The bioreductive activation of the antitumor quinone mitomycin C (MMC) by NAD(P)H: quinone oxidoreductase 1 (NQO1) is complicated by the ability of MMC to also act as a mechanismbased inhibitor of NQO1 in a pH dependent manner. Inhibition of NQO1 by MMC has been studied in purified enzyme preparations and in cultured cells but has not determined in vivo. In the studies presented here, NQO1 activity was measured in mouse tissues following treatment with MMC or the potent mechanism-based human NQO1 inhibitor 5-methoxy-1,2-dimethyl-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936). NQO1 activity was significantly decreased at 1, 2, and 4 h following MMC (10 or 20 mg/kg) treatment in kidney and lung but was unchanged in brain, heart, liver, and bladder. ES936 (1 mg/kg) treatment led to a significant and much more potent inhibition of NQO1 in all murine tissues analyzed except for bladder. To extrapolate these in vivo results from mice to humans, the species-specific kinetics of NQO1 inactivation by MMC was determined in vitro using mouse, rat, and human recombinant NQO1 proteins. Results showed the inactivation kinetics of mouse and human proteins by MMC were similar. Treatment of human and murine endothelial cells with MMC or ES936 showed similar inhibition of NQO1 activity. The aforementioned results clearly demonstrate that MMC can serve as a substrate for NQO1 in vivo; however, the metabolism resulting in enzyme inactivation is possibly tissue-specific. Furthermore, the kinetic similarities for inactivation between murine and human forms of NQO1 show these results are apropos to clinical use of MMC.Although it is clear that NAD(P)H:quinone oxidoreductase 1 (NQO1; EC 1.6.99.2) is capable of reducing the antineoplastic antibiotic mitomycin C (MMC) in vitro (Siegel et al., 1990b, the role of this enzyme in determining the toxicity of MMC remains controversial. A correlative relationship has been shown between NQO1 activity levels and sensitivity to MMC in the NCI tumor cell line panel (Fitzsimmons et al., 1996). Nevertheless, transfection and expression of NQO1 in cell lines has produced varied results (Powis et al., 1995;Belcourt et al., 1996;Gustafson et al., 1996;Winski et al., 1998), as have xenograft studies (Malkinson et al., 1992;Nishiyama et al., 1995;Phillips et al., 2001). The lack of an apparent relationship between NQO1 levels and MMC toxicity is not surprising for a number of reasons. MMC can be metabolically activated by multiple enzyme systems including NADPH:cytochrome P450 reductase (Keyes et al., 1984;Pan et al., 1984), cytochrome b 5 reductase (Hodnick and Sartorelli, 1993), xanthine oxidase/dehydrogenase (Pan et al., 1984;Gustafson and Pritsos, 1992), and probably others not yet identified. MMC bioactivation by NQO1 is pH dependent in vitro (Siegel et al., 1990b), and this pH dependence seems to be due to inactivation of NQO1 by MMC at more neutral pH. Furthermore, evidence suggests that the relationship between NQO1 levels and the toxicity of bioreductive substrates is best described by ...