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ABB Archives of Biochemistry and Biophysics 476 (2008) 107–112 www.elsevier.com/locate/yabbi

Review

Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies? 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 2007 Available online 7 February 2008

Abstract Diets rich in polyphenols are epidemiologically associated with lower risk of developing some age-related diseases in humans. This apparent disease-protective effect of polyphenols is often attributed to their powerful antioxidant activities, as established in vitro. However, polyphenols can also exert pro-oxidant activities under certain experimental conditions. Neither pro-oxidant nor anti-oxidant activities have yet been clearly established to occur in vivo in humans, nor are they likely given the limited levels of polyphenols that are achievable in vivo after consumption of foods and beverages rich in them. Other actions of polyphenols may be more important in 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. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Polyphenols; Cell culture; Antioxidant; Pro-oxidant; Epigallocatechin gallate; Hydrogen peroxide; Ascorbate; Dulbecco’s modified Eagle’s medium; Green tea; Red wine

Aerobic organisms produce a wide range of oxygen radicals and other reactive oxygen species (ROS)1, both for useful purposes (e.g. defence, redox signalling) and by ‘‘accidents of chemistry” (reviewed in [1]). These ROS are metabolised by a series of antioxidant defences, some synthesised in vivo and other diet-derived [1]. The purpose of the ‘‘antioxidant defence network” [2] is not to remove all ROS, but to control their levels so as to allow useful functions whilst minimising oxidative damage (Fig. 1) [1– 4]. But how important are the diet-derived antioxidants such as vitamins C and E to humans? In general, increased intakes of these vitamins do not decrease levels of oxidative damage very much (if at all) in well-nourished humans who are already consuming the recommended dietary allowances [1,5–7]. Indeed, it has been suggested that the main *

Corresponding author. Fax: +65 6775 2207. E-mail address: [email protected] 1 Abbreviations used: ROS, reactive oxygen species; LDL, low-density lipoproteins; DMEM, Dulbecco’s Modified Eagle’s Medium. 0003-9861/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2008.01.028

biological function of a-tocopherol in humans is not as an antioxidant [8]. Polyphenols as antioxidants Foods and beverages rich in flavonoids and other polyphenols have been associated with decreased risk of agerelated diseases in several (but not all) epidemiological studies [9–15]. Flavonoids have powerful antioxidant activities in vitro, being able to scavenge [16–23] a wide range of reactive oxygen, nitrogen, and chlorine species, such as superoxide O2  , hydroxyl radical OH , peroxyl radicals RO2  , hypochlorous acid (HOCl), and peroxynitrous acid (ONOOH). Flavonoids can also chelate metal ions, often decreasing the pro-oxidant activity of metal ions [20,22]. They can inhibit the ability of myeloperoxidase to oxidise low-density lipoproteins (LDL), a potential anti-atherosclerotic effect [24]. Because considerable evidence indicates that increased oxidative damage is associated with, and may contribute to the development of, all major age-related

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

diseases [1–3], many have attributed the apparent diseaseprotective effects of flavonoids to their antioxidant ability (e.g. reviewed in [20]). Polyphenols as pro-oxidants Polyphenols oxidise readily in beverages [25–27] such as green tea. They can also oxidise in cell culture media (see below) and even in the oral cavity; holding or chewing green tea in the mouth generates substantial levels of H2O2 [28]. Often, these pro-oxidant effects involve interactions of polyphenols with transition metal ions [1,29–35]. Oxidation of polyphenols produces O2  , H2O2 and a complex mixture of semiquinones and quinones, all of which are potentially cytotoxic [26,31,36,37]. It has been argued that polyphenols may exert antioxidant and other cytoprotective effects in the gastrointestinal tract because of the high levels that can be present [38–40]. However, since there are often unabsorbed transition metal ions (especially iron [41,42]) in the gastrointestinal tract, pro-oxidant effects could conceivably occur there as well. Indeed, such effects have been demonstrated in the gastrointestinal tracts of certain insects consuming high levels of phenols [43,44]. However, in practice pro-oxidant effects can also be beneficial, since, by imposing a mild degree of oxidative stress, the levels of antioxidant defences and xenobiotic-metabolising enzymes might be raised, leading to overall cytoprotection [45], as illustrated in Fig. 1. Are polyphenols pro-oxidants or antioxidants in vivo in humans? No data are available on whether polyphenols are antioxidant or pro-oxidant in vivo in the human stomach, intestines, and colon, where they can be present at significant levels [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, isoprostane metabolites, 8-hydroxy-20 -deoxyguanosine [8OHdG]), essentially testing for systemic antioxidant or pro-oxidant activity. The results have been reviewed in detail elsewhere [40] and are quite variable, but overall no evidence for systemic pro-oxidant effects of polyphenols has emerged. A few studies report that administration of high doses of epigallocatechin gallate to animals leads to the formation of cysteine conjugates detectable in the urine, indicative of some degree of oxidation in vivo [36]. However, these effects may not be important at lower doses and may not be relevant to humans [36]. Similarly, only limited and variable evidence for antioxidant effects of flavonoids in humans has been obtained (reviewed in [40]). This is not, to the author, very surprising; although flavonoids can be absorbed through the gastrointestinal tract, maximal plasma concentrations achieved are low, usually not more that 1 lmol/L, in part because of rapid metabolism by human tissues [47–49]. Many of the products of metabolism, such as methylated and glucuronidated forms, have decreased antioxidant (or pro-oxidant) abilities because of the blocking of the phenolic hydroxyl groups involved in such activities [23,48]. Therefore, plasma flavonoid concentrations in vivo seem insufficient to exert systemic antioxidant effects. Another point to consider in interpreting the published human studies is that several groups have studied flavonoid-rich foods (e.g. pomegranate [50] or chocolate/cocoa [51,52]) or beverages (e.g. green tea) rather than pure flavonoids, and such foods contain other constituents that might be able to modulate oxidative damage. But are such foods and beverages effective as antioxidants in vivo? Again, the data are mixed. Some studies showed antioxidant effects (e.g. [37,48,51–53]), others no effects (e.g. [54–57]) and yet others some indication of mild pro-oxidant effects (e.g. [58]). One must be careful in studies with foodstuffs, since

