342 B. Halliwell / Cardiovascular Research 73 (2007) 341–347even the humble ethanol molecule itself [2,17–28]. Are are pro-oxidant in vivo are (in my view) equivocal andfruits and vegetables beneficial because of these other inconclusive. Nor is the evidence that they are antioxidants acomponents? Or is it that fruits and vegetables act (in whole great deal better.or in part) by antioxidant actions, but that the activeantioxidants are not vitamin C, α-tocopherol or β-carotene? 4. Enter the flavonoidsSome studies suggested yes to the latter question [29–31].For example, Verhagen et al.  found that urinary Flavonoids and other polyphenols have powerful antiox-excretion of 8-hydroxy-2′-deoxyguanosine (8OHdG), a idant activities in vitro, being able to scavenge a wide rangeputative biomarker of oxidative damage to DNA and DNA of reactive species, including hydroxyl radicals, peroxylprecursors [32,33], was decreased by feeding human radicals hypochlorous acid and (sometimes) superoxide Uvolunteers Brussels sprouts, but not by giving them α- radical, O2 − (reviewed in ). Flavonoids can also inhibittocopherol, ascorbate, or β-carotene . biomolecular damage by peroxynitrite in vitro [47–49], Several other authors have shown that consumption of although they are less good at doing this in the presence of −antioxidant-rich foods decreases levels of oxidative damage physiological levels of HCO3 /CO2 [49,50]. Peroxynitrite −in vivo in humans (reviewed in ). Others have found little reacts fast with CO2/HCO3 to form reactive products thateffect (e.g. ), and some registered increases in bio- flavonoids appear to scavenge less well. Many flavonoids Downloaded from http://cardiovascres.oxfordjournals.org/ by guest on January 15, 2013markers of oxidative protein damage, such as 2-aminoadipic chelate transition metal ions such as iron and copper,and γ-glutamyl semialdehydes . One must be cautious in decreasing their ability to promote reactive species formationall such studies to rule out confounding effects of refeeding [46,51,52].fasted individuals, as opposed to the effects of antioxidants Two observations drew attention to the potential biologicalin the food, on biomarkers of oxidative damage. Thus importance of flavonoids. First, phenolics in red wine wereVissers et al.  showed that olive oil administration to shown to be able to inhibit the oxidation of LDL in vitro andhuman volunteers decreased the propensity of low-density this was suggested as an explanation of the “French paradox”lipoproteins (LDL) subsequently isolated from their blood to [19,53]. Second, the Zutphen study, an epidemiological studyundergo oxidation in vitro, but feeding oil without anti- in the Netherlands, suggested an inverse correlation betweenoxidants had the same effect. In 2000, we reported  that the incidence of coronary heart disease and stroke and thedark soy sauce has powerful antioxidant abilities in vitro. dietary intake of flavonoids, especially quercetin . SinceRecently, we attempted to see if dark soy sauce decreased then, several other epidemiological studies have confirmedoxidative damage in vivo in human volunteers, and indeed it similar associations, although a few have not, and there is littlewas able to decrease levels of F2-isoprostanes . We evidence that flavonoids protect against cancer . Someadministered the soy sauce with rice, using a placebo col- suggestions of protection against neurodegenerative diseaseouring on the same amount of rice as a control. The rice meal have been made [22,23,43,56–58], although it is unclear to(devoid of antioxidants) also had effects on F2-isoprostanes what extent flavonoids can enter the human brain .and urinary 8OHdG excretion , although the soy sauce Thus could flavonoids be major contributors to themeal did better than the placebo in lowering F2-isoprostane disease-protective effects of fruits and vegetables? If so, islevels. Similarly, Richelle et al.  and Lee et al.  this due to antioxidant effects? Many polyphenols aresuggested that fasting may raise plasma F2-isoprostane absorbed, although rarely completely, and most of thelevels. At the moment, the balance of evidence does suggest remainder are broken down in the colon to generate highthat antioxidant effects contribute to the benefits of a high levels of monophenols [60,61]. Are the amounts ofintake of fruits and vegetables (reviewed in [35,43]) but the polyphenols absorbed sufficient to exert significant antiox-extent of their contribution is uncertain. More work needs to idant effects? Several studies administering flavonoid-richbe done on the effect of diet on oxidative damage, using foods and beverages and measuring biomarkers of oxidativesuitable controls. damage suggest yes, but others no (discussed in [35,62]). “Feeding effects” alluded to earlier could account for some3. Pro-oxidant effects of the apparent positive