Free Radical Biology & Medicine 51 (2011) 2319–2327

Contents lists available at SciVerse ScienceDirect

Free Radical Biology & Medicine

j ourna l homepage: www.e lsev ie r .com/ locate / f reeradb iomed

Original Contribution

Polyphenols activate Nrf2 in astrocytes via H2O2, semiquinones, and quinones

Hilla Erlank a, Anat Elmann a,1, Ron Kohen b, Joseph Kanner a,⁎,1

a Department of Food Science, ARO, Volcani Center, Bet-Dagan 50250, Israelb Department of Pharmaceutics, School of Pharmacy, Hebrew University of Jerusalem, Jerusalem, Israel

Abbreviations: BHT, butylhydroxytoluene; EpRE, eleGSH, glutathione; Keap1, kelch-like ECH-associated proNrf2, nuclear factor erythroid 2p45-related factor 2; NQductase 1; ROS, reactive oxygen species; tBHQ, tert-buty⁎ Corresponding author.

E-mail address: jkanner@bezeqint.net (J. Kanner).1 These authors contributed equally to this work.

0891-5849/$ – see front matter © 2011 Elsevier Inc. Alldoi:10.1016/j.freeradbiomed.2011.09.033

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 May 2011Revised 27 September 2011Accepted 28 September 2011Available online 12 October 2011

Keywords:AstrocytesPolyphenolstBHQCurcuminResveratrolNrf2EpRENQO1H2O2

QuinoneFree radicals

Polyphenols, which occur both in edible plants and in foodstuff, have been reported to exert a wide range ofhealth effects; however, the mechanism of action of these molecules is not fully understood. One importantcellular pathway affected by polyphenols is the activation of the transcription factor Nrf2 via the electrophileresponse element, which mediates generation of phase 2 detoxifying enzymes. Our study found that Nrf2 nu-clear translocation and the activity of NAD(P)H quinone oxidoreductase (NQO1) were increased significantlyafter treatment of astrocytes with tert-butylhydroquinone (tBHQ), resveratrol, or curcumin, at 20–50 μM.Incubation of tBHQ, resveratrol, and curcumin in the growth medium in the absence of astrocytes causedthe accumulation of H2O2. Treatment of cells with either glutathione or metmyoglobin was found to decreaseNrf2 translocation and NQO1 activity induced by polyphenols by up to 40 and 60%, respectively. Addition ofboth glutathione and metmyoglobin to growth medium decreased Nrf2 translocation and NQO1 activity byup to 100 and 80%, respectively. In conclusion, because metmyoglobin, in the presence of polyphenolsand glutathione, is known to interact with H2O2, semiquinones, and quinones, the up-regulation of theantioxidant defense of the cells through activation of the Nrf2 transcription factor, paradoxically, occurs viathe generation of H2O2 and polyphenol-oxidized species generated from the exogenous microenvironmentof the cells.

© 2011 Elsevier Inc. All rights reserved.

Polyphenols, which occur both in edible plants and in foodstuff,form a substantial part of the human diet. Their total dietary intakecould be as high as 1000 mg/day, which is much higher than thedietary intake of all other classes of phytochemicals and antioxidantvitamins [1]. It is well accepted that diets rich in polyphenols havehealth benefits, but the absorption of polyphenols in humans is limitedand the mechanism of action of these molecules in the human body isnot fully understood [2,3]. Some reported biological effects of polyphe-nols include antioxidant activity [4,5], amelioration of cardiovasculardiseases [2], prevention of several degenerative age-related diseases[6], and prevention of several kinds of cancer [7].

Polyphenols are a large and diverse family of compounds synthe-sized by plants as secondary metabolites. The benzoic ring, which con-tains three double bonds, decreases very much the bond strengthbetween hydrogen and oxygen in the linked hydroxyl group, turning itto a very active antioxidant [8,9]. The FDA-approved synthetic food an-tioxidants are polyphenols such as galates, BHT2 (butylhydroxytoluene),

ctrophile responsive element;tein 1; MtMb, metmyoglobin;O1, NAD(P)H:quinine oxidore-lhydroquinone.

rights reserved.

or tBHQ (tert-butylhydroquinone), a metabolite of butylhydroxy anis-ole. In cell and tissue culture systems, typical dietary plant polyphenoliccompounds act as antioxidants, with protective properties [10] butunder some circumstances they were found to be pro-oxidants and cy-totoxic [3,11,12]. Although polyphenols are strong reducing agents,under in vitro conditions, in the presence of oxygen and metal ions,they could act as pro-oxidants, very much like ascorbic acid [13]. It hasbeen reported that polyphenols undergo autoxidation and oxygen isconsumed, generating O2

•−, hydrogen peroxide (H2O2), semiquinones,and quinones [11,14,15]. The ability of apple extracts to inhibit prolifer-ation of tumor cells in vitro was attributed to polyphenol antioxidants[16]. Our studies, for the first time, demonstrated that this inhibitionwas caused indirectly by H2O2 generated through interaction of thepolyphenols with the cell culture medium [11,12]. Production of H2O2

by polyphenols in culturemediawas demonstrated by other researchers[15,17].

H2O2 is now clearly recognized as a part of the normal cell signal-ing that is involved in responses to specific genes involved in cell rep-lication, regulation of metabolism, apoptosis, and necrosis [18,19].H2O2 is an activator of the transcription factor nuclear factor ery-throid 2p45-related factor-2 (Nrf2), which by translocation to the nu-cleus induces the activity of the electrophile response element (EpRE)[20,21]. H2O2 is electronically neutral and can freely diffuse throughcellular membranes [22]. Compared to more aggressive ROS mole-cules such as hydroxyl radicals, which react with all molecules they

2320 H. Erlank et al. / Free Radical Biology & Medicine 51 (2011) 2319–2327

encounter, H2O2 is a rather mild oxidant that primarily targets cyste-ines in diverse proteins and, therefore, can act as a second messengerinducing various transcription factors [18,19].

Naturally, cells and tissues are empowered with a panel of antiox-idants and detoxifying enzymes, which are responsible for inactivat-ing or eliminating ROS and elecrophilic compounds, therebyprotecting cellular macromolecule damage caused by these species[5,23,24]. The proximal promoter regions for the antioxidant and de-toxification genes contain the EpRE sequence, which is the preferredtarget of the nuclear transcription factor Nrf2. Nrf2 is sequestered inthe cytoplasm as an inactive complex with its cytosolic repressor,Kelch-like ECH-associated protein 1 (Keap1). In response to oxidativeor electophilic insults that oxidize two SH groups, Nrf2 is dissociatedfrom the inhibitory protein Keap1 and is translocated to the nucleusto bind to the response element EpRE, leading to transcriptionalactivation of the antioxidant and cytoprotective genes [25]. Nrf2-regulated geneproducts are phase 2 enzymes such asNAD(P)H:quinoneoxidoreductase 1 (NQO1), glutathione peroxidase, glutathione S-transferase (GST), glutathione–cysteine ligase, heme oxygenase 1(HO-1), and others [26].

