Environmental Toxicology and Pharmacology 11 (2002) 321–333

The pro-oxidant chemistry of the natural antioxidants vitamin C,vitamin E, carotenoids and flavonoids

Ivonne M.C.M. Rietjens a,b,*, Marelle G. Boersma a, Laura de Haan a,Bert Spenkelink a, Hanem M. Awad a, Nicole H.P. Cnubben b,c,

Jelmer J. van Zanden a,b, Hester van der Woude a,b, Gerrit M. Alink a,b,Jan H. Koeman a

a Di�ision of Toxicology, Wageningen Uni�ersity, Tuinlaan 5, 6703 HE, Wageningen, The Netherlandsb WU/TNO Center for Food Toxicology, P.O. Box 8000, 6700 EA, Wageningen, The Netherlands

c TNO Nutrition and Food Research, P.O. Box 360, 3700 AJ, Zeist, The Netherlands

Received 2 August 2001; received in revised form 17 December 2001; accepted 19 December 2001

Abstract

Natural antioxidants like vitamin C, vitamin E, carotenoids, and polyphenols like flavonoids, are at present generallyconsidered to be beneficial components from fruit and vegetables. The anti-oxidative properties of these compounds are oftenclaimed to be responsible for various beneficial health effects of these food ingredients. Together these studies provide the basisfor the present rapidly increasing interest for the use of natural antioxidants as functional food ingredients and/or as foodsupplements. However, at higher doses or under certain conditions antioxidant-type functional food ingredients may exert toxicpro-oxidant activities. The present manuscript gives an overview of especially this pro-oxidative chemistry and toxicity ofwell-known natural antioxidants including vitamin C, vitamin E, carotenoids and flavonoids. © 2002 Elsevier Science B.V. Allrights reserved.

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1. Introduction

Natural antioxidants like vitamin C and E,carotenoids, and polyphenols like flavonoids, are atpresent generally considered to be beneficial compo-nents from fruit and vegetables. The anti-oxidativeproperties of these compounds are often claimed to beresponsible for the protective effects of these foodcomponents against cardiovascular disease, certainforms of cancer and/or photosensitivity diseases (Petoet al., 1981; Block et al., 1992; Ziegler, 1991; Middletonand Kandaswami, 1994; Rice-Evans et al., 1996;Mayne, 1996; Virtamo, 1999; Stocker, 1999; Pietta,2000). In addition, beneficial health effects in ageinghave also been related to antioxidant action (Block etal., 1992; Diplock, 1996; Diplock et al., 1998; Omenn,

1996). Together these studies provide the basis for thepresent rapidly increasing interest for the use of naturalantioxidants as functional food ingredients and/or asfood supplements. However, many of the reportedhealth claims have been derived from observationalepidemiological studies in which specific diets wereshown to be associated with reduced risks on specificforms of cancer, cardiovascular disease, increased ac-tion of the immune system, and the reduction of stress.Identification of the actual ingredient in a specific dietresponsible for the beneficial health effects remains animportant bottle-neck for translating observational epi-demiology to development of a functional food ingredi-ent. Furthermore, in addition to concerns for scientificsupport for the health claims and identification of theactive ingredient(s), important toxicological concernsarise. Paracelsus (1493–1541) already reported toxicityto be a matter of dose, and toxicological risks may arisewhen daily doses of a compound rise above a certainthreshold limit. For specific food enrichment with forexample vitamin A (retinoids), vitamin D, folic acid,

* Corresponding author. Tel.: +31-317-483971; fax: +31-317-484931.

E-mail address: ivonne.rietjens@algemeen.tox.wau.nl (I.M.C.M.Rietjens).

1382-6689/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.PII: S1382 -6689 (02 )00003 -0

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selenium, copper and zinc, the margin between theamount functionally required for optimal health andthe toxic dose is known to be small and enrichment offood by these ingredients is at present not allowed. Forthe other vitamins and minerals, including for examplethe antioxidant vitamins C and E, it is generally as-sumed that product enrichments will not lead to in-creases in doses of consumption by more than 4–5times the daily intake. This may change upon increas-ing the use of functional food ingredients in a widerange of food items and/or upon the use of isolatedcompounds as food supplements. For antioxidant-typefunctional food ingredients, at higher doses toxic pro-oxidant actions may become of importance. The objec-tive of the present manuscript is to give an overview ofespecially this pro-oxidative chemistry and toxicity ofwell-known natural antioxidants including vitamin Cand E, carotenoids and flavonoids.

