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Ten misconceptions about antioxidants

Aalt Bast and Guido R.M.M. Haenen
Department of Toxicology, Faculty of Health Medicine and Life Sciences, Maastricht University, Maastricht, The Netherlands

Oxidative damage is a common cellular event involved in numerous diseases and drug toxicities. Antioxidants prevent or delay oxidative damage, and therefore there has been extensive research into the discovery of natural and newly designed antioxidants. Initial excitement regarding the potential health benets of antioxidants has diminished. Currently, it is even claimed that antioxidants increase mortality. The antioxidant pendulum appears to swing from healthy to toxic and from general panacea to insignicant ingredient. Owing to the polarity of views towards antioxidants, nutritional recommendation ranges from advice to increase antioxidant status in plasma to the notion that it is a useless measurement. Such views, lacking sufcient scientic support, lead to misconceptions, which in our opinion hinder the rational use of food supplements and impedes the design and development of new antioxidant drugs. As a result, good opportunities might easily be missed. The antioxidant controversy Few scientic subjects have generated as many controversial opinions as antioxidants have. The topic is discussed not only in the scientic literature but also in the lay press. A Google search combining the words antioxidant and health gives over 82 million hits, exemplifying the popularity of the subject. Amid the debate, claims about antioxidants can be strongly exaggerated and irrelevant and awed arguments are even advanced to substantiate such claims. For example, a mixture of compounds is marketed as a life extension formula because the compounds are antioxidants and it is claimed that long-term use of a soap that contains the antioxidant vitamin E is excellent for stretch marks, wrinkles, and blemishes. The soap is also suitable for people with a sensitive skin, and benecial for eczema or psoriasis. An accurate riskbenet analysis of antioxidants cannot be achieved in this way. A basic fact is that antioxidants are part of our daily diet in fruits, vegetables, beverages, spices, and herbs (Box 1), and antioxidant intake is the focus of increasing attention. More recently, designer foods have been enriched with antioxidants. Antioxidants are commonly taken as supplements, and there are also drugs with a clear antioxidant prole on the market [1]. For several decades, we have noticed that the antioxidant pendulum appears to swing
Corresponding author: Bast, A. (a.bast@maastrichtuniversity.nl). Keywords: oxidants; vitamin E; flavonoids; health; nutrition; redox; oxygen radicals. 0165-6147/$ see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tips.2013.05.010

vigorously from only healthy to extremely toxic, and from natural antioxidants are best to antioxidants cannot act. The squabbling parties do not seem to listen to counterarguments. Erroneous statements are not corrected, and thus the pendulum oscillates to the extremes. This inevitably hampers research in the eld and confuses both scientists and consumers. As a consequence, we might fail to spot opportunities for which antioxidants may aid in optimizing health. Here we discuss ten misconceptions and try to restore the balance. Misconception 1: antioxidants cure any disease Antioxidants react with reactive oxygen species (ROS) and thus neutralize their chemical reactivity. It was suggested that this mechanism prevents the (cellular) damage induced by ROS. This led to the claim that ROS-mediated diseases could be treated. ROS are involved in numerous diseases [2]. Subsequently, it was suggested that antioxidants could prevent and treat many diseases [3], which boosted antioxidant research. ROS play a role in chronic obstructive pulmonary disease (COPD), for example. Patients with COPD even exhale the oxidant (ROS) hydrogen peroxide. Another example is the suggested causative role of ROS in ischemia reperfusion damage in brain, heart, and kidney. In atherosclerosis, low-density lipoprotein (LDL) is oxidized and is subsequently taken up by macrophages, leading to foam cell formation and eventually cardiovascular complications. Compounds that can give rise to oxygen radicals (nitrofurantoin, doxorubicin) can also induce tissue damage in lung and heart, respectively. Many studies on protection by antioxidants have been conducted. The expectations for antioxidants were set too high and it was apparent that these compounds cannot remedy everything. Moreover, unrealistic health claims disappointed consumers and scientists. Initial enthusiasm turned into disbelief, and some antioxidants, such as vitamin C, were even considered to be toxic [4,5]. Regarding healthful effects, the positive attitude towards antioxidants was primarily based on in vitro experiments. Effective chemical scavenging activity of antioxidants in vitro led to extrapolation to a protective potential in vivo. Identifying the bioavailability of antioxidants has been largely neglected, although polyphenolic antioxidants appeared to have low bioavailability. In addition, bioaccessibility, corresponding to the fraction that becomes available for absorption, must be considered; in other words, the antioxidant has to be liberated from the
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Box 1. Redox balance between oxidants and antioxidants
What is an oxidant? From a chemical point of view, an oxidant takes up electrons. Biologically, oxidants damage biomolecules. They are reactive species that originate from various biological processes. Examples of oxidizing reactive species are the superoxide anion radical (O2), the hydroxyl radical (OH), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl). Reactive oxygen species are formed as byproducts of mitochondrial respiration and are also generated during inflammation from NADPH oxidase on phagocytes. Xanthine oxidase, the metabolism of arachidonic acid, cytochrome P450, nitric oxide synthase, and myeloperoxidase are also possible sources of reactive species. The biotransformation of drugs, such as the redox cycling drugs doxorubicin and nitrofurantoin, leads to the formation of reactive species. What is an antioxidant? From a chemical point of view, an antioxidant is a compound that prevents or delays the oxidation of another compound. Of two compounds, the one that becomes oxidized functions as an antioxidant for the other. In this sense, antioxidants are not very special. Biologically, however, the definition of an antioxidant requires that it should be active in protecting physiological targets (e.g., fatty acids, proteins, and DNA) at relatively low concentrations. Dietary antioxidants include vitamins E and C. Drugs such as the anesthetic propofol and the recently developed antifibrotic agent pirfenidone act as antioxidants. The mucolytic agent N-acetylcysteine acts as a precursor for the endogenous antioxidant glutathione.

