Using C6 glioma cells in this study we investigated in detail how exposure time and cell concentration affect the cytotoxic potency of H(2)O(2) in vitro. Median cytotoxic concentrations (EC(50)) decreased from 500 to 30 μM with increasing incubation time from 1 to 24h. Twenty-four hours proved to be sufficient to determine incipient cytotoxic concentrations of H(2)O(2). The incipient EC(50) values were linearly related to the cell concentration. A cell concentration-independent median cytotoxic cell dose (ED(50)) of 430 nmol/mg cell protein or 860 nmol/10(7) cells was derived. Median cytotoxic H(2)O(2) concentrations were completely eliminated from the culture medium at a rate proportional to both the H(2)O(2) and the cell concentrations. In contrast to EC(50) 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 H(2)O(2) elimination decelerates and, thus, exposure to H(2)O(2) applied as a bolus approaches a continuous exposure to a steady H(2)O(2) concentration. Taken together, our results indicate that the cytotoxic potency of H(2)O(2) 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 H(2)O(2), and the cell concentration. We recommend expressing the toxic potency of H(2)O(2) in vitro by the incipient toxic cell dose (e.g., nmol H(2)O(2)/mg cell protein or nmol H(2)O(2)/10(7) cells), in particular for comparative purposes.
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