2. Vitamin C

It is commonly believed that the beneficial effects ofvitamin C (ascorbate) (Fig. 1) increase with the amountof vitamin C consumed. Vitamin C is presently mar-keted as an antioxidant supplement, and claimed toincrease resistance to diseases and oxidative stress. Sev-eral prospective studies have investigated the effect ofvitamin C intake from fruits and vegetables and cancerdevelopment. Outcomes obtained have varied from aninverse association to no effect (Virtamo, 1999). Obser-vational epidemiological studies suggest that antioxi-dant vitamins, including vitamin C, at sufficientconcentration inhibit heart disease and cancer (Flagg etal., 1995). Some recent studies have shown a significantreduction in the risk of lung cancer by increased dietaryvitamin C uptake (Bandera et al., 1997; Ocke et al.,1997; Yong et al., 1997). An association betweenplasma vitamin C levels and lung cancer risk has been

less firmly established. Vitamin C plasma levels havebeen reported to be not predictive of subsequent lungcancer or overall cancer mortality (Stahelin et al.,1991), or to show an only modest or non-significantbeneficial effect, which may be most relevant for cancerof the digestive tract, particularly of the esophagus andthe stomach (Stahelin et al., 1991; Comstock et al.,1997). Another study has concluded that vitamin C hasnot demonstrated substantial efficacy in cancer chemo-prevention (Lippman et al., 1998). As a result, the invivo beneficial effects of vitamin C supplementation canbe questioned.

In addition to its well-known antioxidant properties,ascorbate, depending on the environment and condi-tions in which the molecule is active, can also act as apro-oxidant (Halliwell, 1990). In vitro induction of lipidperoxidation by ascorbate-iron systems is a standardtest for inducing oxidative stress and testing antioxidantactivity of other antioxidants. In this model system,chelation of Fe2+ by ascorbate creates an active cata-lyst for the production of reactive oxygen species.When Fe3+ is present, vitamin C can convert Fe3+

into Fe2+, which subsequently reacts with oxygen orhydrogen peroxide resulting in formation of superoxideanions and hydroxyl radicals (Fig. 1) (Samuni et al.,1983; Halliwell et al., 1987; Higson et al., 1988).

Also of importance in relation to the claimed anticar-cinogenic and pro-oxidative action of vitamin C is thatvitamin C has been reported to induce cell death,nuclear fragmentation and internucleosomal DNAcleavage in human myelogenous leukemia cell lines, allin line with the ability of high concentrations of vitaminC to induce apoptosis in various tumor cell lines (Sak-agami and Satoh, 1997; Sakagami et al., 2000). Theapoptosis-inducing activity of vitamin C has been as-cribed to its pro-oxidant action and is inhibited bycatalase, antioxidants like N-acetylcysteine and GSH,Ca2+ depletion and Fe3+, but stimulated by H2O2,Cu2+ and iron chelators (Sakagami and Satoh, 1997;

Fig. 1. Pro-oxidant chemistry of vitamin C.

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Fig. 2. Pro-oxidant action of vitamin E.

Sakagami et al., 2000). Especially the observations thatFe3+ inhibits ascorbate mediated apoptosis, and thatiron chelators stimulate ascorbate mediated apoptosis,point at a pro-oxidant mechanism different from thegenerally accepted mechanism for ascorbate-Fe medi-ated induction of lipid peroxidation and oxidative stressdepicted in Fig. 1. In agreement with this observation isthat reactive oxygen species are found to inhibit apop-tosis by the induction of NF�B (Sen and Packer, 1996).This inhibiting effect of oxidative stress on apoptosismay provide a mechanistic model for the vitamin Cinduced stimulation of apoptosis. An alternative mech-anism suggested for the ascorbate-induced apoptosis isthat induction of hypoxia-inducible factor 1-� stabilizesp53 resulting in growth arrest or apoptosis (Sakagamiet al., 2000). Because the activity of p53 is generallyrelated to tumor suppression, this would imply that thevitamin C induced apoptosis might be a beneficialrather than toxic effect.

