Original Contribution Polyphenols activate Nrf2 in astrocytes via H 2 O 2 , 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, Israel b Department of Pharmaceutics, School of Pharmacy, Hebrew University of Jerusalem, Jerusalem, Israel abstract article info Article history: Received 8 May 2011 Revised 27 September 2011 Accepted 28 September 2011 Available online 12 October 2011 Keywords: Astrocytes Polyphenols tBHQ Curcumin Resveratrol Nrf2 EpRE NQO1 H 2 O 2 Quinone Free radicals Polyphenols, which occur both in edible plants and in foodstuff, have been reported to exert a wide range of health effects; however, the mechanism of action of these molecules is not fully understood. One important cellular pathway affected by polyphenols is the activation of the transcription factor Nrf2 via the electrophile response 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 significantly after 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 caused the accumulation of H 2 O 2 . Treatment of cells with either glutathione or metmyoglobin was found to decrease Nrf2 translocation and NQO1 activity induced by polyphenols by up to 40 and 60%, respectively. Addition of both glutathione and metmyoglobin to growth medium decreased Nrf2 translocation and NQO1 activity by up to 100 and 80%, respectively. In conclusion, because metmyoglobin, in the presence of polyphenols and glutathione, is known to interact with H 2 O 2 , semiquinones, and quinones, the up-regulation of the antioxidant defense of the cells through activation of the Nrf2 transcription factor, paradoxically, occurs via the generation of H 2 O 2 and polyphenol-oxidized species generated from the exogenous microenvironment of 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 intake could be as high as 1000 mg/day, which is much higher than the dietary intake of all other classes of phytochemicals and antioxidant vitamins [1]. It is well accepted that diets rich in polyphenols have health benefits, but the absorption of polyphenols in humans is limited and the mechanism of action of these molecules in the human body is not fully understood [2,3]. Some reported biological effects of polyphe- nols include antioxidant activity [4,5], amelioration of cardiovascular diseases [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 strength between hydrogen and oxygen in the linked hydroxyl group, turning it to a very active antioxidant [8,9]. The FDA-approved synthetic food an- tioxidants are polyphenols such as galates, BHT 2 (butylhydroxytoluene), or tBHQ (tert-butylhydroquinone), a metabolite of butylhydroxy anis- ole. In cell and tissue culture systems, typical dietary plant polyphenolic compounds act as antioxidants, with protective properties [10] but under 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 has been reported that polyphenols undergo autoxidation and oxygen is consumed, generating O 2 •- , hydrogen peroxide (H 2 O 2 ), 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 inhibition was caused indirectly by H 2 O 2 generated through interaction of the polyphenols with the cell culture medium [11,12]. Production of H 2 O 2 by polyphenols in culture media was demonstrated by other researchers [15,17]. H 2 O 2 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]. H 2 O 2 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].H 2 O 2 is electronically neutral and can freely diffuse through cellular membranes [22]. Compared to more aggressive ROS mole- cules such as hydroxyl radicals, which react with all molecules they Free Radical Biology & Medicine 51 (2011) 2319–2327 Abbreviations: BHT, butylhydroxytoluene; EpRE, electrophile responsive element; GSH, glutathione; Keap1, kelch-like ECH-associated protein 1; MtMb, metmyoglobin; Nrf2, nuclear factor erythroid 2p45-related factor 2; NQO1, NAD(P)H:quinine oxidore- ductase 1; ROS, reactive oxygen species; tBHQ, tert-butylhydroquinone. ⁎ 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. All rights reserved. doi:10.1016/j.freeradbiomed.2011.09.033 Contents lists available at SciVerse ScienceDirect Free Radical Biology & Medicine journal homepage: www.elsevier.com/locate/freeradbiomed