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participants have higher rates of lipid peroxidation than others. These rancids might show a higher risk of cardiovascular disease and would be the individuals who might benet from additional antioxidants [14]. Similarly, the overall null effect of vitamin E on total stroke occurrence might simply be due to a broad denition of stroke and this it might not be possible to capture the differences in pathophysiology underlying various ischemic and hemorrhagic events. A meta-analysis of studies that investigated the effect of vitamin E on stroke indeed showed that it increased the risk of hemorrhagic stroke by 22% and reduced the risk of ischemic stroke by 10% [15]. Because of the unfavorable riskbenet ratio, that is, a relatively small risk reduction for ischemic stroke and the generally more severe outcome for hemorrhagic stroke, the authors cautioned against indiscriminate widespread use of vitamin E [15]. Articles on antioxidants and mortality [9,16] have received much attention. For example, it has recently been argued that antioxidant use is more likely to cause than to prevent cancer [16]. Blockage of oxidant-driven apoptosis in cancer cells by antioxidants was presented as a potentially hazardous phenomenon. Because antioxidants do not have to have a benecial effect per se, it is important to identify groups such as rancids who might benet from antioxidants. We should not place too much credence in unbalanced alarming news. Misconception 3: the more the better As the founding father of orthomolecular medicine, Linus Pauling advocated the use of the antioxidant vitamin C in doses of 1000 mg [17] to optimize health, which vastly exceeds the recommended dietary allowance of 75 90 mg/day [18]. A solid scientic justication with clinical studies supporting the assumed health benet of mega doses of vitamin C is, however, lacking. Moreover, the Renaissance physician Paracelsus noted more than 500 years ago that every compound has negative effects at a high dose. This also holds for antioxidants. Administration of high doses of antioxidants might explain the increased toxicity that is sometimes reported. For example, supplementation with 20 mg of b-carotene in male smokers increased the incidence of lung cancer by 18% [19]. Note that the estimated average daily intake of b-carotene is only 2 7 mg [20,21]. In hindsight it is astonishing that at the time of supplementation studies, essential information on the biotransformation of b-carotene was lacking [22]. Clearly, the more the better is not the case. Identication of an optimal dose with a high benetrisk ratio is required, along with adequate knowledge of the biotransformation of antioxidants. Misconception 4: at high doses, antioxidants become pro-oxidant Antioxidants have the ability to donate electrons. This reducing power is essential in neutralizing radicals and other reactive species. In the presence of transition metal ions, electron donation may lead to a pro-oxidant effect. The effect of ascorbic acid on iron-induced lipid peroxidation in vitro is a very illustrative example of this effect [23]. Iron itself induces mild lipid peroxidation and in combination,