Also of interest with respect to the balance betweenbeneficial and toxic effects of vitamin C supplementa-tion are the results from an in vivo study in which 30healthy volunteers received dietary supplements of 500mg of vitamin C per day for 6 weeks (Podmore et al.,1998). The parameter used to assess the anti- andpro-oxidant effects of vitamin C was the level ofmodified DNA bases detected in peripheral bloodlymphocytes. Whereas the level of 8-oxoguanine wasfound to decrease upon supplementation relative toplacebo, the level of 8-oxoadenine increased. Since both8-oxoguanine and 8-oxoadenine may represent muta-genic lesions this observation, although mechanisticallyunexplained, reflects that even for vitamin C supple-mentation the ultimate balance between anti- and pro-oxidative effects is more complex than commonlybelieved.

Recently, the fact that vitamin C has proven to beineffective in cancer chemoprevention has been relatedto vitamin C mediated formation of genotoxins fromlipidhydroperoxides, even in the absence of transition

metal ions (Lee et al., 2001). Vitamin C was reported tobe even more efficient than transition metal ions atinitiating the decomposition of for example 13(S)-hy-droperoxy-(Z,E)-9,11-octadecadienoic acid to �,�-un-saturated aldehydic bifunctional electrophiles. Themechanism suggested for this vitamin C mediated pro-oxidant action is similar to the formation of �,�-unsat-urated aldehyde genotoxins observed with transitionmetals, since vitamin C is suggested to result in perox-ide decomposition by means of one electron donation(Lee et al., 2001). Lee et al. (2001) also indicate thattheir finding that vitamin C generates bifunctional elec-trophiles explains why hydroperoxide dependent lipidperoxidation in vitro is enhanced by vitamin C, andcould help to explain why vitamin C has not demon-strated substantial efficacy in cancer chemopreventiontrials.

3. Vitamin E

Vitamin E is a term used to describe a family oftocopherols of which �-tocopherol (Fig. 2) is the mostabundant and important member (IUPAC-IUB JCBN,1982). Observational epidemiologic studies provide thebasis for relating intake of vitamin E rich food todecreased incidence of risk of mortality due to cardio-vascular diseases (Stampfer et al., 1993; Stephens et al.,1996; Kushi et al., 1996). However, generally resultsfrom large-scale intervention studies are inconclusivereporting adverse as well as beneficial effects or noeffects at all of daily supplementation with �-toco-pherol (Virtamo, 1999; Stocker, 1999). Vitamin E in-takes provided protection against LDL cholesteroloxidation and reduced risk of heart disease (Hatchcock,1997). Human intervention studies in which smokingmale volunteers were exposed during 5–8 years to dailysupplementation with vitamin E did not reveal anyeffect on the overall mortality of male smokers, but didshow increased mortality resulting from hemorrhagic

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stroke (The alpha-tocopherol, beta-carotene cancer pre-vention study group, 1994).

