Experimental validation of deuterium oxide-mediated antitumoral activity as it relates to apoptosis in murine malignant astrocytoma cells

Object. Deuterium oxide (D₂O), or heavy water, affects a variety of biological activities different from those of water. The authors examined the antitumoral effect of D₂O on brain neoplasms and demonstrated D₂O-mediated cytotoxicity by using a Rous sarcoma virus-induced murine malignant astrocytoma cell line, RSVM. The mechanism of the observed cytotoxicity may involve D₂O-induced apoptosis and cell-cycle modulation. Methods. The authors performed an assay with methylthiazol tetrazolium bromide and a trypan blue dye exclusion test to confirm in vitro D₂O-mediated cytotoxicity for RSVM cells. At D₂O concentrations of 10 to 50%, the cytotoxic effect was dose and time dependent. Flow cytometry analysis revealed programmed cell death (apoptosis) and the accumulation of RSVM cells during the G₂/M phase. By applying the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method, fluorescein isothiocyanate-annexin V and propidium iodide double staining, and caspase-family protease activity analysis, the authors demonstrated both DNA fragmentation and enhancement of caspase activity after a 48-hour treatment with D₂O, thus indicating that D₂O induces apoptosis in RSVM cells. Apoptotic DNA fragmentation was completely abolished by the caspase inhibitor Z-VAD-FMK (benzyloxycarbonilVal-Ala-Aps-fluoromethylketone). The findings indicate that the caspase activation pathway may be involved in D₂O-induced apoptosis. Conclusions. The authors found that D₂O is cytotoxic to malignant astrocytoma cells. The mechanism of D₂O-mediated cytotoxicity involved the induction of apoptosis and cell accumulation during the G₂/M phase. This D₂O-induced apoptosis is modulated through the caspase activation pathway.