food matrix [6]. After absorption, rst-pass metabolism can be very extensive, and currently there is more emphasis on the biological relevance of antioxidant metabolites [7]. We now realize that high chemical reactivity of the parent compound in vitro is not conclusive evidence that the compound can cure any disease associated with ROS. We are again observing overenthusiasm towards antioxidants with the discovery of their effects on gene expression by mechanisms other via an inuence on the DNA sequence (i.e., epigenetics). Presented as a new unifying mechanism to putatively explain all the activities of antioxidants [8], it seems that history repeats itself. Misconception 2: antioxidants increase mortality A recent meta-analysis of selected randomized clinical trials concluded that antioxidant supplementation increased all-cause mortality [9]. However, this conclusion was refuted after re-examination showed that none of the studies had mortality as a primary outcome [10]. Despite the obvious criticism, the general unjustied and unbalanced notion that antioxidants could be highly unsafe remains. Instead of the polarized view whereby antioxidants are either good or bad, a high benetrisk ratio would be a more appropriate approach to evaluate antioxidants. Such a ratio weighs both the pros and cons, and thus provides a more realistic and balanced view. Observational studies have suggested a protective effect of vitamin E intake on coronary heart disease [11]. However, many large, randomized, placebo-controlled trials reported disappointing results for the effect of vitamin E on risk of cardiovascular disease [12,13]. A critical evaluation of these studies suggested that a detailed analysis of the participants diets might lead to a different conclusion. It was suggested that some apparently healthy
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ascorbic acid promotes the lipid peroxidation process by reducing the transition metal ion. However, at a relatively high concentration, ascorbic acid inhibits lipid peroxidation. Therefore, at low concentrations ascorbic acid behaves as a pro-oxidant but at high concentrations it becomes an antioxidant [24]. This contradicts the idea that a high concentration of an antioxidant always has a prooxidant effect. The pro-oxidant activity of vitamin C in vivo has been the subject of well-publicized studies [4,5]. Daily supplementation with 500 mg vitamin C for 6 weeks elevated plasma vitamin C levels by 60% [5]. The authors suggested that vitamin C has pro-oxidant properties after observing levels of oxidized DNA bases measured in peripheral blood lymphocytes [5]. Criticism included the notion that DNA oxidation did not occur in vivo but rather during DNA isolation. Moreover, the suggested increase in lymphocyte vitamin C levels was not checked [25]. A separate study demonstrated that vitamin C could react with lipid hydroperoxides, leading to DNA damaging agents [4]. The in vitro study methodology did not take into account three important points: (i) the decomposition of lipid hydroperoxides by glutathione peroxidases in vivo, (ii) the excessively high concentration of lipid peroxides, and (iii) the fact that the formation of lipid hydroperoxides in vivo will occur only after vitamin C has been exhausted [25]. An authoritative review by Carr et al. concluded that the data on vitamin C and DNA oxidation in vivo are inconsistent and conicting, but some of the discrepancies can be explained by aws in experimental design and methodology [25]. There seems to be confusion between the high dose relationship to toxicity according to Paracelsus and the notion of pro-oxidant behavior. Nevertheless, this misconception persists. Misconception 5: any antioxidant will do In general, antioxidants act by delaying or preventing the oxidation of other compounds. Therefore, the word
Table 1. Balanced view of biomarkersa
Biomarker Lipids F2-isoprostanes [60]: prostaglandin-like products of primarily arachidonic acid Malondialdehyde (MDA) [61]: reactive breakdown product of PUFAs formed during lipid peroxidation Lipid dienes [62]: product of PUFAs formed during lipid peroxidation Ex vivo LDL oxidation [63]: antioxidants prevent oxidation of LDL isolated after antioxidant supplementation DNA 8-Hydroxy-20 -deoxyguanosine [64]: damaged nucleotide formed by radical damage to DNA Comet [65]: DNA damage and strand breaks Proteins Protein carbonyls [66]: protein oxidation products Nitrotyrosine [67]: formed by reaction of reactive nitrogen species with tyrosine Protein activity [68]: the activity of many proteins is reduced after reaction with reactive species
a

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antioxidant is used as a general descriptive term because antioxidants are in fact distinct chemical entities with different modes of action for their effects. Antioxidants can be thiols, phenols, and amines and may be either hydrophilic or lipophilic. In analogy to the term pharmacophore, we introduced the term nutricophore to indicate the functional moiety in a molecule and emphasize the diverse chemical properties of antioxidants [26,27]. This diversity gives every antioxidant its unique (bio-)chemical prole, which is reected in different sites of action and biological activities. Different antioxidants display different biological effects, and for specic pathological conditions the right antioxidant needs to be selected. In protecting LDL against oxidation, for example, the lipophilic and hydrophilic features of the antioxidant hydroxytyrosol are both involved in its activity, which is exhibited at the lipidwater interface. Misconception 6: theoretically, antioxidants cannot behave as such This misconception emanates from the fact that the reaction rate of radicals with endogenous (bio-)molecules is very high. This means that in order to protect, antioxidants have to react even faster with radicals, which, it is argued, is impossible. This is correct for extremely reactive hydroxyl radicals. However, lipid peroxyl radicals in membranes, which have a much longer half-life than hydroxyl radicals, can be neutralized by vitamin E [28]. Even for hydroxyl radicals, site-specic scavenging, for example by binding to iron and neutralizing the radical at the site of formation, may provide protection. In other words, primary protection, that is, preventing the formation of hydroxyl radicals, is possible. Antioxidants can also act indirectly. They can have very specic activities, such as inhibition of NADPH oxidase, regulation of redox-sensitive signal transduction pathways including transcription factors, and inhibition of poly(ADPribose)polymerase (PARP-1) [29]. As well as direct scavenging, indirect antioxidant action through anti-inammatory

Advantages Highly specic Frequently applied method Simple Semi-functional marker

Disadvantages Requires specialized analytical skills MDA is broken down by aldehyde dehydrogenases Low sensitivity and specicity Amphiphilic antioxidants are lost during LDL isolation Prone to artifacts Well-standardized procedure required High background and poor selectivity Nitration of tyrosine during sample preparation Low sensitivity

Specic and sensitive Semi-functional marker Relatively stable Specic Semi-functional marker

The biological half-life of radicals and other reactive species is too short for direct detection. Therefore, evidence has to rely on indirect measurements. This is often based on the products generated when a biological target has been hit, that is, lipids, proteins, and DNA [69]. PUFA, polyunsaturated fatty acid.