The inconsistency in the effects of vitamin E may berelated to the complex function and chemical behaviorof vitamin E, being able to have an anti-oxidant, neu-tral or pro-oxidant effect. This dualistic behavior is bestdescribed by the mechanism depicted in Fig. 2. Thepathway depicted implies that increased levels of �-to-copherol result, upon subsequent oxidative stress, inincreased levels of �-tocopherol radicals. These �-toco-pherol radicals can initiate processes of for examplelipid peroxidation by themselves. When antioxidantnetworks are balanced, this pro-oxidant action of vita-min E radicals is inhibited by co-antioxidants whichcan reduce the radical back to vitamin E. Increasingonly the levels of �-tocopherol, may, especially underconditions of increased oxidative stress, result in in-creased levels of �-tocopherol radicals which can nolonger be efficiently detoxified by the co-antioxidants.This provides the possibility for the pro-oxidant toxic-ity of the �-tocopherol radical. This biochemical ratio-nale explains why foods containing comparably smalllevels of vitamin E but also co-antioxidants providegreater health benefits than vitamin E supplements(Stocker, 1999). The observed improvement of �-toco-pherol levels in red blood cell membranes in vivo uponprolonged green tea catechin consumption also sup-ports this idea of antioxidant networks and the hypoth-esis that the levels of co-antioxidants are important formaintaining high levels of �-tocopherol (Palozza andKrinsky, 1992; Brown and Rice-Evans, 1998; Fremontet al., 1998). These results clearly point at the impor-tance of balanced antioxidant networks and the risksrelated to unbalanced networks when only one memberof such an antioxidant network is increased.

In addition to its use as a functional food ingredientor supplement, vitamin E is often used in anti-sunproducts and other skin cosmetics. In view of this it isof importance that vitamin E has been shown to be acomplete tumor promotor in a mouse skin model(Mitchel and McCann, 1993). Upon treatment of theskin with the tumor initiator dimethylbenz[a]anthracene(DMBA), subsequent promotion by topical applicationof vitamin E showed that vitamin E can act as acomplete tumor promotor in DMBA treated mouseskin with an efficiency approaching that of the standardtumor promotor 12-O-tetradecanoylphorbol-13-acetate(TPA) (Mitchel and McCann, 1993). The authors statethat it may be prudent to avoid repetitive or prolongedtopical exposure of human skin to antioxidants likevitamin E, especially in the case of co-exposure tochemical carcinogens or tumor initiators.

In addition, several other studies have reported vita-min E to be a tumor initiating and tumor promotingcarcinogen (Toth and Patil, 1983; Temple and El-Khatib, 1987; Kline and Sanders, 1989; Nitta et al.,

1991a,b). For example, vitamin E has been reported toenhance the induction of intestinal tumorigenesis by1,2-dimethylhydrazine (Toth and Patil, 1983; Templeand El-Khatib, 1987). The chronic subcutaneous ad-ministration of vitamin E to mice and rats was reportedto result in tumor induction (Nitta et al., 1991a) andcontinuous oral administration of vitamin E to micewas reported to significantly increase liver cancer inci-dence (Nitta et al., 1991b). In spite of this, some largeintervention studies report the complete absence of anysignificant effect of vitamin E supplementation on bothlung tumor incidence but also on overall mortality, andeven revealed increased mortality resulting from hemor-rhagic stroke (The alpha-tocopherol, beta-carotene can-cer prevention study group, 1994), again illustrating thecontroversial results observed upon vitamin Esupplementation.

The mechanism of tumor promotion by vitamin E isat present unknown, but may also be related to in-creased formation of �-tocopherol radicals which, whennot efficiently scavenged by other antioxidants, may actas reactive radical species themselves. Alternatively, itmay be related to the Cu2+-dependent pro-oxidantaction of vitamin E. In in vitro systems �-tocopherolinduced oxidative DNA damage in the presence ofCu2+, caused by vitamin E mediated copper-dependentreactive oxygen species formation (Yamashita et al.,1998). Furthermore, Van Haaften et al. (2001) showedthat vitamin E inhibits GST P1-1 activity with an IC50

of less than 1 �M. This inhibitory potency might resultin an increased risk for skin tumorigenesis because ithas been shown that mice lacking GST P1-1, which isnormally abundant in the skin, have an increased riskfor skin tumorgenesis (Henderson et al., 1998).

