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

• Published in: Toxicology letters Vol. 205; no. 2; pp. 173 - 182
• Main Authors: Elbling, Leonilla, Herbacek, Irene, Weiss, Rosa-Maria, Gerner, Christopher, Heffeter, Petra, Jantschitsch, Christian, Trautinger, Franz, Grusch, Michael, Pangratz, Heinrich, Berger, Walter
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier Ireland Ltd 28.08.2011; Elsevier
• Subjects: Algorithms; Biological and medical sciences; Blotting, Western; Catechin - analogs & derivatives; Catechin - toxicity; Cell Count; Cell Culture Techniques; Cell Line; Cell number/volume ratio; Cell Survival - drug effects; Data Interpretation, Statistical; Dose-Response Relationship, Drug; Epigallocatechin-3-gallate; Free radicals; Humans; Hydrogen peroxide; Hydrogen Peroxide - metabolism; Hydrogen Peroxide - toxicity; Keratinocytes - cytology; Keratinocytes - drug effects; Keratinocytes - metabolism; Medical sciences; Mutagens - toxicity; Oxidation-Reduction; Oxidative Stress - drug effects; Polyphenolic compounds; Time Factors; Toxicity Tests - methods; Toxicity Tests - standards; Toxicology; Hydrogen peroxide; Free radicals; Cell number/volume ratio; EGCG; Epigallocatechin-3-gallate; CN; MTT; TB; DCFH-DA; CVI; ROS; Polyphenolic compounds; JC-1; Toxicity; Genotoxicity; Clearance; Algorithm; In vitro; Free radical; Polyphenol; Epigallocatechin gallate; DNA; Phenols; Keratinocyte; Oxidation; Skin; Cell volume
• ISSN: 0378-4274; 1879-3169
• DOI: 10.1016/j.toxlet.2011.06.001

► Green tea EGCG as oxidant agent. ► Cyto- and genotoxicity of EGCG depend on ratio of cell number to working volume. ► Experimental settings determine clearance of autooxidation-derived hydrogen peroxide. ► An algorithm allowing experimental standardization. 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 IC50 values differing up to orders of magnitude and could even exert contradictory effects. This effect was based on cell-mediated clearance of autooxidation-derived H2O2 from the supernatant. In order to estimate EGCG/H2O2 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 H2O2 clearance curves from cell supernatants as well as comparable EGCG/H2O2 effects at different settings. Our results demonstrate the importance of standardized experimental settings when investigating cytotoxic and/or beneficial effects of autooxidizing compounds.