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Table 2. Antioxidant activity: examples of enzymatic and non-enzymatic antioxidants, antioxidant capacity assays, and indirect antioxidant activity
Enzymatic antioxidants Superoxide dismutase (SOD) [70] Glutathione peroxidase (GPx) [71] Peroxiredoxin (PRDX) [72] Glutathione reductase (GR) [73] Catalase (Cat) [74] Thioredoxin (TRX) [75] Thioredoxin reductase (TR) [76] Non-enzymatic antioxidants Vitamin E [77] Carotenoids [78] Flavonoids [79] Hydroxytyrosol [80] Vitamin C [81] Glutathione [82] Antioxidant capacity assay ORAC [83] TEAC [84] DPPH radical scavenging [85] FRAP [86] Indirect antioxidant activity Inhibition of NADPH oxidase [87] Inhibition of xanthine oxidase [88] Iron chelation [89] Inhibition of poly(ADP-ribose) polymerase-1 [90] Activation of the transcription factor Nrf2 [91] Antioxidant activity 2 O2+2 H+!H2O2+O2 2 GSH+ROOH!GSSG+ROH+H2O 2 R0 -SH+ROOH!R0 -S-S-R0 +H2O+ROH GSSG+NADPH+H+!NADP++2 GSH 2 H2O2!2 H2O+O2 Protein disulde ! protein dithiol TRX(S-Se)+NADPH+H+!NADP++TRX(SH SeH) Major reactive species scavenged Lipid peroxyl radical Singlet oxygen Superoxide radical Lipid peroxyl radical Vitamin E radicals (regeneration) Hydro(gen)peroxide (as cofactor) Antioxidant activity Prevention of lipid peroxidation induced by an articial radical Amount of ABTS radicals scavenged Amount of DPPH radicals scavenged Capacity to reduce iron(III) to iron(II) Antioxidant activity Reduced production of superoxide radicals Reduced production of superoxide radicals Reduced production of hydroxyl radicals Increased genomic stability, mitigation of the inammatory response Induction of antioxidant genes, e.g., heme oxygenase (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), glutamatecysteine ligase (Gcl), UDP-glucuronosyltransferase (UGT1A1, UGT1A6), glutathione transferase (GST pi) Essential groups in the active center (human origin) Cu and Zn (SOD1, SOD3), Mn (SOD2) Selenocysteine (GPx14, 6), cysteine (GPx5, 7, 8) Cysteine Vicinal disulde, cofactor FAD Iron-containing heme group Vicinal cysteine and selenocysteine Vicinal disulde, cofactor FAD Characteristics Lipophilic Lipophilic Lipophilic to hydrophilic Amphiphilic Hydrophilic Hydrophilic Remarks Well-standardized procedure required A well standardized procedure has to be applied A well standardized procedure has to be applied Fe(III) reduction promotes radical formation Compounds Apocynin, avonoid metabolites Allopurinol 7-monohydroxyethylrutoside (MonoHER) Deferoxamine Caffeine, theobromine, theophylline, niacin, avonoids Sulforophane, oltipraz

activity or induction of antioxidant protective factors can occur (Table 1). Without antioxidants, life in an aerobic environment would be impossible. Antioxidants do act. Their action has been established by determining their effect on biomarkers reecting oxidative damage. Examples are given in Table 1. Misconception 7: antioxidant status measures health Several methods [e.g., the Trolox equivalent antioxidant capacity (TEAC), oxygen radical absorbance capacity (ORAC) and ferric reducing ability of plasma (FRAP)] are used to determine overall antioxidant status in biological and food samples (Table 2). Antioxidant status in blood plasma was promoted as an ideal biomarker because it reects the antioxidant capacity of all antioxidants present in a sample. The United States Department of Agriculture (USDA) even suggested a denite daily portion of ORAC units as dietary intake. This advice and the ORAC database for selected foods was withdrawn in 2010 because it was rightly regarded as biologically invalid [Oxygen Radical Absorbance Capacity (ORAC) of selected foods, Release 2, http://www.ars.usda.gov/services/docs.htm?docid=15866]. It should be noted that discrete underlying mechanisms that use different radical or oxidant sources and differences
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in experimental procedures affect the outcome of the various antioxidant capacity assays developed. Standardized procedures should be used. Antioxidant status measurements lack specicity [30]. However, this could also be an advantage because the measurement comprises the overall activity of several antioxidants at the same time. Antioxidant status might be useful as an indicator of disease severity; however, it does not take into account the unique biochemical prole of specic antioxidants. This was also elaborated in misconception 5. Misconception 8: once used, antioxidants are inactive It seems quite logical that an antioxidant, once oxidized, loses its antioxidant power. This assumption, however, is not entirely correct. A good example in this context is dihydrolipoic acid, which when oxidized to lipoic acid is still quite active as a HOCl scavenger [31]. Moreover, antioxidants function in a network and regeneration of oxidized antioxidants frequently occurs [32]. In addition, evidence is emerging that glucuronidated as well as methylated metabolites of antioxidants have biological activities [33]. b-Glucuronidase from macrophages or neutrophils can deconjugate glucuronidated avonoids, thereby liberating the free phenolic structures [34]. This