Finally, similar controversial results have been re-ported for the effects of vitamin E (and other antioxi-dants) on the cardiotoxicity of doxorubicin, or the lungtoxicity of bleomycin. Both these anticancer drugs arebelieved to generate their dose-limiting toxic effects bymeans of generation of reactive oxygen species. Co-ad-ministration of vitamin E has been investigated as ameans to overcome the dose-limiting toxic side effects.In addition to studies reporting protective effects(Myers et al., 1976, 1977; Sonneveld, 1978; Wang et al.,1980) and/or the absence of effects (Weitzman et al.,1980; Legha et al., 1982) also potentiation of oxidativedoxorubicin and bleomycin toxicity by vitamin E hasbeen reported: Shinozawa et al. (1988) reported thattreatment of ICR mice with vitamin E and doxorubicinresulted in significant reduction in the survival time ascompared to treatment with doxorubicin alone. Themechanism behind the potentiating pro-oxidant effectof antioxidants like vitamin E in the case of thesetumor drugs may be related to the fact that generationof reactive oxygen species by the drugs may be in partmediated by drug-Fe2+ complexes reducing O2, result-

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ing in superoxide anion radical formation and drug-Fe3+ complexes. Subsequent reduction of the drug-Fe3+ complex by antioxidants may support thecatalytic redoxcycle of the drug-Fe complex.

Altogether, supplementation with an antioxidant likevitamin E may not always exert the protective effectaimed for. The ultimate balance between potentiatingand protective effects may depend on the subtle redoxequilibrium within the cells and the balance within thecomplete cellular antioxidant network.

4. Carotenoids

The antioxidant and radical scavenging ability ofcarotenoids, including �- and �-carotene, lycopene, andothers (Fig. 3) is well documented (Miller et al., 1996;

Woodall et al., 1997; Mortensen and Skibsted, 1997;Stahl et al., 1998). The antioxidant potency of thesecompounds in a variety of different in vitro assays hasrecently been shown to correlate quantitatively withtheir computer calculated ionization potential, thusproviding quantitative structure activity relationships(Soffers et al., 1999). However, experimental studieswith an important member of this series of antioxidantmolecules, i.e. �-carotene, at present provide perhapsthe best example of unexpected health risks related tothe use of an antioxidant as beneficial health supple-ment. Observational epidemiologic studies indicate thatdiets high in carotenoid-rich fruits and vegetables aswell as increased serum levels of �-carotene are associ-ated with a decreased risk of lung cancer (Peto et al.,1981; Ziegler, 1991; Mayne, 1996). Based on theseobservations large human intervention trials with heavy

Fig. 3. Chemical structures of carotenoid antioxidants.

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smokers receiving �-carotene supplements were under-taken (Omenn et al., 1996; The alpha-tocopherol, beta-carotene cancer prevention study group, 1994). TheFinnish intervention study (The alpha-tocopherol, beta-carotene cancer prevention study group, 1994) was thefirst to report increased, instead of the expected reducedlevels of lung cancer incidence in the population ofheavy smokers receiving �-carotene supplements forseveral years. As a result, another large interventiontrial (Omenn et al., 1996) stopped the active interven-tion 21 months earlier than planned. Interim efficacyresults of this study showed the same tendency ofincreased risk of lung cancer and also increased overallmortality in the group receiving the �-carotene supple-ments versus placebo (Omenn et al., 1996). Similar tothe effects of �-carotene on lung cancer risk in heavysmokers, this study also reported an increased lungcancer risk due to �-carotene supplementation in as-bestos-exposed workers.

The mechanism by which �-carotene increases lungcancer risks in both heavy smokers and asbestos work-ers is at present unclear, although some hypotheses andinitial results have been reported. One possible explana-tion suggests the formation of reactive oxidative �-carotene metabolites. Especially the high oxygenpressure in the lungs may favor pro-oxidativemetabolism of �-carotene (Palozza et al., 1995) and theinteraction between reactive oxygen species, derivedfrom tobacco smoke or induced in the lung uponasbestos exposure, may result in �-carotene(auto)oxidation leading to toxic �-carotene metabolites(Mayne et al., 1996; Omaye et al., 1997; Lotan, 1999;Wang et al., 1999). Also induction of cytochrome P450activity may result in formation of increased �-caroteneoxidation. A more extended hypothesis explaining howincreased �-carotene oxidation may result in the in-creased lung tumor risks is summarized in Fig. 4. Thehypothesis (Omaye et al., 1997; Lotan, 1999; Wang etal., 1999) suggests that oxidative �-carotene metabolitesstructurally resemble retinal and affect retinoid signal-ing resulting in reduced retinoid levels and suppressionof RAR� gene expression, the latter representing atumor suppressor gene. Furthermore, the whole processalso induces increased expression of c-jun and c-fosgenes resulting in higher levels of activator protein-1(AP-1). Increased expression of c-Jun and c-Fosproteins has been reported for several mitogenic stimuliand tumor-promoting agents, and is indeed observed intobacco-smoke exposed ferrets supplemented with high-dose �-carotene (Wang et al., 1999).