A wide variety of phytochemicals have been shown to exertchemopreventive effects by protecting cellular antioxidation ordetoxification capacities through activation of Nrf2 signaling. tBHQ[27,28], resveratrol [29], and curcumin [30] have been reported to in-duce antioxidant and detoxifying enzymes via Nrf2/EpRE signaling.Several studies adopted tBHQ as a positive control for activation ofNrf2 signaling in cells [27,28]. tBHQwas found to induce the synthesisof NQO1 and GST in mouse liver and intestinal mucosa [31]. More re-cently, it was found that through activation of Nrf2, tBHQ preventsthe deposition of amyloid β-protein after oxidative stress, in NT2Nneurons, a cell line model for Alzheimer disease [32], and provideseffective prophylaxis against cerebral ischemia in vivo [33].

Resveratrol has been reported to elevate the expression and activityof several enzymes connected to the EpRE [34]. Moreover, resveratrolincreased the phosphorylation and nuclear translocation of Nrf2 andalso induced the activity as well as the expression of NQO1 at bothprotein and mRNA levels in human leukemia K562 cells [29].

The induction of antioxidant or detoxifying enzymes by curcumin isalso mediated via Nrf2/EpRE signaling. Mice injected intraperitoneallywith curcumin showed a twofold increase in total brain glutathionelevels after treatment with buthionine sulfoximine [35]. Dietary ad-ministration of curcumin elevated hepatic GST and NQO1, resulting inincreased detoxification of benzo(a)pyrene-treated mice [36]. Oral ad-ministration of curcumin also enhanced the nuclear translocation andthe EpRE binding of Nrf2, inducing the expression of HO-1 in the liverof male ICR mice, protecting the animals against dimethylnitrosamine-induced hepatotoxicity [37].

Transgenicmice [38] that consumed juices or extracts of polyphenol-rich berries had increased γ-glutamylcysteine-synthesized promoterand glutathione in muscles. The data demonstrate that polyphenolsfrom various classes activate the Nrf2/EpRE pathway in ex vivo and invivo model systems. However, the mechanism by which this action isinduced by these molecules is not fully understood [2,3,39].

Astrocytes are multifunctional cells that are important for themaintenance of the normal functions of the central nervous system.One of their responsibilities is to increase the enzymatic redox activ-ity executed by generating a set of phase 2 enzymes [40]. It was foundthat astrocytes in coculture with neurons at a concentration of 1%of the number of neurons protected the neuronal cells significantlyfrom ROS stress [41].

The objectives of this research were to examine whether thepolyphenols tBHQ, resveratrol, and curcumin are able to induce theactivation of the Nrf2/EpRE pathway in astrocytes; whether this biolog-ical activity can be attributed to either their reduced or their oxidizedmetabolites; and if such activation could be affected by the exogenousmicroenvironment of the cells.

Materials and methods

Chemicals and reagents

Bovine serum albumin, β-nicotinamide adenine dinucleotide re-duced (NADPH), BHT, curcumin, dichlorophenolindophenol (DCPIP),flavine adenine dinucleotide reduced (FAD), ferrous ammonium sul-fate, glutathione reduced (GSH), luminol, myoglobin from horse skel-etal muscle, poly-D-lysine (PDL), protease inhibitors, resveratrol,tBHQ, Triton X-100, and xylenol orange were purchased from Sigma(St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM),Leibovitz-15 medium, glutamine, antibiotics (10,000 IU/ml penicillinand 10,000 g/ml streptomycin), trypsin 0.25% (w/v)–0.53 mM EDTAsolution, soybean trypsin inhibitor, and fetal bovine serum (FBS)were purchased from Biological Industries (Beit Haemek, Israel).4′,6′-Diamidino-2-phenylindol dihydrochloride (DAPI) and dimethylsulfoxide (DMSO) were obtained from AppliChem GmbH (Darmstadt,Germany). β-Mercaptoethanol, H2O2, Tris, Tris–hydrochloride, andmonoclonal mouse anti-actin were obtained from MP Biomedicals(Solon, OH, USA). Acrylamide mix 30%, Laemmli sample buffer, sodi-um dodecyl sulfate (SDS), skim-milk powder, and Precision Plusdual-color standard protein assay dye reagent were purchased fromBio-Rad Laboratories (Hercules, CA ,USA). Protein fraction-enriched(Pro-FEK) commercial kit was obtained from ITSI Biosciences(Johnstown, PA, USA). Cytotoxicity detection kit (lactate dehydroge-nase (LDH)) was obtained from Amresco (Solon, OH, USA). The an-tibody against Nrf2 was purchased from Santa Cruz Biotechnology(Santa Cruz, CA, USA). Fluorescein (FITC)-conjugated anti-rabbitIgG, horseradish peroxidase-conjugated anti-rabbit IgG, and anti-mouse IgG secondary antibodies along with goat serum wereobtained from Jackson ImmunoResearch Laboratories (West Grove,PA, USA). All organic solvents were of AR grade, purchased fromBio-Lab Ltd. (Jerusalem, Israel).

Preparation of primary cultures of astrocytes

Cultures of primary rat astrocytes were prepared from cerebralcortices of 1- to 2-day-old neonatal Wistar rats. Briefly, while inLeibovitz-15 medium, meninges and blood vessels were carefully re-moved, brain tissues were dissociated by trypsinization with 0.5%trypsin (10 min, 37 °C, 5% CO2), and cells were washed first withDMEM containing soybean trypsin inhibitor (100 μg/ml) and 10%FBS and then with DMEM containing 10% FBS. Cells were seeded intissue culture flasks precoated with PDL (20 μg/ml in 0.1 M boratebuffer, pH 8.4) and incubated at 37 °C in humidified air with 5%CO2. The medium was changed on the second day and every secondday thereafter. At the time of primary cell confluence (day 10), mi-croglial and progenitor cells were discarded by shaking the flasksfor 24 h on a horizontal shaking platform. Astrocytes were replatedin 24-well PDL-coated plastic plates at a density of 1×105/well, inDMEM (without phenol red) containing 10% FBS, 2 mM glutamine,100 U/ml penicillin, and 100 μg/ml streptomycin.