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can occur at a site of inammation at which these cells are present and appears to be an elegant way of targeting these molecules. Misconception 9: natural antioxidants are superior Chemophobia is widespread [35] and natural antioxidants, frequently denoted by the prex bio-, are regarded to be superior to chemically synthesized antioxidants. For consumers, the word bio means natural and is thought to be synonymous with safe. The same school of thought associates the word chemical with danger. The conviction of this myth is widespread and forms the basis for organic farming, in which no pesticides, herbicides, or articial fertilizers are used [35]. Interestingly, R,R,R-a-tocopherol, the natural form of atocopherol, is more effective as a protector against membrane lipid peroxidation than other synthetically derived enantiomers of a-tocopherol [36]. However, synthetic R,R,R-a-tocopherol is identical to the natural form, simply because it is the same molecule. This is also true for biovitamin C which is bioequivalent to chemically synthesized L-ascorbic acid. Misconception 10: antioxidant drugs do not work Many drugs have antioxidant activity even if they are not specically designed as antioxidants, which may contribute to their therapeutic effects [1]. The signicance of this antioxidant action depends both on the compound used and the pathology involved. The antioxidant scavenging activity of the angiotensin-converting enzyme (ACE) inhibitor captopril and of the dihydropyridine-type calcium antagonists is probably less clear because the concentrations required for this activity are scarcely reached. It appears to be difcult to establish direct and acute effects of scavenging antioxidant compounds in vivo. The human physiology is already equipped with an elaborate antioxidant network. The antioxidant action of network-augmenting compounds such as N-acetylcysteine (a glutathione precursor [37]) and propofol (an anesthetic that can act as a substitute for a-tocopherol) is not appraised sufciently. Although N-acetylcysteine reduces the rate of exacerbation in patients with COPD and its mode of antioxidant action is biochemically well understood [38,39], it is not included in the treatment guidelines for these patients [40]. The effect of antioxidants is particularly clear in situations in which radical formation and subsequent damage are also evident. The toxicity of compounds that produce radicals via redox cycling [41] (e.g., the anti-tumor agent doxorubicin) is attenuated by antioxidants. We extensively studied the effect of the iron chelator/antioxidant 7-monohydroxyethylrutoside (monoHER) in doxorubicin-induced cardiotoxicity [42,43]. The protective effect of monoHER on cardiotoxicity is very prominent in mice but less clear in humans. A species-dependent difference in monoHER biotransformation explains this difference between mice and humans [43]. It was found that monoHER increases the cytostatic efcacy of doxorubicin [44,45]. Other antioxidants also inhibit cell proliferation, albeit via various mechanisms [46]. However, there are also suggestions that antioxidants can negatively affect the antitumor action of drugs [16].

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Interestingly, although the physiological relevance of the well-described antioxidant action of compounds is frequently disputed, the relatively unsubstantiated notion that antioxidant nutritional supplements cause rather than prevent cancer by disturbing oxidant-induced apoptosis of cancer cells is receiving huge attention [16]. The paradox antioxidants do not work because they cannot display antioxidant activity and that they at the same time cause toxicity and even premature mortality via antioxidant activity, is contradictory. Another way in which antioxidants act is via activation of transcription factors, including Nrf2 [47]. This leads to activation of endogenous antioxidant enzymes via the antioxidant response element and thus indirect action of antioxidants (Table 2). Concluding remarks How can the proper antioxidant balance be obtained? The toxicological risk for oxygen, as for any compound, is dened as the hazard multiplied by the exposure. We recognize oxygen as being one of the most toxic compounds known. The hazardous property of oxygen arises from its intrinsic chemical reactivity. Exposure is life-long and cannot be prevented without consequence for aerobic life forms. To cope with oxygen, antioxidants are vital. Therefore, endogenous antioxidant protection is already high, and there are several physiological phenomena that make use of the reactivity of oxygen. Clear pharmacological responses should not be expected from antioxidants. Adrien Albert dened pharmacological responses as a form of selective toxicity [48]. Drugs act on a specic target, such as an enzyme, a receptor, or a transporter. The preferable action of a drug is specic, that is, it acts on a unique target and induces a strong effect. The clinical efcacy of a drug is therefore relatively easy to measure. This is in contrast to food and food-derived compounds such as food supplements (Figure 1). These compounds have a multitude of actions. Their action is certainly not specic and their effects on human health are difcult to determine [49]. The benecial effect of drugs is linked to the denition of health as described in the founding constitution of the WHO [50] as a state of complete physical, mental, and social well-being and not merely the absence of disease or inrmity. The denition of health is still subject to change and it is increasingly realized that health is dynamic and involves an ability to adapt to changes, rather than a state of perfection [51]. Antioxidants, nutrients, and food supplements have multifarious modes of action (Figure 1). We concur with the suggestion to upgrade the term for antioxidants with this pleiotropic mode of action to bioactives rather than just antioxidants [52]. Rather subtle homeostatic changes resulting from bioactives might increase the ability to adapt under a variety of perturbations [53]. In attempts to capture this phenomenon, new expressions have emerged as linguistic artwork: increasing metabolic exibility, heterostasis, improving metabolic health by triage, tuning up metabolism, enhancing homeostasis, and increasing elasticity [54,55]. The efcacy of antioxidants is best tested in terms of their ability to maintain homeostasis. In this case, biomarkers for the effect of antioxidants can be found by challenging
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A state of complete physical, mental, and social well-being

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Stac
One target Drug

Eect

Dynamic
the ability to adapt
Multarget
Eect1 Eect 2 Increasing metabolic exibility Heterostasis Enhancing homeostasis Eect 3 Increasing elascity Eect 4 Tuning up metabolism
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Bioacve anoxidant

Figure 1. The effect(s) of drugs and bioactive antioxidants in relation to the change from a static definition of health [50] to a more dynamic one [51]. Note that the static concept, in which a drug is designed to act on only one target, has been changed to a dynamic concept of health, in which a bioactive has a multitude of subtle effects via different targets.