Of importance to notice is that these pro-oxidativetumor promoting effects of �-carotene are especiallyobserved upon high dose supplementation in heavysmokers. �-carotene does not exert this tumor riskenhancing effect in former smokers (Omenn et al.,1996). These observations support the hypothesis that a

direct interaction between cigarette smoke and �-carotene is required for the tumor promoting effects of�-carotene in heavy smokers corroborating a role foroxidative �-carotene metabolites (Mayne et al., 1996).In asbestos workers this �-carotene oxidation may bestimulated by the inflammatory process known to beinduced in asbestos-exposed lungs (Mayne et al., 1996),since inflammatory cells isolated from non-smokerswith asbestosis are known to release significantly in-creased amounts of reactive oxygen species comparedto cells recovered from control individuals (Rom et al.,1987).

5. Polyphenols: flavonoids

Natural polyphenols like flavonoids (Fig. 5) and theircorresponding glycosides are important constituents offruits, vegetables, nuts, seeds, tea, olive oil and red wine(Middleton and Kandaswami, 1994; Rice-Evans et al.,1996). The anti-oxidative properties of these com-pounds are often claimed to be responsible for theprotective effects of these food components againstcardiovascular disease, certain forms of cancer and/orphotosensitivity diseases and ageing (Middleton andKandaswami 1994; Rice-Evans et al., 1996). As a result,increased human exposure to polyphenolic flavonoid-type antioxidants can be expected in the near future.

Flavonoids are a wide family of polyphenolic antiox-idants. Efficient antioxidant activity of these moleculesis generally assumed to be related to the presence of (1)a 3�,4�-dihydroxy (=catechol) moiety; (2) the C4�Oketo group; (3) a 3-hydroxyl substituent; and (4) aC2�C3 double bond (Rice-Evans et al., 1996). Clearly,quercetin (Fig. 5), an abundant and much studiedflavonoid, contains all these structural elements.

In addition to their mode of action as antioxidant,flavonoids may inhibit carcinogenesis by modulation ofthe metabolism of food-born carcinogens through inhi-bition and/or induction of phase I and II biotransfor-mation enzymes, and by the suppression of theabnormal proliferation of early, preneoplastic lesions.Inhibition of cell proliferation may result from inhibi-tion of various enzymes involved in cellular responsesto growth factors, including protein kinase C, tyrosinekinase, phosphatidylinositol 3-kinase and/or the effectof flavonoids on expression of various tumor-relatedgenes including antioxidant protein genes or the tumorsuppressor gene p53 (Deschner et al., 1991; Scambia etal., 1991; Matter et al., 1992; Avila et al., 1994; Middle-ton and Kandaswami, 1994; Agullo et al., 1997;Kameoka et al., 1999). Together the health claims onbeneficial effects of flavonoids provide the basis for thepresent ‘hausse’ in the ideas on possibilities for the useof flavonoids as so-called functional food ingredients,as food supplements, or as active compounds in so-

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Fig. 4. Hypothesis for the pro-oxidative mechanism for tumor enhancement by �-carotene.

called nutraceuticals. At present flavonoids present oneof the most important categories of bioactive foodcomponents. Also for flavonoids increased future hu-man exposure regimens induce the question on theirpro-oxidant chemistry.