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the mere act of eating in a fasted individual can alter parameters of oxidative damage. For example, dark soy sauce has powerful antioxidant abilities in vitro [59,60]. Recently, we attempted to see if dark soy sauce decreases oxidative damage in vivo in human volunteers, and indeed it was able to decrease levels of F2-isoprostanes [61]. We administered the soy sauce with rice, using a control consisting of a placebo colouring on the same amount of rice. The rice meal (devoid of antioxidants) also had effects on F2-isoprostanes and urinary 8OHdG excretion [61], although the soy sauce did better than the placebo in lowering F2-isoprostane levels. Similarly, Richelle et al. [62] and Lee et al. [63] suggested that fasting may raise plasma F2-isoprostane levels. As another example [64], olive oil administration to human volunteers decreased the propensity of LDL subsequently isolated from their blood to undergo oxidation in vitro, but feeding oil without antioxidants had the same effect. So overall, in vivo we have no evidence of systemic prooxidant effects of flavonoids in humans, and little or no clear evidence of antioxidant effects. Remember also that flavonoids are not only anti- and pro-oxidants. They have many other biological effects including the ability to inhibit cyclooxygenases, lipoxygenases, metalloproteinases and NADPH oxidases (reviewed in [1,40,65–69] and other papers in the current volume). These other actions may be more important in vivo than antioxidant effects, although again many of them have been demonstrated in vitro only at unphysiologically-high levels of polyphenols. Antioxidants in cell culture Cell culture has often been used to study the cellular effects of reactive species and of antioxidants, and many useful data have resulted. However, one must be cautious, for two reasons. First, normal culture conditions are a state of hyperoxia [70,71]. Most cells in the human body are exposed to O2 concentrations in the range of 1–10 mm Hg (obvious exceptions include corneocytes, corneal and respiratory tract lining cells). Yet culture under 95% air/5% CO2 is about 150 mm Hg of O2. Rates of production of ROS by cellular enzymes (e.g. xanthine oxidase) or by leakage from electron transport chains (especially in mitochondria) appear to be O2-limited at 10 mm Hg and so production of ROS will increase if O2 levels are raised [71–73]. In other words, cells in culture are under an oxidative stress, which can alter their properties in multiple ways [70], including sometimes promoting proliferation [1,74]. A second problem is that cell culture media are frequently deficient in antioxidants, especially tocopherols and ascorbate [75]. Vitamin E is rarely added because it is insoluble in water, and vitamin C because it is unstable (discussed below). Thus cells are deprived of these antioxidants, a situation which can lead to over-interpretations of the beneficial effects of added antioxidants. In other words, antioxidants may appear to have beneficial effects