effects. Are significant antioxidant effects likely in vivo? Plasma levels of unconjugated flavo- Some authors have claimed that “antioxidants” can noids rarely exceed 1 μM and the metabolites tend to have lowerstimulate oxidative damage in vivo, especially ascorbate, antioxidant activity because radical-scavenging –OH groups arealleged in several studies to increase oxidative DNA damage blocked by methylation, sulphation, or glucuronidation(reviewed in ). Indeed, it was suggested that mega-doses [60,61]. Since plasma total antioxidant capacities (TAC) areof ascorbate might kill cancer cells in vivo by oxidizing to often in the range of 1 mM or more (reviewed in ), it seemsproduce H2O2 . We found small and transient increases difficult to imagine how an additional 1 μM polyphenol couldin oxidative DNA damage in human volunteers fed mixtures exert a powerful antioxidant effect in vivo. Some studies haveof ascorbate, β-carotene and α-tocopherol, but there was shown effects of flavonoid-rich foods in raising plasma TAC inwide variation between experiments . Overall, the humans. But one must be cautious here; many such foods canavailable data that ascorbate, β-carotene or α-tocopherol increase plasma uric acid levels, and urate is detected by
B. Halliwell / Cardiovascular Research 73 (2007) 341–347 343several TAC assays [62–64]. Since elevated urate may be a risk against gastric, and possibly colonic, cancer, although againfactor for some diseases, the alleged “antioxidant benefit” may it must not be assumed that any protective effect of fla-not be what it seems . Finally, flavonoids and other phenols vonoid-rich foods is attributable to antioxidant actions ofare complex molecules and have multiple potential actions other the flavonoids, or to flavonoids at all, rather than to otherthan antioxidant ones, including inhibiting telomerase, gluta- components in the foods. However, ingestion of green teamate dehydrogenase, cyclooxygenase, lipoxygenase, xanthine was reported to rapidly decrease prostaglandin E2 concen-oxidase, matrix metalloproteinases, angiotensin-converting trations in human rectal mucosa, consistent with inhibitionenzyme, proteasome, cytochrome P450 and sulphotransferase of cyclooxygenase activity, a potential anti-cancer mecha-enzyme activities, affecting signal transduction pathways and nism . The levels of individual flavonoids in faecalinteracting with sirtuins [25,35,43,56,57,65–73]. Flavonoids water are fairly low (μM or less), but monophenols (manymay also interact with cellular drug transport systems, compete derived from polyphenol breakdown) are present at muchwith glucose for transmembrane transport, interfere with higher concentrations . By contrast to the colon, poly-regulation of the cell cycle, inhibit protein glycation, modulate phenols are likely to be present in the stomach and intes-paraoxonase, myeloperoxidase and thyroid peroxidase activi- tines at high (≥ mM) concentrations after consumption ofties, increase endothelial nitric oxide production and affect polyphenol-rich foods and beverages.platelet function [74–82]. Again, it is uncertain whether some of Why should antioxidant protective effects of polyphe- Downloaded from http://cardiovascres.oxfordjournals.org/ by guest on January 15, 2013these effects occur in vivo, given the low concentration of nols be important to the stomach and intestines? Thebioavailable polyphenols. gastrointestinal (GI) tract is constantly exposed to reactive species. Some are released by the GI tract itself, eg.5. Do polyphenols work pre-absorption? superoxide and H2O2 production by NADPH oxidases and “dual oxidases” in epithelial cells [85–87]. Some reactive It has been proposed  that antioxidant and other species are present in food and beverages, and yet othersprotective effects of flavonoids and other phenolic com- are generated by chemical reactions of dietary componentspounds could occur before absorption, i.e. within the within the stomach [83,88]. Sources of reactive speciesstomach, intestines and colon (Fig. 1). This could account include H2O2 in beverages , the mixtures of ascorbatefor the suggested ability of flavonoid-rich foods to protect and Fe2+ in the stomach (dietary iron, dietary ascorbate,Fig. 1. Dietary antioxidants and the gastrointestinal (GI) tract. ⁎Except when supplements are taken. This figure refers to normal dietary intake. +, There isconsiderable intersubject variability in the efficiency of GI uptake of vitamin E. ▵Much H2O2 may be removed in the oral cavity by catalase and peroxidases insaliva, and by H2O2 diffusion into the oral and oesophageal epithelium followed by its rapid catabolism. Adapted from  with permission from Oxford U U UUniversity Press. RNS Reactive nitrogen species, OH hydroxyl radical, RO alkoxyl radical, RO2 peroxyl radical, RS reactive species, LOX lipoxygenase,COX-2 cyclooxygenase-2.
