Are Polyphenols Antioxidants or Pro-oxidants What Do We Learn

8/12/2019 Are Polyphenols Antioxidants or Pro-oxidants What Do We Learn

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Review

Are polyphenols antioxidants or pro-oxidants? What do we learnfrom cell culture and in vivostudies?

Barry Halliwell *

Department of Biochemistry, National University of Singapore, University Hall, Lee Kong Chian Wing,

UHL #05-02G, 21 Lower Kent Ridge Road, Singapore 119077, Singapore

Received 13 November 2007, and in revised form 26 December 2007Available online 7 February 2008

Abstract

Diets rich in polyphenols are epidemiologically associated with lower risk of developing some age-related diseases in humans. Thisapparent disease-protective effect of polyphenols is often attributed to their powerful antioxidant activities, as established in vitro. How-ever, polyphenols can also exert pro-oxidant activities under certain experimental conditions. Neither pro-oxidant nor anti-oxidant activ-ities have yet been clearly established to occur in vivo in humans, nor are they likely given the limited levels of polyphenols that areachievable in vivo after consumption of foods and beverages rich in them. Other actions of polyphenols may be more importantin vivo. Many studies of the biological effects of polyphenols in cell culture have been affected by their ability to oxidise in culture media,and awareness of this problem can avoid erroneous claims.2008 Elsevier Inc. All rights reserved.

Keywords: Polyphenols; Cell culture; Antioxidant; Pro-oxidant; Epigallocatechin gallate; Hydrogen peroxide; Ascorbate; Dulbeccos modified Eaglesmedium; Green tea; Red wine

Aerobic organisms produce a wide range ofoxygen rad-icals and other reactive oxygen species (ROS)1, both foruseful purposes (e.g. defence, redox signalling) and byaccidents of chemistry(reviewed in[1]). These ROS aremetabolised by a series of antioxidant defences, some syn-thesised in vivo and other diet-derived[1]. The purpose ofthe antioxidant defence network [2] is not to removeall ROS, but to control their levels so as to allow usefulfunctions whilst minimising oxidative damage (Fig. 1) [1

4]. But how important are the diet-derived antioxidantssuch as vitamins C and E to humans? In general, increasedintakes of these vitamins do not decrease levels of oxidativedamage very much (if at all) in well-nourished humans whoare already consuming the recommended dietary allow-ances [1,57]. Indeed, it has been suggested that the main

biological function of a-tocopherol in humans is not asan antioxidant[8].

Polyphenols as antioxidants

Foods and beverages rich in flavonoids and other poly-phenols have been associated with decreased risk of age-related diseases in several (but not all) epidemiologicalstudies[915]. Flavonoids have powerful antioxidant activ-

itiesin vitro, being able to scavenge[1623]a wide range ofreactive oxygen, nitrogen, and chlorine species, such assuperoxide O2

, hydroxyl radical OH, peroxyl radicalsRO2

, hypochlorous acid (HOCl), and peroxynitrous acid(ONOOH). Flavonoids can also chelate metal ions, oftendecreasing the pro-oxidant activity of metal ions [20,22].They can inhibit the ability of myeloperoxidase to oxidiselow-density lipoproteins (LDL), a potential anti-athero-sclerotic effect[24]. Because considerable evidence indicatesthat increased oxidative damage is associated with, and maycontribute to the development of, all major age-related

0003-9861/$ - see front matter 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.abb.2008.01.028

* Corresponding author. Fax: +65 6775 2207.E-mail address:[email protected]

1 Abbreviations used:ROS, reactive oxygen species; LDL, low-densitylipoproteins; DMEM, Dulbeccos Modified Eagles Medium.

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diseases [13], many have attributed the apparent disease-protective effects of flavonoids to their antioxidant ability(e.g.reviewed in[20]).

Polyphenols as pro-oxidants

Polyphenols oxidise readily in beverages[2527]such asgreen tea. They can also oxidise in cell culture media (seebelow) and even in the oral cavity; holding or chewinggreen tea in the mouth generates substantial levels ofH2O2[28]. Often, these pro-oxidant effects involve interac-

tions of polyphenols with transition metal ions [1,2935].Oxidation of polyphenols produces O2, H2O2and a com-

plex mixture of semiquinones and quinones, all of whichare potentially cytotoxic [26,31,36,37]. It has been arguedthat polyphenols may exert antioxidant and other cytopro-tective effects in the gastrointestinal tract because of thehigh levels that can be present [3840]. However, sincethere are often unabsorbed transition metal ions (especiallyiron [41,42]) in the gastrointestinal tract, pro-oxidanteffects could conceivably occur there as well. Indeed, sucheffects have been demonstrated in the gastrointestinal tractsof certain insects consuming high levels of phenols [43,44].