Treatment of cells

Twenty-four hours after plating, the original medium of the cellswas aspirated and fresh medium was added to the cells. Glutathioneand metmyoglobin were solubilized in growth medium. Dilutions ofthe various tested materials were done first in DMSO (resveratrol,tBHQ) or ethanol (curcumin) and then in the growth medium. Alldilutions were made freshly from stock solutions just before eachexperiment and were used immediately. A combined stock solutionof glutathione with each of the polyphenols (3:1) was prepared justbefore the experiment. Each treatment was performed in triplicate.

Fig. 1. tBHQ induces Nrf2 nuclear translocation in astrocytes. Astrocyteswere treatedwithDMSO (control) or (A) various concentrations of tBHQ for 2.5 h or (B) 20 μM tBHQ for theindicated time points. Nuclear proteinswere extracted, and equal amountswere separatedby SDS–PAGE and immunoblotted with specific Nrf2 antibody.

Fig. 2. Resveratrol induces Nrf2 nuclear translocation in astrocytes. Astrocytes weretreated with DMSO (control) or (A) various concentrations of resveratrol for 2.5 h or(B) 25 μM resveratrol for the indicated time points. Nuclear proteins were extracted,and equal amounts were separated by SDS–PAGE and immunoblotted with specificNrf2 antibody.

2321H. Erlank et al. / Free Radical Biology & Medicine 51 (2011) 2319–2327

Immunofluorescence

Astrocytes were seeded into 24-well plates containing glass cover-slips. After treatment, the cells were washed with 0.05% Tween 20 inPBS. Then, these cells were fixed with 4% formaldehyde in PBS con-taining 0.075 mM D-lysine and permeabilized with cold acetone. Thecells were washed again and incubated with 6% goat serum in PBSovernight at room temperature. After the cells were washed, theywere incubated for 3 h at room temperature with rabbit anti-humanNrf2 antibody in PBS containing 1% goat serum. The cells werewashed again and further incubated for 1 h with FITC-conjugatedgoat anti-rabbit IgG as a secondary antibody. The cells were counter-stained with DAPI to visualize the nuclei. For Nrf2 and DAPI detection,stained cells were viewed using a fluorescence microscope (Leica,Germany, DLMB) with a Fluotar lens. Photographs were taken usinga DC 200 camera (40×/0.75 magnification) and processed usingIM1000 software (Leica, Germany).

Western blot analysis

After treatment, cells were washed twice with ice-cold PBS. Sepa-ration of the nuclear proteins from the cytosolic and membranal pro-teins was conducted using a protein fraction-enriched (Pro-FEK)commercial kit, to which a protease inhibitor cocktail was added. Pro-tein content was determined by Bradford reagent using bovine serumalbumin as a standard. Samples were boiled in SDS sample buffer con-taining 10% β-mercaptoethanol for 5 min. Equal amounts of nuclearproteins (~40 μg) were separated by electrophoresis in 8% SDS–polyacrylamide gel and were transferred onto nitrocellulose mem-branes. After blotting, the membranes were blocked with 5% nonfatdry milk in PBS and incubated with rabbit anti-human Nrf2 antibodyovernight at 4 °C followed by incubation with horseradishperoxidase-conjugated goat anti-rabbit IgG as a secondary antibody.To ensure equal loading of protein samples, membranes werestripped off and reprobed with monoclonal mouse anti-actin, fol-lowed by incubation with horseradish peroxidase-conjugated don-key anti-mouse IgG secondary antibody. Detection was performedusing enhanced chemiluminescence.

Determination of NQO1 activity

After treatment, cultured cells were washed twice in PBS, and0.5 ml of ice-cold homogenization buffer (50 mM potassium phos-phate, pH 7.4, 1.15% KCl) was added to each well. The cells werescraped off and the entire plate was frozen for 24 h at −80 °C.Thawed cells were sonicated using a probe sonicator (Sonics VibraCell) before they were centrifuged at 10,000 g, 4 °C, for 20 min. Thesupernatants were collected and stored at −80 °C for later use to de-termine NQO1 enzymatic activities. Protein concentrations were de-termined by Bradford reagent using bovine serum albumin as astandard. NQO1 activity was determined by a continuous spectropho-tometric assay to measure the reduction of its substrate, DCPIP, as de-scribed previously [33]. Briefly, 0.1–10 μg of protein from each samplewas incubated with 1 ml of the assay buffer (40 μM DCPIP, 0.2 mMNADPH, 5 μM FAD, 25 mM Tris–HCl (pH 7.8), 0.1% (v/v) Tween 20,and 0.023% bovine serum albumin). The rate of DCPIP reduction wasmonitored over 1 min at 600 nm with an extinction coefficient (ε)of 2.1 mM−1 cm−1. The NQO1 activity was calculated as the decreasein absorbance per minute per milligram of total protein of the sample.

Determination of H2O2 concentration by the FOX2 method

Ten microliters of triphenylphosphine (TPP) solution (10 mM TPPin methanol) was added to 90 μl of fresh culture medium or of condi-tioned medium from treated astrocytes (18,000 cell equivalents).After 30 min of incubation the samples were diluted with FOX2

reagent (250 μM Fe(NH4)2(SO4)2, 100 μM xylenol orange, 25 mMH2SO4, 4.4 mM BHT, 90% (v/v) methanol). After 30 min incubationat room temperature the samples were centrifuged (2 min, 5200 g)and the absorption was measured at 560 nm.

Statistical analysis

Statistical analyses were performed with one-way ANOVAfollowed by multiple comparison tests using GraphPad InStat 3 forWindows (GraphPad Software, San Diego, CA, USA).

Results

Polyphenols induce Nrf2 nuclear translocation in astrocytes

To optimize the experimental conditions, time and dose responseof the induction of Nrf2 translocation from the cytosol to the nucleusby tBHQ and resveratrol were determined. Figs. 1 and 2 show that theoptimal conditions for induction by tBHQ or resveratrol were 2.5 h of

2322 H. Erlank et al. / Free Radical Biology & Medicine 51 (2011) 2319–2327

incubation with 20 or 25 μM, respectively. Curcumin (30 μM) showedoptimal induction after 4 h of incubation (data not shown). Similarresults were obtained when astrocytes were treated with the above-mentioned polyphenols and were immunostained for Nrf2. Fig. 3Ashows representative photographs of the cells, and Fig. 3B showsquantitative analysis of the photographs. The indicated concentra-tions were not toxic to astrocytes, as examined by LDH cytotoxicitytest (data not shown).