344 B. Halliwell / Cardiovascular Research 73 (2007) 341–347and ascorbate normally present in gastric juice ), and than would be expected from the rate of its generation byingested haem proteins, which can promote oxidation of either compound alone .dietary lipids . Other reactive species that can be Since there are transition metal ions in the GI tract, it ispresent in foods include lipid peroxides, cytotoxic possible that polyphenols could oxidize there as well. Thisaldehydes, and isoprostanes [91–95]. Nitrite is present at might even be good for you, generating a pro-oxidanthigh levels in saliva and in foods . It is converted to challenge that raises levels of xenobiotic-metabolizing andHNO2 by gastric acid, and HNO2 can then form nitrosating antioxidant defence enzymes in the GI tract.and DNA-deaminating species . Activation of immunecells naturally present in the GI tract by diet-derived bacteria 7. A caution about supplementsand toxins can also increase ROS production . Flavonoids and other phenolic compounds might exert Flavonoid-rich foods appear good for us, although todirect protective effects in the gastrointestinal tract, by what extent (if any) the flavonoids contribute to this benefitscavenging reactive species and/or preventing their forma- is uncertain. Other possible protective components in foodstion. For example, polyphenols can inhibit haem protein- were listed in Section 1. So should we consume flavonoidinduced peroxidation in the stomach [88,99] and decrease supplements or the flavonoid-enriched foods (e.g. cocoa,DNA base deamination or nitrosamine formation by HNO2- chocolate) now coming onto the market in some countries? I Downloaded from http://cardiovascres.oxfordjournals.org/ by guest on January 15, 2013derived reactive nitrogen species [97,100]. Phenols might would be cautious until we know more [2,77,111]. To me,also increase levels of toxin-metabolizing or antioxidant dietary polyphenols are typical xenobiotics, metabolized asdefence enzymes in the GI tract, and chelate transition metal such and rapidly removed from the circulation. They may beions . Dietary iron is usually not completely absorbed, beneficial in the gut in the correct amounts. But too muchespecially among subjects on Western diets. Unabsorbed may not be good and thus, I suggest that one should bedietary iron enters the faeces, where it could represent a pro- content with eating a good diet for now.oxidant challenge to the colon and rectum . Indeed,diets rich in fat and low in fibre may aggravate this pro- Referencesoxidant effect . Phenolic compounds, by chelating iron,may help to alleviate pro-oxidant actions of colonic iron.  Bjelakovic G, Nikolova D, Simonetti RG, Gluud C. Antioxidant supplements for prevention of gastrointestinal cancers: a systematic review and meta-analysis. Lancet 2004;364:1219–28.6. Flavonoids as pro-oxidant xenobiotics  Halliwell B. Polyphenols; antioxidant treats for healthy living or covert toxins. J Sci Food Agric 2006;86:1992–5. Why do hot beverages often contain high levels of H2O2?  Lawlor DA, Davey Smith G, Kundu D, Bruckdorfer KR, Ebrahim S.Simply because the polyphenols within them oxidize readily at Those confounded vitamins: what can we learn from the differenceshigh temperatures [89,103–105]. Polyphenols can also oxidize between observational versus randomised trial evidence? Lancet 2004;363:1724–7.readily in cell culture media, and several claims of the  Lee DH, Folsom AR, Harnack L, Halliwell B, Jacobs Jr DR. Doescytotoxic effects of flavonoids on malignant, and other, cells in supplemental vitamin C increase cardiovascular disease risk inculture may have been led astray by this artefact. Flavonoids women with diabetes? Am J Clin Nutr 2004;80:1194–200.oxidize especially readily in Dulbeccos Modified Eagles  Miller III ER, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ,medium (DMEM), but also do so in most other cell culture Guallar E. Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann Intern Med 2005;142:37–46.media, at a slower rate [106,107]. Oxidation generates H2O2,  Neuhouser ML, Patterson RE, Thornquist MD, Omenn GS, King IB,quinones and semiquinones that can contribute to (and Goodman GE. Fruits and vegetables are associated with lower lungsometimes entirely account for) cytotoxicity [106–110]. For cancer risk only in the placebo arm of the beta-carotene and retinolexample, the apparent toxicity of green tea to PCI2 cells efficacy trial (CARET). Cancer Epidemiol Biomarkers Prevappeared entirely due to oxidation products generated in the 2003;12:350–8.  Halliwell B, Gutteridge JMC. Free radicals in biology and medicine.culture medium . 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