However, in practice pro-oxidant effects can also be ben-eficial, since, by imposing a mild degree of oxidative stress,the levels of antioxidant defences and xenobiotic-metabol-ising enzymes might be raised, leading to overall cytopro-tection[45], as illustrated inFig. 1.

Are polyphenols pro-oxidants or antioxidants in vivo in

humans?

No data are available on whether polyphenols are anti-oxidant or pro-oxidantin vivoin the human stomach, intes-tines, and colon, where they can be present at significantlevels[38,39,46,47]. As for effects after absorption into the

body, multiple well-designed human studies have been done

using reliable biomarkers of oxidative damage in plasma(F2-isoprostanes) and urine (F2-isoprostanes, isoprostanemetabolites, 8-hydroxy-20-deoxyguanosine [8OHdG]),essentially testing for systemic antioxidant or pro-oxidantactivity. The results have been reviewed in detail elsewhere[40]and are quite variable, but overall no evidence for sys-temic pro-oxidant effects of polyphenols has emerged. Afew studies report that administration of high doses of epi-gallocatechin gallate to animals leads to the formation ofcysteine conjugates detectable in the urine, indicative ofsome degree of oxidationin vivo[36]. However, these effects

may not be important at lower doses and may not be rele-vant to humans[36].Similarly, only limited and variable evidence for antiox-

idant effects of flavonoids in humans has been obtained(reviewed in[40]). This is not, to the author, very surpris-ing; although flavonoids can be absorbed through the gas-trointestinal tract, maximal plasma concentrationsachieved are low, usually not more that 1 lmol/L, in partbecause of rapid metabolism by human tissues [4749].Many of the products of metabolism, such as methylatedand glucuronidated forms, have decreased antioxidant (orpro-oxidant) abilities because of the blocking of the pheno-lic hydroxyl groups involved in such activities [23,48].

Therefore, plasma flavonoid concentrations in vivo seeminsufficient to exert systemic antioxidant effects.

Another point to consider in interpreting the publishedhuman studies is that several groups have studied flavo-noid-rich foods (e.g.pomegranate[50] or chocolate/cocoa[51,52]) or beverages (e.g.green tea) rather than pure flavo-noids, and such foods contain other constituents that mightbe able to modulate oxidative damage. But are such foodsand beverages effective as antioxidants in vivo? Again, thedata are mixed. Some studies showed antioxidant effects(e.g. [37,48,5153]), others no effects (e.g.[5457]) and yetothers some indication of mild pro-oxidant effects (e.g.

[58]). One must be careful in studies with foodstuffs, since

Fig. 1. Balance of antioxidants and reactive species in vivo.

108 B. Halliwell/ Archives of Biochemistry and Biophysics 476 (2008) 107112


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the mere act of eating in a fasted individual can alterparameters of oxidative damage. For example, dark soysauce has powerful antioxidant abilities in vitro [59,60].Recently, we attempted to see if dark soy sauce decreasesoxidative damage in vivo in human volunteers, and indeedit was able to decrease levels of F2-isoprostanes [61]. We

administered the soy sauce with rice, using a control con-sisting of a placebo colouring on the same amount of rice.The rice meal (devoid of antioxidants) also had effects onF2-isoprostanes and urinary 8OHdG excretion [61],although the soy sauce did better than the placebo in low-ering F2-isoprostane levels. Similarly, Richelle et al. [62]and Lee et al.[63]suggested that fasting may raise plasmaF2-isoprostane levels. As another example [64], olive oiladministration to human volunteers decreased the propen-sity of LDL subsequently isolated from their blood toundergo oxidation in vitro, but feeding oil without antiox-idants had the same effect.

So overall, in vivo we have no evidence of systemic pro-

oxidant effects of flavonoids in humans, and little or noclear evidence of antioxidant effects. Remember also thatflavonoids are not only anti- and pro-oxidants. They havemany other biological effects including the ability to inhibitcyclooxygenases, lipoxygenases, metalloproteinases andNADPH oxidases (reviewed in [1,40,6569] and otherpapers in the current volume). These other actions maybe more important in vivo than antioxidant effects,although again many of them have been demonstratedin vitro only at unphysiologically-high levels ofpolyphenols.