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when added to cultured cells, but this is because a deficiency is being corrected rather than being a real beneficial effect of ‘extra antioxidants’. Deficiencies in selenium in some cell culture media have been reported [76,77]. This could decrease or prevent oxidative stress-triggered rises in the activities of selenium-dependent antioxidant enzymes, such as the glutathione peroxidase family and thioredoxin reductase [77,78]. One factor that has bedevilled studies of the cellular effects of flavonoids and other polyphenols is their instability in commonly-used culture media, especially Dulbecco’s Modified Eagle’s Medium (DMEM) [70,79]. Oxidation products include H2O2 and quinones/semiquinones, which can often react with and deplete cellular GSH [37,70,84]. Fig. 2 shows a striking example; epigallocatechin gallate (EGCG) added to DMEM begins oxidizing immediately and rapidly generates cytotoxic levels of H2O2. Such effect may have led to artefacts in interpretations of the cellular effects of high concentrations of added polyphenols. Table 1 summarises some published examples. Not all the cellular effects of polyphenols are due to such artefacts (e.g. some of those observed when lower levels, e.g. the lM range, are added to cells), but it is necessary to consider the potential for error when determining what the true cellular effects really are. Oxidation artefacts can also lead to false-positive results in in vitro genotoxicity testing using cultured cells, where the generated H2O2 (or other oxidation products) rather than the compound under test is causing the chromosomal aberrations detected [102,108–110]. Why do these effects occur? As well as its normal iron content (due to contamination and iron-containing proteins in foetal calf serum), DMEM contains added ferric nitrate, i.e. there is ‘‘free” pro-oxidant iron, which would be expected to catalyse autoxidation reactions [1]. Surprisingly however, iron ion chelating agents such as desferriox-

Fig. 2. Generation of H2O2 on addition of epigallocatechin gallate (EGCG) to Dulbecco’s modified Eagle’s medium. The final concentrations of EGCG in the medium are shown. SD values are not shown to avoid cluttering the figures. Note the rapid rate of H2O2 production from EGCG as soon as it is added to DMEM (see legend to Table 2). Adapted from [79].

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Table 1 Examples 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 myeloid leukaemia cells or human fibroblasts Induction of apoptosis by green tea in PC12 cells

Due to generation of H2O2 by ascorbate oxidation in cell culture media Due to generation of H2O2 by oxidation of tea components in cell culture media Due to H2O2, quinones, and semiquinones generated by oxidation of L-DOPA and dopamine in the culture medium Due to oxidation to produce H2O2 in the culture medium Entirely or largely due to oxidation of gallic acid to produce H2O2 in culture medium

[80–82]

Induction of cell death by L-DOPA and dopamine in PC12 and M14 cells Toxicity of apple phenolics to cancer cells Cytotoxicity of gallic acid Addition of grape seed extract to CaCo-2 cell culture medium generates H2O2 due to oxidation of phenolics in the medium Effects of polyphenols on c-jun phosphorylation in bronchial epithelial cell lines Epigallocatechin gallate induces apoptosis in human oral cell lines Toxicity of myricetin to Chinese hamster lung fibroblast V79 cells Cell culture media found to generate ROS as detected by spin traps and fluorescent dyes Ascorbate observed to inhibit cell proliferation and fibronectin synthesis in human skin fibroblasts Stimulation of SIRT1 activity by polyphenols in HT29 cells Cyanidin-3-rutoside toxic to HL60 cells. EGCG and green tea extract cause oxidative stress responses in S. cerevisiae Cytotoxicity of EGCG to oral carcinoma cell lines Activation of NF-jB in macrophages by coffee Toxicity of catechols to PC12-AC cells Toxicity of EGCG to Jurkat T cells Cytotoxicity and genotoxicity of green tea extract to H260 and RAW264.7 cells Toxicity of extracts of the oriental fungus Ganoderma lucidum to human lymphocytes Toxicity of quercetin, catechin and ascorbate to pancreatic b-cells Toxicity of 4-methylcatechol to murine tumor cells Toxicity of EGCG to ovarian cancer calls in DMEM Stimulatory effects of garcinol on growth of intestinal cells

[83] [84] [85] [86,87] [88]

Shown to involve H2O2, although H2O2 was not specifically identified as coming from the culture medium Due to production of H2O2 in the culture medium Due to H2O2 production, although H2O2 was not specifically identified as coming from the culture medium

[89] [90] [91] [92]

Inhibition by catalase suggests may be due to H2O2 generation in the culture medium Results confounded by instability of polyphenols in the culture medium Shown to involve peroxide, although H2O2 was not specifically identified as coming from the culture medium Involves H2O2 production in the medium

[80,93]

Involves both H2O2 and quinones, although these did not account for all the effects Due to H2O2; coffee contains substantial H2O2 levels [98] Involves H2O2, mainly generated in the extracellular space Involves H2O2 generation in the culture medium Involves H2O2 generation, although H2O2 was not specifically identified as coming from the culture medium Involves H2O2 generation

[97]

Involves H2O2 generation in the cell culture medium Involves oxidation to form H2O2 and quinones/semiquinones in the cell culture medium Due to H2O2 formation, probably both intracellularly and in the culture medium Involves ROS production in the culture medium; low levels of H2O2 often stimulate cell proliferation [1,74]

