arewe now? J. Neurochem. 97, 1634–1658

28 Halliwell, B. (2009) The wanderings of a free radical. Free Radic. Biol.Med. 46, 531–542

129

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

29 Halliwell, B. et al. (2005) Health promotion by flavonoids, tocopherols,tocotrienols, and other phenols: direct or indirect effects? Antioxidantor not? Am. J. Clin. Nutr. 81, 276S–286S

30 Møller, P. and Loft, S. (2002) Oxidative DNA damage in human whiteblood cells in dietary antioxidant intervention studies. Am. J. Clin.Nutr. 76, 303–310

31 Loke, W.M. et al. (2008) Pure dietary flavonoids quercetin and (�)-epicatechin augment nitric oxide products and reduce endothelin-1acutely in healthy men. Am. J. Clin. Nutr. 88, 1018–1025

32 Rytter, E. et al. (2010) Biomarkers of oxidative stress in overweightmen are not influenced by a combination of antioxidants. Free Radic.Res. 44, 522–528

33 Meagher, E.A. et al. (2001) Effects of vitamin E on lipid peroxidation inhealthy persons. JAMA 285, 1178–1182

34 Jacob, R.A. et al. (2003) Moderate antioxidant supplementation has noeffect on biomarkers of oxidant damage in healthy men with low fruitand vegetable intakes. J. Nutr. 133, 740–743

35 Bjelakovic, G. et al. (2008) Systematic review: primary and secondaryprevention of gastrointestinal cancers with antioxidant supplements.Aliment. Pharmacol. Ther. 28, 689–703

36 Xu, H. et al. (2010) An international trial of antioxidants in theprevention of preeclampsia (INTAPP). Am. J. Obstet. Gynecol. 202,239.e1–e10

37 Seet, R.C. et al. (2010) Markers of oxidative damage are not elevated inotherwise healthy individuals with the metabolic syndrome. DiabetesCare 33, 1140–1142

38 Mas, E. et al. (2010) The omega-3 fatty acids EPA and DHA decreaseplasma F2-isoprostanes: results from two placebo-controlledinterventions. Free Radic. Res. 44, 983–990

39 Lehr, H.A. et al. (1999) Do vitamin E supplements in diets forlaboratory animals jeopardize findings in animal models of disease?Free Radic. Biol. Med. 26, 472–481

40 Loke, W.M. et al. (2010) Specific dietary polyphenols attenuateatherosclerosis in apolipoprotein E knockout mice by alleviatinginflammation and endothelial dysfunction. Arterioscler. Thromb.Vasc. Biol. 30, 749–757

41 Halliwell, B. (2001) Role of free radicals in the neurodegenerativediseases. Therapeutic implications for antioxidant treatment. Drugs& Aging 18, 685–726

42 Halliwell, B. (2007) Oxidative stress and cancer: have we movedforward? Biochem. J. 401, 1–11

43 Butterfield, D.A. (2010) Oxidative stress in Alzheimer disease: synergybetween the Butterfield andMarkesbery laboratories.NeuromolecularMed. DOI: 10.1007/s12017-010-8123-9

44 Cocheme, H.M. and Murphy, M.P. (2010) Can antioxidants be effectivetherapeutics? Curr. Opin. Investig. Drugs 11, 426–431

45 Day, B.J. (2009) Catalase and glutathione peroxidase mimics.Biochem. Pharmacol. 77, 285–296

130

46 Batinıc-Haberle, I. et al. (2010) Superoxide dismutase mimics:chemistry, pharmacology and therapeutic potential. Antioxid. RedoxSignal. 13, 877–918

47 Wanagat, J. et al. (2010) Mitochondrial oxidative stress andmammalian healthspan. Mech. Ageing Dev. 131, 527–535

48 Anisimov, V.N. et al. (2008) Mitochondria-targeted plastoquinonederivatives as tools to interrupt execution of the ageing program. 5.SkQ1 prolongs lifespan and prevents development of traits ofsenescence. Biochemistry (Mosc.) 73, 1329–1342

49 Doughan, A.K. and Dikalov, S.I. (2007) Mitochondrial redox cycling ofmitoquinone leads to superoxide production and cellular apoptosis.Antioxid. Redox Signal. 9, 1825–1836

50 Halliwell, B. (2000) The gastrointestinal tract: a major site ofantioxidant action? Free Radic. Res. 33, 819–830

51 Gorelik, S. et al. (2008) The stomach as a ‘bioreactor’: when red meatmeets red wine. J. Agric. Food Chem. 56, 5002–5007

52 American Type Culture Collection Standards DevelopmentOrganization Workgroup ASN-0002 (2010) Cell line misidentification:the beginning of the end. Nat. Rev. Cancer 10, 441–448

53 Long, L.H. et al. (2010) Instability of, and generation of hydrogenperoxide by, phenolic compounds in cell culture media. Arch.Biochem. Biophys. 501, 162–169

54 Harman, D. (2009) About ‘origin and evolution of the free radicaltheory of aging: a brief personal history, 1954–2009’. Biogerontology10, 783

55 Dai, D-F. et al. (2009) Overexpression of catalase targeted tomitochondria attenuates murine cardiac aging. Circulation 119,2789–2797

56 Gruber, J. et al. (2008) Themitochondrial free radical theory of ageing –

where do we stand? Front. Biosci. 13, 6554–657957 Rattan, S.I. and Demirovic, D. (2009) Hormesis can and does work in

humans. Dose Response 8, 58–6358 Pun, P.B. et al. (2010) Ageing in nematodes: do antioxidants extend

lifespan in Caenorhabditis elegans? Biogerontology 11, 17–3059 Lapointe, J. et al. (2009) Reversal of the mitochondrial phenotype and

slow development of oxidative biomarkers of aging in long-livedMclk1+/� mice. J. Biol. Chem. 284, 20364–20374

60 Droge, Q. and Schipper, H.M. (2007) Oxidative stress and aberrantsignaling in aging and cognitive decline. Aging Cell 6, 361–370

61 Bergeron, F. et al. (2010) HO� radicals induce an unexpected highproportion of tandem base lesions refractory to repair by DNAglycosylases. Proc. Natl. Acad. Sci. U.S.A. 107, 5528–5533

62 Johansson, C. et al. (2010) An ECVAG trial on assessment of oxidativedamage to DNA measured by the comet assay. Mutagenesis. 25, 125–

13263 Zainol, M. et al. (2009) Introducing a true internal standard for the

Comet assay to minimize intra- and inter-experiment variability inmeasures of DNA damage and repair. Nucleic Acids Res. 37, e150