title: EGCG-meditated cyto- and genotoxicity in HaCat keratinocytes is impaired by cell-mediated clearance of auto-oxidation-derived H2O2: an algorithm for experimental setting correction.
abstract: Several lines of evidence suggest that besides antioxidant also prooxidant properties are crucially involved in cytotoxic and protective activities of the major green tea catechin epigallocatechin-3-gallate (EGCG) in vitro (Elbling et al., 2011). Furthermore recent data suggest that EGCG induces oxidative stress also in vivo (Li et al., 2010). Here we set out to identify factors modulating cellular effects of EGCG in vitro. Using the HaCat keratinocytes model, we demonstrate that the cytotoxic, genotoxic and signal-activating effects of EGCG are significantly dependent on the ratio of cell number to working volume. Treatment with identical EGCG concentrations at altered experimental settings resulted in IC(50) values differing up to orders of magnitude and could even exert contradictory effects. This effect was based on cell-mediated clearance of autooxidation-derived H(2)O(2) from the supernatant. In order to estimate EGCG/H(2)O(2) concentrations equally effective under different settings, we have rationally derived and experimentally verified a simple algorithm relating concentration, working volume, cell number and - indirectly - exposure time. Algorithm application resulted in similar H(2)O(2) clearance curves from cell supernatants as well as comparable EGCG/H(2)O(2) effects at different settings. Our results demonstrate the importance of standardized experimental settings when investigating cytotoxic and/or beneficial effects of autooxidizing compounds.
authors.affiliation: Department of Medicine I, Institute of Cancer Research, Medical University Vienna, Borschkegasse 8a, 1090 Vienna, Austria. leonilla.elbling@meduniwien.ac.at
authors.forename: Leonilla
authors.initials: L
authors.lastname: Elbling
authors.forename: Irene
authors.initials: I
authors.lastname: Herbacek
authors.forename: Rosa-Maria
authors.initials: RM
authors.lastname: Weiss
authors.forename: Christopher
authors.initials: C
authors.lastname: Gerner
authors.forename: Petra
authors.initials: P
authors.lastname: Heffeter
authors.forename: Christian
authors.initials: C
authors.lastname: Jantschitsch
authors.forename: Franz
authors.initials: F
authors.lastname: Trautinger
authors.forename: Michael
authors.initials: M
authors.lastname: Grusch
authors.forename: Heinrich
authors.initials: H
authors.lastname: Pangratz
authors.forename: Walter
authors.initials: W
authors.lastname: Berger
mesh_terms: D000465:Algorithms; D015153:Blotting, Western; D002392:Catechin / Q000031:analogs & derivatives* / Q000633:toxicity; D002452:Cell Count; D018929:Cell Culture Techniques; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003627:Data Interpretation, Statistical; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000633:toxicity*; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009153:Mutagens / Q000633:toxicity*; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D013997:Time Factors; D018675:Toxicity Tests / Q000379:methods* / Q000592:standards
chemical_list: D009153:Mutagens; D002392:Catechin; D006861:Hydrogen Peroxide; C045651:epigallocatechin gallate