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Mechanisms of Hypoxic and Aerobic Cytotoxicity of Mitomycin C in Chinese Hamster V79 Cells¹

Radiation Oncology Branch, Clinical Oncology Program [M. C. K., W. D., A. R., J. B. M.] and Laboratory of Molecular Carcinogenesis [S. T., F. J. G.], National Cancer Institute, NIH, Bethesda, Maryland 20892, and Molecular Biology, Hebrew University Medical School, Jerusalem, Israel [A. S.]

Mitomycin C (MMC) induced aerobic and hypoxic cytotoxicity in Chinese hamster V79 cells was studied to evaluate the role of the 1-electron versus 2-electron reductive bioactivation. Superoxide dismutase, catalase, and desferal had no protective effects on the aerobic or hypoxic cytotoxicity of MMC, whereas Tempol and Tempol-H, which are known to interrupt and terminate radical reactions, provided partial protection under aerobic conditions. However, under hypoxic conditions, Tempol provided complete protection whereas Tempol-H was ineffective. Electron paramagnetic resonance and spin-trapping investigations, designed to study the mechanisms of such protective effects, confirmed that MMC is activated by the human NADPH:cytochrome P-450 oxidoreductase to its semiquinone radical and that, under aerobic conditions, the semiquinone radical reduces molecular oxygen. Under hypoxic conditions, the semiquinone of MMC reduces H₂O₂ to produce OH radicals as detected by electron paramagnetic resonance-spin trapping with 5,5-dimethyl-1-pyrroline N-oxide. The 1-electron reduced product of MMC was also found to reduce Tempol to the hydroxylamine, Tempol-H, whereas oxidation of Tempol-H by was negligible.

Cell survival studies and electron paramagnetic resonance observations indicate that the hypoxic cytotoxicity of MMC is mediated by 1-electron activation to its semiquinone intermediate. Under aerobic conditions, the steady state concentration of this intermediate is low due to the facile autooxidation of the semiquinone producing and H₂O₂ which are capable of causing oxidative cytotoxicity. Tempol, which can accept an electron from reducing radical species, completely inhibited the hypoxic cytotoxicity of MMC indicating , the semiquinone of MMC as the species responsible for DNA alkylation and selective hypoxic cytotoxicity of MMC. Our results also indicate that the aerobic cytotoxicity is mediated by other processes in addition to the 1-electron mediated activation.

¹ This research was partly supported by Grant 89-00124 from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel.

² To whom requests for reprints should be addressed.

Received 7/30/91. Accepted 10/ 3/91.

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