Polyphenols produce H2O2, which is diminished by astrocytes

H2O2 concentration was used as an indicator for the reactive oxy-gen species formed during polyphenol oxidation. To estimate the au-toxidation of various polyphenols, H2O2 production by eachpolyphenol was measured after incubation in growth medium. In theabsence of astrocytes, after 30 min of incubation of tBHQ in the growthmedium, H2O2 was generated in concentrations equimolar to those oftBHQ (Fig. 4A). Resveratrol was less active, and H2O2 generated by thispolyphenol was 10% of its molar concentrations (Fig. 4B). Resultssimilar to those of resveratrol were obtained for curcumin (data notshown). Once polyphenols were incubated with astrocytes, signifi-cantly lower concentrations of H2O2 could be detected in the growthmedium, compared to those detected in the absence of astrocytes(Fig. 5).

Fig. 3. Induction of nuclear translocation of Nrf2 in astrocytes by various polyphenols. Astrocurcumin (30 μM). Control cells were treated with equal volumes of DMSO or ethanol. Afterw(A) Representative microphotographs at×400 original magnification of immunofluorescentcontrol cells arrows indicate nuclei lacking Nrf2; in resveratrol-treated cells arrows indicatestained nuclei from the overall number of DAPI-stained nuclei. For resveratrol n=13; for t

Scavenging of the oxidized forms of the polyphenols reduces their abilityto induce Nrf2 nuclear translocation

To distinguish between the biological activities of the reducedforms of the various polyphenols and their respective oxidizedforms, the reducing agent glutathione was used. Incubation of astro-cytes with tBHQ in the presence of glutathione, which binds to the ox-idized form of tBHQ, lowered nuclear levels of Nrf2 (Fig. 6). In thepresence of metmyoglobin, which acts as a pseudo-peroxidase andscavenges H2O2, Nrf2 translocation into the nucleus was also inhib-ited (Fig. 6). Addition of both glutathione and metmyoglobin totBHQ-treated astrocytes was found to further decrease the nuclearlevels of Nrf2 (Fig. 6). Similar results were obtained when astrocyteswere treated with resveratrol or curcumin in the presence of eitherglutathione or metmyoglobin (Fig. 7). Addition of glutathione,metmyoglobin, or both agents inhibited Nrf2 translocation inducedby resveratrol or curcumin by 40–60%.

Polyphenols increase NQO1 activity in astrocytes, and scavenging of theiroxidized forms inhibits their ability to increase the enzymatic activity

The activity of NQO1, which is known to be induced by Nrf2, in-creased by 1.5- to 1.8-fold after treatment of astrocytes with tBHQ,curcumin, or resveratrol (Fig. 8). The effects of glutathione and

cytes were treated for 2.5 h with tBHQ (20 μM) or resveratrol (25 μM), or for 4 h withard, cells were fixed and immunostained against Nrf2, and DNAwas stained with DAPI.staining of Nrf2 (left) and counterstaining of nuclei with DAPI (right) in astrocytes. Innuclei containing Nrf2. (B) Nrf2 translocation was calculated as the percentage of Nrf2-BHQ n=4; for curcumin n=7. *pb0.05 vs control group.

Fig. 4. H2O2 generation by tBHQ and resveratrol in growth medium in the absence ofastrocytes. H2O2 concentration was determined in fresh growth medium containingDMSO (control) or various concentrations of (A) tBHQ or (B) resveratrol at 37 °C,using the FOX2 method.

Fig. 5. H2O2 generation in growth medium, by various polyphenols, in the absence orpresence of astrocytes. The concentrations of H2O2 generated by tBHQ (20 μM), resver-atrol (25 μM), or curcumin (30 μM) were determined after 1 h of incubation in thepresence or absence of astrocytes, using the FOX2 method. *pb0.05, n=3.

Fig. 7. The effects of glutathione and metmyoglobin on nuclear translocation of Nrf2induced by resveratrol or curcumin. Astrocytes were treated with resveratrol, glutathi-one, or metmyoglobin (25, 75, 25 μM, respectively) for 2.5 h or curcumin, glutathione,and metmyoglobin (30, 90, 30 μM, respectively) for 4 h. Control cells were treated withequal volumes of DMSO or ethanol. After treatment, the cells were fixed and immuno-stained against Nrf2, and DNAwas stained with DAPI. Nrf2 translocation was calculatedas the percentage of Nrf2-stained nuclei from the overall number of DAPI-stained nu-clei. Percentage of stained nuclei of control cells was subtracted from all treatments.*pb0.05, n=6.

Fig. 8. Polyphenols induceNQO1 activity in astrocytes. Astrocyteswere treatedwith tBHQ(20 μM, 24 h), curcumin (20 μM, 24 h), or resveratrol (2.5 μM, 18 h). Control cellswere treated with an equal volume of DMSO. NQO1 activity (1 μg cellular proteins) wasdetermined as described under Materials and methods. *pb0.05.

2323H. Erlank et al. / Free Radical Biology & Medicine 51 (2011) 2319–2327

metmyoglobin on polyphenol-induced NQO1 activity in astrocyteswere determined, and Fig. 9 demonstrates that either glutathioneor metmyoglobin significantly decreased NQO1 activity induced bytBHQ, resveratrol, or curcumin. However, maximal inhibition(50–80%) of polyphenol-induced NQO1 activity was achieved in thepresence of both glutathione and metmyoglobin.

Fig. 6. The effects of glutathione and metmyoglobin on Nrf2 translocation induced by tBHQ.tathione (60 μM) and/or metmyoglobin (20 μM). Control cells were treated with an equal voSDS–PAGE and immunoblotted with specific Nrf2 antibody.

Discussion

Polyphenols are naturally occurring compounds present in fruits,vegetables, and many beverages that have been reported to exert awide range of health effects; however, the mechanism by which poly-phenols neutralize and protect against oxidative stress in humans stillremains unclear.

The Nrf2 transcription factor regulates major environmentaland oxidative stress responses [42]. In the cytoplasm, Nrf2 is nega-tively regulated by sequestering the protein Keap1. Electrophilic

Astrocytes were treated (2.5 h) with tBHQ (20 μM) in the absence and presence of glu-lume of DMSO. Nuclear proteins were extracted, and equal amounts were separated by

Fig. 9. Polyphenol induction of NQO1 activity as affected by glutathione and metmyo-globin. Astrocytes were treated with resveratrol, glutathione, and metmyoglobin (25,75, 25 μM, respectively) for 2.5 h or curcumin, glutathione, and metmyoglobin (30,90, 30 μM, respectively) for 4 h. Control cells were treated with equal volumes ofvehicle. NQO1 activity was determined, using 1 μg cellular proteins, as describedunder Materials and methods. *pb0.05.

