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The quinone antitumor agent mitomycin C is preferentially toxic to some cells under hypoxic and acidic conditions. The two-electron reducing enzyme DT-diaphorase may be a major contributor to mitomycin C activation under aerobic conditions, but its role in drug activation under hypoxic and acidic conditions is unclear. In this study, we observed that mitomycin C produced increased DNA cross-linking and cytotoxicity in Chinese hamster ovary cells at pH 6.6, compared with pH 7.2, under aerobic conditions, but drug activity was similar at these pH values under hypoxic conditions. The DT-diaphorase inhibitor dicoumarol completely inhibited the enhanced activity of mitomycin C at acidic pH under aerobic conditions but had no effect on DNA cross-linking or cytotoxicity under hypoxic conditions. These finding suggest that the enhanced activity of mitomycin C at acidic pH, in air, is due to increased drug activation by DT-diaphorase. However, the role of DT-diaphorase in activating mitomycin C under hypoxic conditions appears to be limited, even at acidic pH.

The role of the two-electron reducing enzyme DT-diaphorase in the activation of mitomycin C under hypoxic conditions was investigated. Mitomycin C activity was compared in L5178Y murine lymphoblasts, which have low levels of DT-diaphorase activity, and L5178Y/HBM10 cells, which have elevated levels of enzyme activity. The cytotoxic and DNA cross-linking activities of mitomycin C were greater in L5178Y/HBM10 cells than in L5178Y cells. In L5178Y/HBM10 cells, dicoumarol, an inhibitor of DT-diaphorase, decreased cell kill and DNA cross-linking by mitomycin C in air but had no significant effect on these activities under hypoxia. By comparison, in L5178Y cells, dicoumarol had no effect on drug activity under either aerobic or hypoxic conditions. A model for the activation of mitomycin C by both one-electron and two-electron reduction is proposed. Our findings suggest that two-electron reduction by DT-diaphorase has only a limited role in the activation of mitomycin C under hypoxic conditions, although this enzyme appears to be an important contributor to drug activation under aerobic conditions.

Previous studies have demonstrated that the quinone group may play an important role in modulating the alkylating activity of quinone alkylating agents. Introduction of a quinone moiety markedly increased the alkylating activity and cytotoxic activity of the model quinone alkylating agents benzoquinone mustard and benzoquinone dimustard. However, the cytotoxic and DNA-damaging activity of benzoquinone mustard was considerably greater than that of benzoquinone dimustard. In this study, we have investigated the role of the quinone group as a modulator of alkylating activity in these antitumor agents, using extracellular assays to eliminate differences due to cellular drug uptake and metabolism. Evidence was obtained that the alkylating activities of both benzoquinone mustard and benzoquinone dimustard were enhanced by reduction of the quinone group. In addition, when these agents were reduced, they displayed equal alkylating activity. This finding suggests that the difference in the activity of these agents in cells is not due to intrinsic differences in alkylating activities of the activated forms of these agents. Electrochemical studies revealed that benzoquinone dimustard has a lower redox potential than benzoquinone mustard and, thus, is less easily reduced. Inactivation and spectroscopic studies suggested that a major reason for the differences in activity between benzoquinone mustard and benzoquinone dimustard may be the rapid inactivation of the dimustard before its reduction. This effect may be enhanced by the lower redox potential of benzoquinone dimustard, compared with benzoquinone mustard. These findings support the hypothesis that the quinone group can modulate the alkylating activity of quinone alkylating agents; however, the mechanisms by which this modulation occurs may vary for different antitumor agents.