[94] [95] [96]

[99] [100] [101] [102] [103] [104] [105] [106] [107]

Table 2 Levels of hydrogen peroxide in culture media under 95% air/5% CO2 containing 1 mM epigallocatechin gallate (EGCG) Culture Media

Mean H2O2 (lM) ± SD at various times (h) 0h

0.5 h

1.0 h

1.5 h

2.0 h

DMEM F-10 F-12 RPMI with Hepes RPMI without Hepes Mc Coy 5A Williams’ E

75 ± 22 21 ± 10 65 ± 11 33 ± 3 82 ± 3 24 ± 1 66 ± 8

183 ± 8 34 ± 4 89 ± 4 99 ± 12 184 ± 32 79 ± 14 143 ± 15

275 ± 21 35 ± 3 85 ± 3 159 ± 9 257 ± 16 136 ± 10 209 ± 20

317 ± 35 37 ± 2 92 ± 4 212 ± 13 299 ± 23 192 ± 16 278 ± 23

334 ± 34 43 ± 3 121 ± 14 259 ± 17 332 ± 22 251 ± 19 329 ± 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 H2O2 generation at t = 0, indicating that EGCG has oxidized substantially when added to media in the few seconds before H2O2 measurement can be made. Media alone generated no significant levels of H2O2 (<2 lM) after 2 h incubation at 37 °C.

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amine or ortho-phenantholine did not decrease the rate of H2O2 production when gallic acid was added to DMEM [111]. Effects can be even more complex when mixtures of antioxidants are used. Thus both ascorbate [80] and polyphenols [79] generate H2O2 in DMEM, but if both are present the amounts of H2O2 produced are much less than the sum of the amounts with each compound alone [88,111]. Thus one should always be alert when adding polyphenols to cells in culture; one must check for reactions taking place in the culture medium that could lead to artefacts and carefully distinguish effects of oxidation products from ‘‘real” effects of polyphenols. Addition of catalase can be used to scavenge the H2O2, and GSH or N-acetylcysteine to scavenge quinones and semiquinones [84]. Another approach is to use a less ‘‘pro-oxidant” medium, since other culture media seems less good at catalysing polyphenol oxidation than is DMEM [109]. For example, Table 2 shows that F-10 and F-12 media seem far less ‘‘pro-oxidant” than is DMEM. Conclusion Polyphenols are metabolized as ‘‘typical xenobiotics” by the human body, and such metabolism decreases their antioxidant and pro-oxidant abilities. It is now looking unlikely that polyphenols act as antioxidants in vivo, and attention is turning to their other potential effects. Even so, whether polyphenols contribute to human health by any mechanism remains uncertain. Care is needed when studying their effects in cell culture to use biologically-relevant levels, to examine the effects of important metabolites, and to allow for artefactual chemical processes in the cell culture media. Acknowledgment I am grateful to the Biomedical Research Council of Singapore for support (BMRC 01/1/21/18/213). References [1] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, fourth ed., Clarendon Press, Oxford, UK, 2007. [2] J.J. Thiele, C. Schroeter, S.N. Hsieh, M. Podda, L. Packer, Curr. Probl. Dermatol. 29 (2001) 26–42. [3] C.K. Sen, Med. Sci. Sports Exerc. 33 (2001) 368–370. [4] S.G. Rhee, Science (2006) 1882–1883. [5] E.A. Meagher, O.P. Barry, J.A. Lawson, J. Rokach, G.A. FitzGerald, JAMA 285 (2001) 1178–1182. [6] H. Prieme, S. Loft, K. Nyyssonen, J.T. Salonen, H.E. Poulsen, Am. J. Clin. Nutr. 65 (1997) 503–507. [7] T.L. Duarte, J. Lunec, Free Radic. Res. 39 (2005) 671–686. [8] J.M. Zingg, A. Azzi, Curr. Med. Chem. 11 (2004) 1113–1133. [9] R.R. Huxley, H.A. Neil, Eur. J. Clin. Nutr. 57 (2003) 904–908. [10] M.G.L. Hertog, E.J.M. Feskens, P.C.H. Hollman, M.B. Katan, D. Kromhout D, Lancet 342 (1993) 1007–1011. [11] J. Lin, K.M. Rexrode, Hu. F, C.M. Albert, C.U. Chae, E.B. Rimm, M.J. Stampfer, J.E. Manson, Am. J. Epidemiol. 165 (2007) 1305–1313. [12] L. Yochum, L.H. Kushi, K. Meyer, A.R. Folsom, Am. J. Epidemiol. 149 (1999) 943–949.

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