homeostasis [56]. Prevention of insidious pathologies can be detected using stress tests [57]. In this same vein, a search for new ways to detect the subtle effects of antioxidants is needed. A recent example of this is the use of a vascular health index to integrate multiple effects of food supplement intervention on vascular health. The index was calculated by adding the change for 23 separate biomarkers. The signicant increase in this integrated biomarker pointed to a benecial effect of the food supplement on cardiovascular health [58]. The decay of aerobic life by oxygen leads to disease and can be delayed by appropriate antioxidant measures [59]. What a pity it would be if antioxidant-rich diets or antioxidant nutraceuticals were neglected for their putative benecial health effects. What a shame it would be if the design and development of new antioxidant drugs were to cease because misconceptions prevail.
References
1 Bast, A. (1994) Antioxidant pharmacotherapy. Drug News Perspect. 7, 465472 2 Halliwell, B. and Gutteridge, J.M.C. (2007) Free Radicals in Biology and Medicine (4th edn), Oxford University Press 3 Willcox, J.K. et al. (2004) Antioxidants and prevention of chronic disease. Crit. Rev. Food Sci. Nutr. 44, 275295 4 Lee, S.H. et al. (2001) Vitamin C-induced decomposition of lipid hydroperoxides to endogenous genotoxins. Science 292, 20832086 5 Podmore, I.D. et al. (1998) Vitamin C exhibits pro-oxidant properties. Nature 392, 559 6 Anson, N.M. et al. (2009) Bioprocessing of wheat bran improves in vitro bioaccessibility and colonic metabolism of phenolic compounds. J. Agric. Food Chem. 57, 61486155 7 Hollman, P.C. et al. (2011) The biological relevance of direct antioxidant effects of polyphenols for cardiovascular health in humans is not established. J. Nutr. 141, 989S1009S 8 Malireddy, S. et al. (2012) Phytochemical antioxidants modulate mammalian cellular epigenome: implications in health and disease. Antioxid. Redox Signal. 17, 327339
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9 Bjelakovic, G. et al. (2007) Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: systematic review and meta-analysis. J. Am. Med. Assoc. 297, 842857 10 Biesalski, H.K. et al. (2010) Reexamination of a meta-analysis of the effect of antioxidant supplementation on mortality and health in randomized trials. Nutrients 2, 929949 11 Stampfer, M.J. et al. (1993) Vitamin-E consumption and the risk of coronary-disease in women. N. Engl. J. Med. 328, 14441449 12 Vivekananthan, D.P. et al. (2003) Use of antioxidant vitamins for the prevention of cardiovascular disease: meta-analysis of randomised trials. Lancet 361, 20172023 13 Eidelman, R.S. et al. (2004) Randomized trials of vitamin E in the treatment and prevention of cardiovascular disease. Arch. Intern. Med. 164, 15521556 14 Halliwell, B. (2000) The antioxidant paradox. Lancet 355, 11791180 15 Schu rks, M. et al. (2010) Effects of vitamin E on stroke subtypes: metaanalysis of randomised controlled trials. Br. Med. J. 314, c5702 16 Watson, J. (2013) Oxidants, antioxidants and the current incurability of metastatic cancers. Open Biol. 3, 120144 17 Pauling, L. (1970) Vitamin C and the Common Cold. W.H. Freeman 18 Institute of Medicine (2000) Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids. National Academy Press 19 The Alfa-Tocopherol, Beta Carotene Cancer Prevention Study Group (1994) The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancer in male smokers. N. Engl. J. Med. 330, 10291035 20 Kousik, A. et al. (2006) Intake of the major carotenoids and the risk of epithelial ovarian cancer in a pooled analysis of 10 cohort studies. Int. J. Cancer 119, 21482154 21 EFSA Panel on Food Additives and Nutritional Sources Added to Food (2012) Scientic opinion on the re-evaluation of mixed carotenes and beta-carotene as food additive. EFSA J. 10, 2593 22 Wang, X.D. (1994) Absorption and metabolism of beta-carotene. J. Am. Coll. Nutr. 13, 314325 23 Bast, A. et al. (1991) Oxidants and antioxidants: state of the art. Am. J. Med. 91, 2S13S 24 Halliwell, B. (1996) Vitamin C: antioxidant or pro-oxidant in vivo. Free Radic. Res. 25, 439454 25 Carr, A. et al. (1999) Does vitamin C act as a pro-oxidant under physiological conditions? FASEB J. 13, 10071024 26 Rezk, B.M. et al. (2002) The antioxidant activity of ploretin: the disclosure of a new antioxidant pharmacophore in avonoids. Biochem. Biophys. Res. Commun. 295, 913 27 Heijnen, C.G.M. et al. (2002) Protection of avonoids against lipid peroxidation: the structure activity relationship revisited. Free Radic. Res. 36, 575581 28 van Acker, S.A.B.E. et al. (1993) Molecular pharmacology of vitamin E: structural aspects of antioxidant activity. Free Radic. Biol. Med. 15, 311328 29 Weseler, A.R. et al. (2010) Oxidative stress and vascular function: implications for pharmacologic treatment. Curr. Hypertens. Rep. 12, 154161 30 Arts, M.J.T.J. et al. (2004) A new approach to assess the total antioxidant capacity using the TEAC assay. Food Chem. 88, 567570 31 Biewenga, G.P. et al. (1997) The pharmacology of the antioxidant lipoic acid. Gen. Pharmacol. 29, 315331 32 Jacobs, H. et al. (2010) An essential difference between the avonoids monoHER and quercetin in their interplay with the endogenous antioxidant network. PLoS ONE 5, e13880 33 Chanet, A. et al. (2013) Flavanone metabolites decrease monocyte adhesion to TNF-a-activated endothelial cells by modulating expression of atherosclerosis-related genes. Br. J. Nutr. http:// dx.doi.org/10.1017/S0007114512005454 , R. et al. (2010) Deconjugation kinetics of glucuronidated 34 Bartholome phase II avonoid metabolites by b-glucuronidase from neutrophils. Drug Metab. Pharmacokinet. 25, 379387 35 Bast, A. (2003) The risk of eating: natural versus man-made toxins. In Chemistry of Crop Protection (Voss, G. and Ramos, G., eds), pp. 6368, Wiley-VCH , R. and Traber, M.G. (1999) Vitamin E: function and 36 Brigelius-Flohe metabolism. FASEB J. 13, 11451155

