Identification of the factors responsible for the in vitro pro-oxidant and cytotoxic activities of the olive polyphenols oleuropein and hydroxytyrosol

The olive polyphenols oleuropein and hydroxytyrosol were reported recently to produce extracellular hydrogen peroxide (H₂O₂) under standard culture conditions. The precise factors responsible for this production and the conditions promoting or retarding it are critical for the interpretation of the in vitro results. In this study, a systematic evaluation of the components of the most commonly used culture media revealed that sodium bicarbonate is the defining cause for the production of H₂O₂ by these polyphenols. The produced H₂O₂ caused extensive oxidative DNA damage and significant decrease in cell viability of cancer (MDA-MB-231) and normal (MCF-10A, STO) cells alike. Sodium pyruvate and the antioxidant N-acetyl cysteine (NAC) totally reversed these effects. Therefore, we conclusively identified the culture conditions that promote H₂O₂ production by these polyphenols, producing artifacts that may be misinterpreted as a specific anticancer activity. Our findings raise considerable questions regarding the use of culture media with sodium bicarbonate or sodium pyruvate as components, for the in vitro study of these and possibly other plant polyphenols.