Original Contribution Hydrogen peroxide mediates EGCG-induced antioxidant protection in human keratinocytes Leonilla Elbling a, ⁎, Irene Herbacek a , Rosa-Maria Weiss a , Christian Jantschitsch b , Michael Micksche a , Christopher Gerner a , Heinrich Pangratz c , Michael Grusch a , Siegfried Knasmüller a , Walter Berger a, ⁎ a Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, 1090 Vienna, Austria b Department of Dermatology, University of Kiel, Kiel, Germany c Technical University of Vienna, Vienna, Austria abstract article info Article history: Received 14 April 2010 Revised 4 August 2010 Accepted 5 August 2010 Available online 12 August 2010 Keywords: Green tea Polyphenol Hydrogen peroxide EGCG Protection DNA damage Free radicals The beneficial health effects of (-)-epigallocatechin-3-gallate (EGCG), the main catechin of green tea, have been attributed to complex interactions with a focus on antioxidative properties. Susceptibility to autoxidation and production of cytotoxic reactive oxygen species (ROS), mostly H 2 O 2 , have been suggested to occur in vitro but also in vivo. In this study, we address whether autoxidation-derived H 2 O 2 may be involved in the cytoprotective effects of EGCG. To that end we investigated keratinocyte-derived HaCat and HL-60 promyelocytic leukemia cells with significantly different sensitivities to H 2 O 2 (IC 50 117.3 versus 58.3 μM, respectively) and EGCG (134.1 versus 84.1 μM). HaCat cells significantly resisted cytotoxicity and DNA damage based on enhanced H 2 O 2 clearance, improved DNA repair, and reduced intracellular ROS generation. Cumulative versus bolus EGCG and H 2 O 2 treatment and H 2 O 2 pretreatment before subsequent high-dose EGCG and vice versa significantly reduced DNA damage and cytotoxicity in HaCat cells only. Addition of catalase abolished the protective activities of low-dose H 2 O 2 and EGCG. In summary, our data suggest that autoxidative generation of low-dose H 2 O 2 is a significant player in the cell-type-specific cytoprotection mediated by EGCG and support the hypothesis that regular green tea consumption can contribute as a pro-oxidant to increased resistance against high-dose oxidative stressors. © 2010 Elsevier Inc. All rights reserved. Habitual green tea consumption has long been associated with health benefits and cancer prevention, although the overall clinical evidence is inconclusive [2]. Among the bioactive chemicals of green tea leaves, (-)-epigallocatechin-3-gallate (EGCG), the predominant polyphenolic catechin constituent, has been postulated to be the main effective agent [3]. Consequently, many efforts to develop health- promoting dietary supplements and nutraceuticals focus on EGCG. Chemoprevention attributed to EGCG includes antioxidant properties [4] of direct scavenging of reactive oxygen/nitrogen species and chelation of reactive metal ions, as well as interactions with signal transduction pathways [5,6] and binding to target proteins [7,8] resulting in cell cycle arrest and/or apoptosis. EGCG and other dietary chemopre- ventive compounds have also been shown to activate the redox-sensitive Nrf2 signaling pathway [9,10], which regulates phase II detoxification and antioxidant enzymes in response to environmental and oxidative stressors [11]. Green tea catechins, undergoing extensive metabolic biotransformation in vivo [12,13], have been shown to polymerize oxidatively with cogeneration of reactive oxygen species (ROS), including H 2 O 2 , under cell culture conditions [12–14]. Recently, EGCG- induced ROS generation has been verified in an in vivo system [1], which now gives new impulse to the ongoing discussion of whether ROS, apart from the proven H 2 O 2 generation within the oral cavity [15], may also affect inner organs [16]. Several cell-damaging effects of EGCG, classified as in vitro artifacts, have been linked to the generated H 2 O 2 [13,17,18] and oxidative degradation products of EGCG [19,20]. Nevertheless, in vivo liver toxicity has been reported after high-dose green tea exposure following consumption of dietary supplements in humans [21] and administration of tea extracts in animal studies. Potential pro-oxidant activities of EGCG have been implicated in this high-dose-induced hepatotoxicity [22]. Beyond doubt, it is a matter of dosage whether H 2 O 2 acts destructively or functions as a cellular signaling molecule with diverse physiological functions [23–25]. A repetitive low-level H 2 O 2 stress is capable of protecting against apoptosis induced by subsequent acute stress by initiating upregulation of antioxidant enzymes [26] and the enhanced expression and activity of the peroxisome proliferator-activated nuclear receptor (PPAR)-β [27]. Free Radical Biology & Medicine 49 (2010) 1444–1452 Abbreviations: DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; EGCG, (-)- epigallocatechin-3-gallate; EGCG fp , freshly prepared EGCG; EGCG a , aged EGCG; PPAR, peroxisome proliferator-activated nuclear receptor; ROS, reactive oxygen species; TB, trypan blue. ⁎ Corresponding authors. Fax: + 43 1 427765149. E-mail addresses: leonilla.elbling@meduniwien.ac.at (L. Elbling), walter.berger@meduniwien.ac.at (W. Berger). 0891-5849/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2010.08.008 Contents lists available at ScienceDirect Free Radical Biology & Medicine journal homepage: www.elsevier.com/locate/freeradbiomed