Formation of hydrogen peroxide in cell culture media by apple polyphenols and its effect on antioxidant biomarkers in the colon cell line HT-29

Beneficial health effects of diets containing fruits have partly been attributed to polyphenols which display a spectrum of bioactive effects, including antioxidant activity. However, polyphenols can also exert prooxidative effects in vitro. In this study, polyphenol-mediated hydrogen peroxide (H2O2) formation was determined after incubation of apple juice extracts (AEs) and polyphenols in cell culture media. Effects of extracellular H2O2 on total glutathione (tGSH; =GSH + GSSG) and cellular reactive oxygen species (ROS) level of HT-29 cells were Studied by coincubation catalase (CAT). AEs (>= 30 mu g/mL) significantly generated H2O2 in DMEM, depending oil their composition. Similarly, H2O2 was measured for individual apple polyphenols/degradation products (phenolic acids > epicatechin, flavonols > dihydrochalcones). Highest concentrations were generated by compounds bearing the o-catechol moiety. H2O2 formation was found to be pH dependent; addition of CAT caused a complete decomposition of H2O2 whereas superoxide dismutase was less/not effective. At incubation of HT-29 cells with quercetin (1 - 100 mu M), generated H2O2 slightly contributed to antioxidant cell protection by modulation of tGSH- and ROS-level. In conclusion, H2O2 generation in vitro by polyphenols has to be taken into consideration when interpreting results of such cell culture experiments. Unphysiologically high polyphenol concentrations, favoring substantial H2O2 formation, are not expected to be met in vivo, even under conditions of high end nutritional uptake.