2324 H. Erlank et al. / Free Radical Biology & Medicine 51 (2011) 2319–2327

compounds can activate Nrf2 primarily by oxidizing or alkylatingspecific cysteines, causing structural modifications in the two Keap1molecules clenching the Nrf2 and enabling its release [43] (Fig. 10).

One important mechanism by which polyphenols seem to affecthuman metabolism is by the induction of phase 2 detoxifying en-zymes, via an EpRE-mediated response [44-46]. Zoete et al. [44] ex-amined the EHOMO of 30 different polyphenols and their inductionfactor for NQO1 activation in Hepa 1c1c7 cells. They concluded that

Fig. 10. The induction of the Nrf2/EpRE p

the electron-releasing power expressed by the HOMO energy, or theease with which a molecule donates an electron and oxidizes, corre-lates with the induction of NQO1. Interestingly, the results reportedby Zoete et al. [44] are very similar to those published by Lee-Hilz etal. [46], who reported that EHOMO of 21 tested flavonoids correlatedwith their induction factor for the EpRE-mediated gene transcription.

Our results show that tBHQ, curcumin, and resveratrol generatedH2O2 in the cell growth medium during their autoxidation. The re-sults are in line with our previous results on the generation of H2O2

by polyphenols in cell growth medium [11,12] and also those ofothers [15]. tBHQ in cell growth medium generated H2O2 in anamount equimolar to its concentration. The stoichiometric natureof the polyphenol oxidation can be explained by the followingequation:

Ph ðOHÞ2 þ O2→Ph ¼ ðOÞ2 þ H2O2: ð1Þ

Our study demonstrated a significant reduction in exogenousH2O2 by the astrocytic culture. The ability of these cells to detoxify ex-ogenously applied H2O2 was previously determined by Dringer andHamprecht [47] and corresponds with our results. The reduction ofH2O2 seems to occur mostly because of its ability to freely diffusethrough cell membranes [22,47] and its intracellular decompositionby glutathione peroxidase or by thioredoxins. A small part of thisH2O2 flux seems to affect signaling, translocation of Nrf2, and activa-tion of NQO1.

Our previous data [5,48] demonstrated that metmyoglobinstrongly interacted with H2O2, forming oxoferryl myoglobin. Al-though oxoferryl myoglobin may cause oxidative damage undersome circumstances, we clearly demonstrated that in our systems,

athway in astrocytes by polyphenols.

2325H. Erlank et al. / Free Radical Biology & Medicine 51 (2011) 2319–2327

which contained polyphenols [5,48,49], the effect was far less toxicthan in the absence of polyphenols. Oxoferryl myoglobin is very effi-ciently reduced by polyphenolic antioxidants, acting as pseudo-peroxidase, as summarized below:

Ph OH þ O2 þ culture medium→Ph ¼ Oþ H2O2; ð2Þ

MbFe3þ þH2O2→•MbFe4þ–O þ H2O; ð3Þ

•MbFe4þ–Oþ PhðOHÞ2→MbFe3þ þ polymerized phenolics:

It may be suggested that the ferryl myoglobin generated in themedium will oxidize all the polyphenols, leaving the system with ox-idized ineffective polyphenols for further H2O2 generation. However,from the stoichiometric point of view, for example, resveratrol in thepresence of astrocytes generated only 4 μM H2O2/2 h. This amount ofH2O2 will activate MtMb, forming ferryl (4 μM). Taking into consider-ation that a part of ferrylmyoglobin is autoreduced, then about 2 μMferryl will oxidize 4 μM polyphenols, leaving in the system about21 μM reduced polyphenols. As we found that the system needsonly 2 h for a significant induction of Nrf2 signaling (see Fig. 2), themedium remained with enough reduced polyphenols for furtherH2O2 generation.

In culture medium, metmyoglobin decomposes H2O2 generated bypolyphenols much better than catalase [12]. As most of the polyphe-nols interact with membranal proteins and phospholipids, it seemsthat generation of H2O2 occurs on the extracellular membrane andits diffusion into cells is much higher than its accumulation in the cul-ture medium. However, it is possible that in the presence of cells, de-composition of H2O2 also occurs by exogenously secreted proteinssuch as thioredoxins. Previously we showed that catalase decreasedH2O2 levels generated by glucose oxidase, but it could not cause thesame effect when H2O2 was generated by several polyphenols [12].This discrepancy is of note because glucose oxidase generates H2O2

in the medium and polyphenols generate H2O2, site specifically, onthe membrane. To inhibit such H2O2 flux, myoglobin, a cationizedprotein, was found to interact with the negatively charged mem-branes, which makes it a more efficient H2O2 decomposer than cata-lase, which has a low affinity for membranes, because of its negativecharge [12,50].

Our results explain those of Lee et al. (27), who found that catalasecould inhibit Nrf2 activation induced by diethyl maleate but not bytBHQ. Diethyl maleate increases production of H2O2 in the cell medi-um [51]. However, like many other polyphenolic compounds, thepolyphenol tBHQ is better associated with the membrane and uponautoxidation produces H2O2 in the proximity of the membrane[52,53].

Our results clearly show that metmyoglobin significantly preventstBHQ, resveratrol, and curcumin activation of Nrf2; its translocationto the nucleus; and NQO1 activity. As metmyoglobin has no cell per-meativity, its activity is carried out exogenously, meaning in thegrowthmedium. Glutathione, like metmyoglobin, has no cell permea-tivity and therefore also acts exogenously in our system. Glutathioneprevents generation of superoxide and H2O2 by scavenging phenoxylradicals. It also reduces semiquinones by generating GSSG and gluta-thione–phenoxyl conjugates, as can be seen in Reactions (5)–(7)[54,55]. Glutathione alone was found to decrease Nrf2 translocationinduced by tBHQ, resveratrol, and curcumin, most probably by elimi-nating their oxidation products, H2O2 and quinones, and thus pre-venting their diffusion into the cells and the activation of Nrf2.Although resveratrol and curcumin generate low concentrations ofH2O2, as was found also by Long et al. [56], the activation of Nrf2was inhibited by glutathione. Glutathione at high concentration(mM) could react with H2O2, albeit slowly; however, at the

concentration we used (μM) it seems to act spontaneously with phe-noxyl radicals and quinones:

PhO• þ GSH→PhOH þ GS•; ð5Þ

Ph ¼ Oþ GSH→PhO• þ GS•→GS–PhOH; ð6Þ

GS• þ GS•→GSSG: ð7Þ

Nrf2 activation induced by all three polyphenols tested in thisstudy was inhibited more efficiently after the addition of bothmetmyoglobin and glutathione. This effect was well demonstratedby a low NQO1 enzymatic activity. This outcome was achieved mostprobably by an additional beneficial effect on the reduction of H2O2

and oxidized polyphenols, as demonstrated by the following reaction:

MbFe3þ þH2O2 þ 2GSH þ PhðOHÞ2→MbFe3þ þ PhðOHÞ2þ GSSG þ 2H2O: ð8Þ

The generation of a glutathione–polyphenol conjugated moleculeprevented the production of superoxide and H2O2 from the medium.Reaction (8), which was demonstrated by Khalife and Lupidi [57],displays decomposition of H2O2 in the presence of metmyoglobin,polyphenol, and glutathione. Such an interaction could not only affectthe reduction of hypervalent myoglobin but also increase the elimina-tion rate of H2O2 from the medium.