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37 Bast, A. et al. (1995) a-Lipoic acid and N-acetylcysteine: new concepts for old drugs. In Biothiols in Health and Disease (Cadenas, E. and Packer, L., eds), pp. 409425, Marcel Dekker 38 Stey, C. et al. (2000) The effect of oral N-acetylcysteine in chronic bronchitis: a quantitative systematic review. Eur. Respir. J. 16, 253262 39 Decramer, M. et al. (2005) Effects of N-acetylcysteine on outcomes in chronic obstructive pulmonary disease (Bronchitis Randomized on NAC Cost-Utility Study, BRONCUS): a randomised placebocontrolled trial. Lancet 365, 15521560 40 Global Initiative for Chronic Obstructive Lung Disease (2013) Global Strategy for the Diagnosis, Management and Prevention of COPD. GOLD 41 Kappus, H. et al. (1981) Toxic effects associated with oxygen metabolism: Redox cycling drugs. Experientia 37, 12331241 42 Bast, A. et al. (2007) Protection by avonoids against anthracycline cardiotoxicity: from chemistry to clinical trials. Cardiovasc. Toxicol. 7, 154159 43 Jacobs, H. et al. (2011) Differences in pharmacological activities of the antioxidant avonoid monoHER in humans and mice are caused by variations in its metabolic prole. Clin. Pharmacol. Ther. 90, 852859 44 Jacobs, H. et al. (2011) The semisynthetic avonoid monoHER sensitizes human soft tissue sarcoma cells to doxorubicin-induced apoptosis via inhibition of nuclear factor -kB. Br. J. Cancer 104, 437440 45 Bruynzeel, A.M. et al. (2007) The effect of monohydroxyethylrutoside on doxorubicin-induced cardiotoxicity in patients treated for metastatic cancer in phase II study. Br. J. Cancer 97, 10841089 46 Sung, B. et al. (2007) Fisetin, an inhibitor of cyclin-dependent kinase 6, down-regulates nuclear factor-kB-regulated cell proliferation, antiapoptotic and metastatic gene products through the suppression of TAK-1 and receptor-interacting protein-regulated IkBa kinase activation. Mol. Pharmacol. 71, 17031714 47 Reuland, D.J. et al. (2013) The role of Nrf2 in the attenuation of cardiovascular disease. Exerc. Sport Sci. Rev. http://dx.doi.org/ 10.1097/JES.0b013e3182948a1e 48 Albert, A. (1985) Selective Toxicity. The Physico-chemical Basis of Therapy (7th edn), Chapman & Hall 49 Heaney, R.P. (2008) Nutrients, endpoints, and the problem of proof. J. Nutr. 138, 15911595 50 World Health Organisation (1946) Preamble to the Constitution of the World Health Organisation as adopted by the International Health Conference. WHO 51 Editorial (2009) What is health? The ability to adapt. Lancet 373, 781 52 Sies, H. (2010) Polyphenols and health: update and perspectives. Arch. Biochem. Biophys. 501, 25 53 Weseler, A. (2012) Pleiotropic-acting nutrients require integrative investigational approaches: the example of avonoids. J. Agric. Food Chem. 60, 89418946 54 Litvin, M. et al. (2013) Recurrent hypoglycemia: boosting the brains metabolic exibility. J. Clin. Invest. 123, 19221924 55 Ames, B.N. (2010) Prevention of mutation, cancer, and other ageassociated diseases by optimizing micronutrient intake. J. Nucleic Acids 2010, 725071 56 van Ommen, B. et al. (2009) Challenging homeostasis to dene biomarkers for nutrition related health. Mol. Nutr. Food Res. 53, 795804 57 Biesalski, H.K. et al. (2011) 26th Hohenheim Consensus Conference, September 11, 2010. Scientic substantiation of health claims: evidence-based nutrition. Nutrition 27, S1S20 58 Weseler, A.R. et al. (2011) Pleiotropic benet of monomeric and oligomeric avanols on vascular health. A randomized controlled clinical pilot study. PLoS ONE 6, e28460 59 Halliwell, B. (2012) Free radicals and antioxidants: updating a personal view. Nutr. Rev. 70, 257265 60 Morrow, J.D. et al. (1995) Increase in circulating products of lipid peroxidation (F2-isoprostanes) in smokers smoking as a cause of oxidative damage. N. Engl. J. Med. 332, 11981203 61 Nielsen, F. et al. (1997) Plasma malondialdehyde as biomarker for oxidative stress: reference interval and effects of life-style factors. Clin. Chem. 43, 12091214 62 Guichardant, M. and Lagarde, M. (2009) Analysis of biomarkers from lipid peroxidation: a comparative study. Eur. J. Lipid Sci. Technol. 111, 7582