Antioxidants in cell culture

Cell culture has often been used to study the cellulareffects of reactive species and of antioxidants, and manyuseful data have resulted. However, one must be cautious,for two reasons. First, normal culture conditions are a stateof hyperoxia [70,71]. Most cells in the human body areexposed to O2concentrations in the range of 110 mm Hg(obvious exceptions include corneocytes, corneal and respi-ratory tract lining cells). Yet culture under 95% air/5% CO2is about 150 mm Hg of O2. Rates of production of ROS bycellular enzymes (e.g. xanthine oxidase) or by leakage fromelectron transport chains (especially in mitochondria)appear to be O2-limited at 10 mm Hg and so productionof ROS will increase if O2levels are raised[7173]. In otherwords, cells in culture are under an oxidative stress, whichcan alter their properties in multiple ways [70], includingsometimes promoting proliferation[1,74].

A second problem is that cell culture media are fre-quently deficient in antioxidants, especially tocopherolsand ascorbate [75]. Vitamin E is rarely added because itis insoluble in water, and vitamin C because it is unstable(discussed below). Thus cells are deprived of these antiox-idants, a situation which can lead to over-interpretationsof the beneficial effects of added antioxidants. In other

words, antioxidants may appear to have beneficial effects

when added to cultured cells, but this is because a defi-ciency is being corrected rather than being a real beneficialeffect of extra antioxidants. Deficiencies in selenium insome cell culture media have been reported [76,77]. Thiscould decrease or prevent oxidative stress-triggered risesin the activities of selenium-dependent antioxidant

enzymes, such as the glutathione peroxidase family and thi-oredoxin reductase[77,78].One factor that has bedevilled studies of the cellular

effects of flavonoids and other polyphenols is their instabil-ity in commonly-used culture media, especially DulbeccosModified Eagles Medium (DMEM) [70,79]. Oxidationproducts include H2O2 and quinones/semiquinones, whichcan often react with and deplete cellular GSH [37,70,84].Fig. 2 shows a striking example; epigallocatechin gallate(EGCG) added to DMEM begins oxidizing immediatelyand rapidly generates cytotoxic levels of H2O2. Such effectmay have led to artefacts in interpretations of the cellulareffects of high concentrations of added polyphenols.Table

1summarises some published examples. Not all the cellulareffects of polyphenols are due to such artefacts (e.g. someof those observed when lower levels, e.g. the lM range,are added to cells), but it is necessary to consider the poten-tial for error when determining what the true cellular effectsreally are. Oxidation artefacts can also lead to false-posi-tive results in in vitro genotoxicity testing using culturedcells, where the generated H2O2(or other oxidation prod-ucts) rather than the compound under test is causing thechromosomal aberrations detected[102,108110].

Why do these effects occur? As well as its normal ironcontent (due to contamination and iron-containing pro-

teins in foetal calf serum), DMEM contains added ferricnitrate, i.e. there is free pro-oxidant iron, which wouldbe expected to catalyse autoxidation reactions[1]. Surpris-ingly however, iron ion chelating agents such as desferriox-

Fig. 2. Generation of H2O2 on addition of epigallocatechin gallate(EGCG) to Dulbeccos modified Eagles medium. The final concentrationsof EGCG in the medium are shown. SD values are not shown to avoidcluttering the figures. Note the rapid rate of H2O2production from EGCGas soon as it is added to DMEM (see legend to Table 2). Adapted from

[79].

B. Halliwell / Archives of Biochemistry and Biophysics 476 (2008) 107112 109


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Table 1Examples of artefacts caused by oxidation of compounds added to cell culture media (Adapted with considerable updating from [70])

Observation Comment Reference

Induction of cell death by ascorbate in HL-60 or acute myeloidleukaemia cells or human fibroblasts

Due to generation of H2O2by ascorbate oxidation in cell culturemedia

[8082]

Induction of apoptosis by green tea in PC12 cells Due to generation of H2O2by oxidation of tea components in cellculture media

[83]

Induction of cell death by L-DOPA and dopamine in PC12 and M14cells

Due to H2O2, quinones, and semiquinones generated by oxidationof L-DOPA and dopamine in the culture medium

[84]

Toxicity of apple phenolics to cancer cells Due to oxidation to produce H2O2in the culture medium [85]Cytotoxicity of gallic acid Entirely or largely due to oxidation of gallic acid to produce H2O2

in culture medium[86,87]

Addition of grape seed extract to CaCo-2 cell culture mediumgenerates H2O2due to oxidation of phenolics in the medium

[88]

Effects of polyphenols on c-jun phosphorylation in bronchialepithelial cell lines

Shown to involve H2O2, although H2O2was not specificallyidentified as coming from the culture medium

[89]

Epigallocatechin gallate induces apoptosis in human oral cell lines Due to production of H2O2in the culture medium [90]Toxicity of myricetin to Chinese hamster lung fibroblast V79 cells Due to H2O2production, although H2O2was not specifically

identified as coming from the culture medium[91]

Cell culture media found to generate ROS as detected by spin trapsand fluorescent dyes