Upon ingestion, flavonoid glucosides are deglycosylated and theaglycones are metabolized to glucoronide, sulfate, and methyl conju-gates [46,58]. Only a small part of the metabolites are transported tothe bloodstream and flavonoid glucoronides are the major metabo-lites present in the circulation [58]. Several metabolites of quercetinwere shown to effectively activate EpRE-mediated gene expression,thereby inducing detoxification enzymes such as NQO1. However,the induction by the mixed metabolites constituted only 40–50% ofthe maximal level induced by quercetin aglycone alone [46]. Thisseems reasonable because quercetin metabolites generated lowerconcentrations of H2O2 and quinones [59,60].

Hydrogen peroxide is a well-accepted second messenger [19].Among many ROS, H2O2 is more stable and plays the role of a survivalmolecule. The main prosurvival functions are kinase-driven oxidationof cysteines in the active sites of various phosphatases and the regu-lation of transcription factors such as p53, NF-κB, AP-1, and Nrf2. Hy-drogen peroxide concentration and its specific site of generationgreatly influence its activity as a second messenger. Thus, for H2O2

to play a direct role in signaling, its target(s) must be localized nearits site of production especially because of the high cellular enzymaticactivity of peroxidases. The application of an extracellular concentra-tion of H2O2 of 0.1–5.0 μM to cell cultures results in intracellular H2O2

levels of about 0.01–0.5 μM, which directly stimulates cell prolifera-tion and affects cell adaptation (such activation of Nrf2), whereasvery high concentrations induce apoptosis and cell death [58].According to our results, the medium concentration of H2O2 achievedin the presence of cell culture was lower than 5 μM. This most proba-bly resulted in an intracellular concentration in the range of cell adap-tation to ROS. Most recently it was found that Keap1 intermoleculardisulfide formation via cysteine 151 underlies the activation of Nrf2by low concentrations of H2O2 [21].

In conclusion, cytosolic Nrf2 levels decreased and nuclear Nrf2levels as well as NQO1 enzymatic activity increased in astrocytesafter treatment with tBHQ, resveratrol, and curcumin. All polyphe-nols, when present in growth medium, generated H2O2. H2O2 concen-trations in the media were affected to a large extent by the presenceof astrocytes, which reduced it to 4–5 μM. As most of the polyphenolsinteract with cell membranes, reduction of H2O2 seems to occur via itspartial diffusion into the cells. Addition of metmyoglobin and

2326 H. Erlank et al. / Free Radical Biology & Medicine 51 (2011) 2319–2327

glutathione to the medium decreased significantly the translocationof Nrf2 and NQO1 activity, induced by all polyphenols. Because met-myoglobin and glutathione fail to permeate membranes, their modeof action is expressed exogenously.

For a long time, ROS generated in cells had been consideredharmful mediators because of their highly reactive nature. Althoughtraditionally considered lethal to cells, ROS at low concentrationsseem to be involved in redox signaling that may contribute to normalcell function and adaptation as well as disease prevention [61,62].

We hypothesize that low concentrations of polyphenols generateH2O2, at very low concentration, at the level of arterioles and capil-laries, in association with the outer surface of the cells, and by diffu-sion it activates Nrf2 signaling, inducing cell adaptation to oxidativestress. Thus, polyphenols act as nutritional "medicinals," whichmight have a preventative nature, rather than functioning as thera-peutic agents. However, for the same reason, high concentrations ofpolyphenols could enhance the generation of H2O2 and other metab-olites capable of causing cytotoxic events.

Acknowledgment

The authors thank Mrs. Mordechay Sharon, Mrs. Rindner Miriam,and Mrs. Granit Rina for technical assistance.

References

[1] Kuhnau, J. The flavonoids: a class of semi-essential food components: their role inhuman nutrition. World Rev. Nutr. Diet. 24:117–191; 1976.

[2] Manach, C.; Williamson, G.; Morand, C.; Scalbert, A.; Remesy, C. Bioavailabilityand bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies.Am. J. Clin. Nutr. 81:230S–242S; 2005.

[3] Tang, S. Y.; Halliwell, B. Medicinal plants and antioxidants: what do we learn fromcell culture and Caenorhabditis elegans studies? Biochem. Biophys. Res. Commun.394:1–5; 2010.

[4] Bors, W.; Saran, M. Radical scavenging by flavonoid antioxidants. Free Radic. Res.Commun. 2:289–294; 1987.

[5] Kanner, J.; German, J. B.; Kinsella, J. E. Initiation of lipid peroxidation in biologicalsystems. Crit. Rev. Food Sci. Nutr. 25:317–364; 1987.

[6] Stevenson, D. E.; Hurst, R. D. Polyphenolic phytochemicals—just antioxidants ormuch more? Cell. Mol. Life Sci. 64:2900–2916; 2007.

[7] Roginsky, A. On the potential use of flavonoids in the treatment and prevention ofpancreatic cancer. In Vivo 19:61–67; 2005.

[8] Kanner, J.; Frankel, E.; Granit, R.; German, B.; Kinsella, J. E. Natural antioxidants ingrapes and wines. J. Agric. Food Chem. 42:64–69; 1994.

[9] Lapidot, T.; Granit, R.; Kanner, J. Lipid peroxidation by "free" iron ions and myo-globin as affected by dietary antioxidants in simulated gastric fluids. J. Agric.Food Chem. 53:3383–3390; 2005.

[10] Ramassamy, C. Emerging role of polyphenolic compounds in the treatment of neu-rodegenerative diseases: a review of their intracellular targets. Eur. J. Pharmacol.545:51–64; 2006.