Trends in Pharmacological Sciences xxx xxxx, Vol. xxx, No. x

63 Rietjens, S.J. et al. (2007) New insights into controversies on the antioxidant potential of the olive oil antioxidant hydroxytyrosol. J. Agric. Food Chem. 55, 76097614 64 Shigenaga, M.K. and Ames, B.N. (1991) Assays for 8-hydroxy-20 deoxyguanosine: a biomarker of in vivo oxidative DNA damage. Free Radic. Biol. Med. 10, 211216 65 Collins, A.R. (2004) The comet assay for DNA damage and repair. Mol. Biotechnol. 26, 249261 66 Dalle-Donne, I. et al. (2003) Protein carbonyl groups as biomarkers of oxidative stress. Clin. Chim. Acta 329, 2338 67 Van der Vliet, A. et al. (1996) Nitrotyrosine as biomarker for reactive nitrogen species. Methods Enzymol. 269, 175184 68 Aksenov, M. et al. (2000) Oxidative modication of creatine kinase BB in Alzheimers disease brain. J. Neurochem. 74, 25202527 69 Halliwell, B. (2011) Free radicals and antioxidants quo vadis? Trends Pharmacol. Sci. 32, 125130 70 Fukai, T. and Ushio-Fukai, M. (2011) Superoxide dismutases: role in redox signaling, vascular function, and diseases. Antioxid. Redox Signal. 15, 15831606 , R. and Maiorino, M. (2013) Glutathione peroxidases. 71 Brigelius-Flohe Biochim. Biophys. Acta 1830, 32893303 72 Rhee, S.G. et al. (2001) Peroxiredoxin, a novel family of peroxidases. IUBMB Life 52, 3541 73 Meister, A. (1988) Glutathione metabolism and its selective modication. J. Biol. Chem. 263, 1720517208 74 Mueller, S. et al. (1997) Determination of catalase activity at physiological hydrogen peroxide concentrations. Anal. Biochem. 245, 5560 75 Holmgren, A. (1985) Thioredoxin. Annu. Rev. Biochem. 54, 237271 r, E.S. and Holmgren, A. (2000) Physiological functions of 76 Arne thioredoxin and thioredoxin reductase. Eur. J. Biochem. 267, 61026109 77 Niki, E. (2013) Role of vitamin E as lipid-soluble peroxyl radical scavenger: in vitro and in vivo evidence. Free Radic. Biol. Med. http://dx.doi.org/10.1016/j.freeradbiomed.2013.03.022 78 Stahl, W. and Sies, H. (2003) Antioxidant activity of carotenoids. Mol. Aspects Med. 24, 345351 79 Robak, J. and Gryglewski, R.J. (1988) Flavonoids are scavengers of superoxide anions. Biochem. Pharmacol. 37, 837841 80 Rietjens, S.J. et al. (2007) The olive oil antioxidant hydroxytyrosol efciently protects against the oxidative stress-induced impairment of the NO. Response of isolated rat aorta. Am. J. Physiol. Heart Circ. Physiol. 292, H1931H1936 81 Packer, J.E. et al. (1979) Direct observation of a free radical interaction between vitamin E and vitamin C. Nature 278, 737738 82 Mills, G.C. (1960) Glutathione peroxidase and the destruction of hydrogen peroxide in animal tissues. Arch. Biochem. Biophys. 86, 15 83 Huang, D. et al. (2002) High-throughput assay of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate uorescence reader in 96-well format. J. Agric. Food Chem. 50, 44374444 84 van den Berg, R. et al. (1999) Applicability of an improved Trolox equivalent antioxidant capacity (TEAC) assay for evaluation of antioxidant capacity measurements of mixtures. Food Chem. 66, 511517 85 Sharma, O.P. and Bhat, T.K. (2009) DPPH antioxidant assay revisited. Food Chem. 113, 12021205 86 Benzie, I.F. and Strain, J.J. (1996) The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Anal. Biochem. 239, 7076 87 Steffen, Y. et al. (2008) Mono-O-methylated avanols and other avonoids as inhibitors of endothelial NADPH oxidase. Arch. Biochem. Biophys. 469, 209219 88 Cos, P. et al. (1998) Structureactivity relationship and classication of avonoids as inhibitors of xanthine oxidase and superoxide scavengers. J. Nat. Prod. 61, 7176 89 van Acker, S.A.B.E. et al. (1998) Inuence of iron chelation on the antioxidant activity of avonoids. Biochem. Pharmacol. 56, 935943 90 Geraets, L. et al. (2006) Caffeine metabolites are inhibitors of the nuclear enzyme poly (ADP-ribose) polymerase-1 at physiological concentrations. Biochem. Pharmacol. 72, 902910 91 Nguyen, T. et al. (2009) The Nrf2antioxidant response element signaling pathway and its activation by oxidative stress. J. Biol. Chem. 284, 1329113295
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