6. Mechanisms of pro-oxidant action of flavonoids

Flavonoids have been reported to be able to exertpro-oxidant chemistry including the formation insteadof scavenging of radicals (Laughton et al., 1989; Caoet al., 1997). Flavonoids with a phenol-type subs-tituent pattern in their B-ring, like apigenin and narin-genin (Fig. 5) have been reported to result in a 30–50times increase in the formation of reactive oxidantspecies when incubated in the presence of GSH and

enzymes from thymus and bone marrow like peroxi-dases (Galati et al., 1999, 2001). Especially theseflavonoids were found to generate increased lipid per-oxidation and to act as a pro-oxidant at concentrationswhere other flavonoids were still active as antioxidantspreventing this lipid peroxidation (Galati et al., 1999).The redoxcycling GSH-oxidizing pro-oxidant activityof this type of flavonoids seemed to partly correlatewith the high one-electron oxidation potential of theircorresponding phenoxyl radicals (Sudhar and Arm-strong, 1990; Galati et al., 1999; Metodiewa et al.,1999). The mechanism may proceed as depicted in Fig.6. Enzymatic and/or chemical (auto)oxidation of theflavonoid generates the flavonoid semiquinone radical,which may be scavenged by GSH, thereby regeneratingthe flavonoid and generating the thiyl radical of glu-tathione. This thiyl radical may react with GSH to

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Fig. 5. Chemical structure of flavonoids.

type moiety is present in the flavonoid molecule.Flavonoids containing a catechol-type substituent pat-tern in their B-ring did not co-oxidize GSH whenoxidized to their semiquinone. This may be due to theirlower one-electron redox potentials (Galati et al., 1999).Subsequent studies revealed that B-ring catechol-typeflavonoids showed swift formation of their two electronoxidized quinone type metabolites, even upon their oneelectron oxidation by peroxidases (Fig. 7) (Awad et al.,2000, 2001). This implies the formation of electrophilictoxic quinone type metabolites which may be scavengedby GSH not by means of chemical reduction but ratherby conjugate formation. The formation of these GSHflavonoid adducts was recently demonstrated providingevidence for the actual pro-oxidative formation of reac-tive quinone type metabolites from B-ring catecholflavonoids (Boersma et al., 2000; Awad et al., 2000,2001; Galati et al., 2001). The possible pro-oxidanttoxicity of these catechol-containing compounds hasrecently been underlined by studies on the mutagenicityof estrogens. Metabolic activation of estrogens to redoxactive and/or electrophilic metabolites has been pro-posed as one of the mechanisms responsible for the linkbetween estrogen exposure and the risk of developingcancer (Iverson et al., 1996; Bolton et al., 1998, 2000).Especially catechol (ortho-diol)-type of metabolites re-sulting from cytochrome P450 catalyzed hydroxylationof estrogens may be involved. The involvement ofcatechol-type metabolites has also been outlined to playa role in the metabolic activation of polycyclic aromatichydrocarbons (Penning et al., 1999; Murthy and Pen-ning, 1992). Clearly flavonoids like quercetin, luteolin,fisetin and many others already contain the pro-oxida-tive catechol structural element, without the require-ment for an initial bioconversion step.

Oxidation of the catechols to quinones and theirisomeric quinone methides (Fig. 8) generates potentelectrophiles that could alkylate DNA. With respect to

generate a disulfide radical anion which rapidly reducesmolecular oxygen to superoxide anion radicals (Fig. 6)(Galati et al., 1999).

Another mechanism of flavonoid pro-oxidant activityis related to the formation of quinone type oxidationproducts and occurs especially when a 3�,4�-catechol

Fig. 6. Pro-oxidant chemistry of phenol-type flavonoids.

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Fig. 7. Pro-oxidant chemistry of catechol-type flavonoids.

this possible pro-oxidant toxicity it is of interest tonotice that the mutagenic properties of the flavonoidquercetin have been demonstrated in a variety of bacte-rial and mammalian mutagenicity tests, and have beenrelated to its quinone/quinone methide chemistry (Mac-Gregor and Jurd, 1978; Brown, 1980; Middleton andKandaswami, 1994). Interestingly, the structural re-quirements for good antioxidant activity match therequirements essential for pro-oxidant action and qui-none methide formation.