[11] Lapidot, T.; Walker, M. D.; Kanner, J. Can apple antioxidants inhibit tumor cellproliferation? Generation of H2O2 during interaction of phenolic compoundswith cell culture media. J. Agric. Food Chem. 50:3156–3160; 2002.

[12] Lapidot, T.; Walker, M. D.; Kanner, J. Antioxidant and prooxidant effects of pheno-lics on pancreatic beta-cells in vitro. J. Agric. Food Chem. 50:7220–7225; 2002.

[13] Kanner, J.; Mendel, H.; Budowski, P. Pro-oxidant and antioxidant effects ofascorbic-acid and metal-salts in a beta-carotene–linoleate model system. J. FoodSci. 42:60–64; 1977.

[14] Canada, A. T.; Giannella, E.; Nguyen, T. D.; Mason, R. P. The production of reactiveoxygen species by dietary flavonols. Free Radic. Biol. Med. 9:441–449; 1990.

[15] Long, L. H.; Clement, M. V.; Halliwell, B. Artifacts in cell culture: rapid generationof hydrogen peroxide on addition of (−)-epigallocatechin, (−)-epigallocatechingallate, (+)-catechin, and quercetin to commonly used cell culture media.Biochem. Biophys. Res. Commun. 273:50–53; 2000.

[16] Eberhardt, M. V.; Lee, C. Y.; Liu, R. H. Antioxidant activity of fresh apples. Nature405:903–904; 2000.

[17] Maeta, K.; Nomura, W.; Takatsume, Y.; Izawa, S.; Inoue, Y. Green tea polyphenolsfunction as prooxidants to activate oxidative-stress-responsive transcriptionfactors in yeasts. Appl. Environ. Microbiol. 73:572–580; 2007.

[18] Reth, M. Hydrogen peroxide as second messenger in lymphocyte activation. Nat.Immunol. 3:1129–1134; 2002.

[19] Rhee, S. G. Cell signaling: H2O2, a necessary evil for cell signaling. Science 312:1882–1883; 2006.

[20] Shih, A. Y.; Erb, H.; Murphy, T. H. Dopamine activates Nrf2-regulated neuroprotec-tive pathways in astrocytes and meningeal cells. J. Neurochem. 101:109–119; 2007.

[21] Fourquet, S.; Guerois, R.; Biard, D.; Toledano, M. B. Activation of NRF2 by nitrosa-tive agents and H2O2involves KEAP1 disulfide formation. J. Biol. Chem. 285:8463–8471; 2010.

[22] Bienert, G. P.; Schjoerring, J. K.; Jahn, T. P. Membrane transport of hydrogenperoxide. Biochim. Biophys. Acta 1758:994–1003; 2006.

[23] Rhee, S. G.; Yang, K. S.; Kang, S.W.; Woo, H. A.; Chang, T. S. Controlled elimination ofintracellular H2O2: regulation of peroxiredoxin, catalase, and glutathione peroxidasevia post-translational modification. Antioxid. Redox Signal. 7:619–626; 2005.

[24] Talalay, P. Chemoprotection against cancer by induction of phase 2 enzymes.Biofactors 12:5–11; 2000.

[25] Chen, C.; Kong, A. N. Dietary chemopreventive compounds and ARE/EpREsignaling. Free Radic. Biol. Med. 36:1505–1516; 2004.

[26] Wakabayashi, N.; Dinkova-Kostova, A. T.; Holtzclaw, W. D., et al. Protectionagainst electrophile and oxidant stress by induction of the phase 2 response:fate of cysteines of the Keap1 sensor modified by inducers. Proc. Natl. Acad. Sci.U. S. A. 101:2040–2045; 2004.

[27] Lee, J. M.; Moehlenkamp, J. D.; Hanson, J. M.; Johnson, J. A. Nrf2-dependentactivation of the antioxidant responsive element by tert-butylhydroquinone isindependent of oxidative stress in IMR-32 human neuroblastoma cells. Biochem.Biophys. Res. Commun. 280:286–292; 2001.

[28] Ben-Dor, A.; Steiner, M.; Gheber, L.; Danilenko, M.; Dubi, N.; Linnewiel, K.; Zick,A.; Sharoni, Y.; Levy, J. Carotenoids activate the antioxidant response elementtranscription system. Mol. Cancer Ther. 4:177–186; 2005.

[29] Hsieh, T. C.; Lu, X.; Wang, Z.; Wu, J. M. Induction of quinone reductase NQO1 byresveratrol in human K562 cells involves the antioxidant response element AREand is accompanied by nuclear translocation of transcription factor Nrf2. Med.Chem. 2:275–285; 2006.

[30] Dickinson, D. A.; Iles, K. E.; Zhang, H. Q.; Blank, V.; Forman, H. J. Curcumin altersEpRE and AP-1 binding complexes and elevates glutamate–cysteine ligase geneexpression. FASEB J. 17:473–476; 2003.

[31] Prochaska, H. J.; De Long, M. J.; Talalay, P. On the mechanisms of induction ofcancer-protective enzymes: a unifying proposal. Proc. Natl. Acad. Sci. U. S. A. 82:8232–8236; 1985.

[32] Eftekharzadeh, B.; Maghsoudi, N.; Khodagholi, F. Stabilization of transcriptionfactor Nrf2 by tBHQ prevents oxidative stress-induced amyloid beta formationin NT2N neurons. Biochimie 92:245–253; 2010.

[33] Shih, A. Y.; Li, P.; Murphy, T. H. A small-molecule-inducible Nrf2-mediated anti-oxidant response provides effective prophylaxis against cerebral ischemia invivo. J. Neurosci. 25:10321–10335; 2005.

[34] Chen, C. Y.; Jang, J. H.; Li, M. H.; Surh, Y. J. Resveratrol upregulates hemeoxygenase-1 expression via activation of NF-E2-related factor 2 in PC12 cells.Biochem. Biophys. Res. Commun. 331:993–1000; 2005.

[35] Jagatha, B.;Mythri, R. B.; Vali, S.; Bharath,M.M. Curcumin treatment alleviates the ef-fects of glutathionedepletion invitro and in vivo: therapeutic implications for Parkin-son's disease explained via in silico studies. Free Radic. Biol. Med. 44:907–917; 2008.

[36] Garg, R.; Gupta, S.; Maru, G. B. Dietary curcumin modulates transcriptional regu-lators of phase I and phase II enzymes in benzo[a]pyrene-treated mice: mecha-nism of its anti-initiating action. Carcinogenesis 29:1022–1032; 2008.

