Cytotoxic potency of H2O2 in cell cultures: Impact of cell concentration and exposure time

Using C6 glioma cells in this study we investigated in detail how exposure time and cell concentration affect the cytotoxic potency of H2O2 in vitro. Median cytotoxic concentrations (EC50) decreased from 500 to 30 μM with increasing incubation time from 1 to 24 h. Twenty-four hours proved to be sufficient to determine incipient cytotoxic concentrations of H2O2. The incipient EC50 values were linearly related to the cell concentration. A cell concentration-independent median cytotoxic cell dose (ED50) of 430 nmol/mg cell protein or 860 nmol/107 cells was derived. Median cytotoxic H2O2 concentrations were completely eliminated from the culture medium at a rate proportional to both the H2O2 and the cell concentrations. In contrast to EC50 values the corresponding areas under the concentration versus time curve (AUC) were independent of the cell concentration and amounted to 1800 μM × min. With decreasing cell concentration the H2O2 elimination decelerates and, thus, exposure to H2O2 applied as a bolus approaches a continuous exposure to a steady H2O2 concentration. Taken together, our results indicate that the cytotoxic potency of H2O2 administered to cultured cells as a bolus is characterized by the AUC, which depends on its initial concentration, the ability of the cells to eliminate H2O2, and the cell concentration. We recommend expressing the toxic potency of H2O2 in vitro by the incipient toxic cell dose (e.g., nmol H2O2/mg cell protein or nmol H2O2/107 cells), in particular for comparative purposes.