[92]

Ascorbate observed to inhibit cell proliferation and fibronectin

synthesis in human skin fibroblasts

Inhibition by catalase suggests may be due to H2O2generation in

the culture medium

[80,93]

Stimulation of SIRT1 activity by polyphenols in HT29 cells Results confounded by instability of polyphenols in the culturemedium

[94]

Cyanidin-3-rutoside toxic to HL60 cells. Shown to involve peroxide, although H2O2was not specificallyidentified as coming from the culture medium

[95]

EGCG and green tea extract cause oxidative stress responses in S.cerevisiae

Involves H2O2production in the medium [96]

Cytotoxicity of EGCG to oral carcinoma cell lines Involves both H2O2and quinones, although these did not accountfor all the effects

[97]

Activation of NF-jB in macrophages by coffee Due to H2O2; coffee contains substantial H2O2levels[98] [99]Toxicity of catechols to PC12-AC cells Involves H2O2, mainly generated in the extracellular space [100]Toxicity of EGCG to Jurkat T cells Involves H2O2generation in the culture medium [101]Cytotoxicity and genotoxicity of green tea extract to H260 and

RAW264.7 cellsInvolves H2O2generation, although H2O2was not specificallyidentified as coming from the culture medium

[102]

Toxicity of extracts of the oriental fungus Ganoderma lucidumtohuman lymphocytes

Involves H2O2generation [103]

Toxicity of quercetin, catechin and ascorbate to pancreaticb-cells Involves H2O2generation in the cell culture medium [104]Toxicity of 4-methylcatechol to murine tumor cells Involves oxidation to form H2O2 and quinones/semiquinones in

the cell culture medium[105]

Toxicity of EGCG to ovarian cancer calls in DMEM Due to H2O2formation, probably both intracellularly and in theculture medium

[106]

Stimulatory effects of garcinol on growth of intestinal cells Involves ROS production in the culture medium; low levels ofH2O2 often stimulate cell proliferation[1,74]

[107]

Table 2

Levels of hydrogen peroxide in culture media under 95% air/5% CO 2 containing 1 mM epigallocatechin gallate (EGCG)

Culture Media Mean H2O2(lM) SD at various times (h)

0 h 0.5 h 1.0 h 1.5 h 2.0 h

DMEM 75 22 183 8 275 21 317 35 334 34F-10 21 10 34 4 35 3 37 2 43 3F-12 65 11 89 4 85 3 92 4 121 14RPMI with Hepes 33 3 99 12 159 9 212 13 259 17RPMI without Hepes 82 3 184 32 257 16 299 23 332 22Mc Coy 5A 24 1 79 14 136 10 192 16 251 19Williams E 66 8 143 15 209 20 278 23 329 15

Data are means SD, n = 3. Data selected from [110].Levels of H2O2 were significantly greater than in medium alone (P< 0.05) at all time points for all media. Note the rapid H 2O2 generation at t= 0,indicating that EGCG has oxidized substantially when added to media in the few seconds before H 2O2measurement can be made. Media alone generated

no significant levels of H2O2(


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amine or ortho-phenantholine did not decrease the rate ofH2O2 production when gallic acid was added to DMEM[111]. Effects can be even more complex when mixtures ofantioxidants are used. Thus both ascorbate [80]and poly-phenols[79]generate H2O2in DMEM, but if both are pres-ent the amounts of H2O2produced are much less than the

sum of the amounts with each compound alone[88,111].Thus one should always be alert when adding polyphe-nols to cells in culture; one must check for reactions takingplace in the culture medium that could lead to artefacts andcarefully distinguish effects of oxidation products fromreal effects of polyphenols. Addition of catalase can beused to scavenge the H2O2, and GSH or N-acetylcysteineto scavenge quinones and semiquinones [84]. Anotherapproach is to use a less pro-oxidant medium, sinceother culture media seems less good at catalysing polyphe-nol oxidation than is DMEM[109]. For example,Table 2shows that F-10 and F-12 media seem far less pro-oxi-dantthan is DMEM.

Conclusion

Polyphenols are metabolized as typical xenobioticsbythe human body, and such metabolism decreases their anti-oxidant and pro-oxidant abilities. It is now looking unli-kely that polyphenols act as antioxidants in vivo, andattention is turning to their other potential effects. Evenso, whether polyphenols contribute to human health byany mechanism remains uncertain. Care is needed whenstudying their effects in cell culture to use biologically-rele-vant levels, to examine the effects of important metabolites,

and to allow for artefactual chemical processes in the cellculture media.

Acknowledgment

I am grateful to the Biomedical Research Council ofSingapore for support (BMRC 01/1/21/18/213).

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