[37] Farombi, E. O.; Shrotriya, S.; Na, H. K.; Kim, S. H.; Surh, Y. J. Curcumin attenuatesdimethylnitrosamine-induced liver injury in rats through Nrf2-mediated induc-tion of heme oxygenase-1. Food Chem. Toxicol. 46:1279–1287; 2008.

[38] Myhrstad, M. C.; Carlsen, H.; Dahl, L. I.; Ebihara, K.; Glemmestad, L.; Haffner, K.;Moskaug, J. O.; Blomhoff, R. Bilberry extracts induce gene expression throughthe electrophile response element. Nutr. Cancer 54:94–101; 2006.

[39] Gutteridge, J. M.; Halliwell, B. Antioxidants: molecules, medicines, and myths.Biochem. Biophys. Res. Commun. 393:561–564; 2010.

[40] Cotrina, M. L.; Nedergaard, M. Astrocytes in the aging brain. J. Neurosci. Res. 67:1–10; 2002.

[41] Shih, A. Y.; Johnson, D. A.; Wong, G.; Kraft, A. D.; Jiang, L.; Erb, H.; Johnson, J. A.;Murphy, T. H. Coordinate regulation of glutathione biosynthesis and release byNrf2-expressing glia potently protects neurons from oxidative stress. J. Neurosci.23:3394–3406; 2003.

[42] Leiser, S. F.; Miller, R. A. Nrf2 signaling, a mechanism for cellular stress resistancein long-lived mice. Mol. Cell. Biol. 30:871–884; 2009.

[43] Dinkova-Kostova, A. T.; Talalay, P. Direct and indirect antioxidant properties of in-ducers of cytoprotective proteins.Mol. Nutr. Food Res. 52 (Suppl. 1):S128–138; 2008.

[44] Zoete, V.; Rougee, M.; Dinkova-Kostova, A. T.; Talalay, P.; Bensasson, R. V. Redoxranking of inducers of a cancer-protective enzyme via the energy of their highestoccupied molecular orbital. Free Radic. Biol. Med. 36:1418–1423; 2004.

[45] Liu, H.; Dinkova-Kostova, A. T.; Talalay, P. Coordinate regulation of enzymemarkers for inflammation and for protection against oxidants and electrophiles.Proc. Natl. Acad. Sci. U. S. A. 105:15926–15931; 2008.

[46] Lee-Hilz, Y. Y.; Boerboom, A. M.; Westphal, A. H.; Berkel, W. J.; Aarts, J. M.;Rietjens, I. M. Pro-oxidant activity of flavonoids induces EpRE-mediated geneexpression. Chem. Res. Toxicol. 19:1499–1505; 2006.

[47] Dringen, R.; Hamprecht, B. Involvement of glutathione peroxidase and catalase inthe disposal of exogenous hydrogen peroxide by cultured astroglial cells. BrainRes. 759:67–75; 1997.

[48] Kanner, J.; Harel, S. Initiation of membranal lipid peroxidation by activatedmetmyoglobin and methemoglobin. Arch. Biochem. Biophys. 237:314–321; 1985.

[49] Kanner, J.; Lapidot, T. The stomach as a bioreactor: dietary lipid peroxidation inthe gastric fluid and the effects of plant-derived antioxidants. Free Radic. Biol.Med. 31:1388–1395; 2001.

[50] Kohen, R.; Kakunda, A.; Rubinstein, A. The role of cationized catalase and catio-nized glucose oxidase in mucosal oxidative damage induced in the rat jejunum.J. Biol. Chem. 267:21349–21354; 1992.

[51] Iantomasi, T.; Favilli, F.; Catarzi, S.; Vincenzini, M. T. GSH role on platelet-derivedgrowth factor receptor tyrosine phosphorylation induced by H2O2. Biochem.Biophys. Res. Commun. 280:1279–1285; 2001.

2327H. Erlank et al. / Free Radical Biology & Medicine 51 (2011) 2319–2327

[52] Koren, E.; Kohen, R.; Ginsburg, I. Polyphenols enhance total oxidant-scavengingcapacities of human blood by binding to red blood cells. Exp. Biol. Med. (Maywood)235:689–699; 2010.

[53] Youdim, K. A.; Shukitt-Hale, B.; MacKinnon, S.; Kalt, W.; Joseph, J. A. Polyphenolicsenhance red blood cell resistance to oxidative stress: in vitro and in vivo. Biochim.Biophys. Acta 1523:117–122; 2000.

[54] Galati, G.; Moridani, M. Y.; Chan, T. S.; O'Brien, P. J. Peroxidative metabolism ofapigenin and naringenin versus luteolin and quercetin: glutathione oxidationand conjugation. Free Radic. Biol. Med. 30:370–382; 2001.

[55] Cuddihy, S. L.; Parker, A.; Harwood, D. T.; Vissers, M. C.; Winterbourn, C. C.Ascorbate interacts with reduced glutathione to scavenge phenoxyl radicals inHL60 cells. Free Radic. Biol. Med. 44:1637–1644; 2008.

[56] Long, L. H.; Hoi, A.; Halliwell, B. Instability of, and generation of hydrogen perox-ide by, phenolic compounds in cell culture media. Arch. Biochem. Biophys. 501:162–169; 2010.

[57] Khalife, K. H.; Lupidi, G. Reduction of hypervalent states of myoglobin and hemo-globin to their ferrous forms by thymoquinone: the role of GSH, NADH andNADPH. Biochim. Biophys. Acta 1780:627–637; 2008.

[58] Spencer, J. P.; Abd-el-Mohsen, M. M.; Rice-Evans, C. Cellular uptake and metabo-lism of flavonoids and their metabolites: implications for their bioactivity. Arch.Biochem. Biophys. 423:148–161; 2004.

[59] Stone, J. R.; Yang, S. Hydrogen peroxide: a signaling messenger. Antioxid. RedoxSignal. 8:243–270; 2006.

[60] Shirai, M.; Yamanishi, R.; Moon, J. H.; Murota, K.; Terao, J. Effect of quercetin and its con-jugated metabolite on the hydrogen peroxide-induced intracellular production of reac-tiveoxygen species inmousefibroblasts.Biosci. Biotechnol. Biochem.66:1015–1021; 2002.

[61] Groeger, G.; Quiney, C.; Cotter, T. G. Hydrogen peroxide as a cell-survival signalingmolecule. Antioxid. Redox Signal. 11:2655–2671; 2009.

[62] Hultqvist, M.; Olsson, L. M.; Gelderman, K. A.; Holmdahl, R. The protective role ofROS in autoimmune disease. Trends Immunol. 30:201–208; 2009.