Based on these positive mutagenicity results in avariety of bacterial as well as mammalian test systems,several studies have investigated the possible carcino-genicity of especially quercetin. Several animal studiesreported no tumor initiating activity (Hirono et al.,1981; Morino et al., 1982; Stoewsand et al., 1984;Hirose et al., 1983; Ito et al., 1989). In contrast, Pa-mukcu et al. (1980) reported induction of intestinal andbladder tumors by quercetin in male and female rats. Astudy from the National Toxicology Program (NTP,1991) reported some evidence of carcinogenic activityof quercetin in male F344/N rats, based on an increasedincidence of renal tubular cell carcinomas. Erturk et al.(1985) reported bladder tumors in rats exposed toquercetin. Dunnick and Haily (1992) reported quercetin

to show carcinogenic activity in the kidney of maleF344/N rats.

The relevance for the human in vivo situation as wellas the mechanism behind this quercetin-mediated toxiceffect remains a matter of debate (Ito, 1992). Ito (1992)suggested a possible factor of special interest to be therole of �2u globulin nephropathy in chemically inducedrenal carcinogenicity, a nephropathy which is observedselectively in male rats only. Such a hypothesis wouldbe in line with the observations that increased numbersof benign tumors are often observed in male but notfemale rats (NTP, 1991; Dunnick and Haily, 1992).

Another mechanism which may be of importance forcarcinogenicity upon exposure to quercetin is the hy-pothesis that overloading the organism with quercetinmay deplete the cofactor for catechol O-methyltrans-ferases, S-adenosyl-L-methionine (SAM), because cate-chol O-methyltransferase metabolism represents animportant metabolic pathway for catechol typeflavonoids (Zhu et al., 1994; Williamson et al., 2000).Cofactor depletion may affect the methylation of cate-chol estrogens, thereby providing increased possibilitiesfor estrogen mediated carcinogenesis, because accumu-lation of catechol-type estrogens in the kidney maystimulate their oxidation to DNA alkylating elec-

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trophilic quinones (Zhu and Liehr, 1996; Bolton et al.,1998, 2000).

Altogether, the pro-oxidative carcinogenicity ofquercetin and possible interference of hormonal factorsin this carcinogenicity needs to be re-evaluated.

7. Conclusions

In nowadays society where consumers are used tofind everything for sale on the market or the internet,the wish to buy good health has rapidly emerged.Rapid development and use of functional foods andfood supplements is the result of this demand. How-ever, many health claims have been derived from obser-vational epidemiological studies in which specific dietswere shown to be associated with reduced risks onspecific forms of cancer, cardiovascular disease, in-creased action of the immune system and the reductionof stress. In spite of this, identification of the actualingredient in a specific diet responsible for the beneficialhealth effects remains an important bottle-neck fortranslating observational epidemiology to development

of a functional food ingredient. Furthermore, in addi-tion to concerns for scientific support for health claimsand identification of the active functional ingredients,important toxicological concerns arise. Paracelsus(1493–1541) already reported toxicity to be a matter ofdose, and toxicological risks may arise at the momentdaily doses of a compound rise above a certainthreshold limit, or, in the case of initiating carcinogens,with every molecule of a compound ingested. Thisraises the question about the dose-effect responses forbeneficial versus toxic effects of functional food ingredi-ents, which are often unknown. For the antioxidantvitamins E and C it is generally assumed that productenrichments will not lead to increase in doses of con-sumption by more than 4–5 times the daily intake.Safe-history approaches may no longer be valid whenbarriers between food and pharma start to disappearand daily intakes increase even higher than 4–5 timesthese values, for example because consumers may think‘the more the better’. The present review focussing onespecially the pro-oxidative aspects and chemistry ofantioxidants has illustrated that much remains to belearned about the exact impact of these compounds on

Fig. 8. Quinone/quinone methide chemistry of quercetin.

I.M.C.M. Rietjens et al. / En�ironmental Toxicology and Pharmacology 11 (2002) 321–333 331

biological systems. This includes beneficial as well astoxic health effects with special emphasis on dose-re-sponse curves and the mechanisms of beneficial but alsopro-oxidative toxic action.

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