Reductive activation of mitomycin C by NADH:cytochrome b5 reductase.
Mitomycin C requires bioreduction in order to exert its cytotoxic action. Activation of mitomycin C to an electrophile was equally supported by NADPH and NADH in EMT6 tumor cell sonicates under hypoxia. No alkylation was observed under aerobic conditions. Purified NADH:cytochrome b5 reductase catalyzed the reduction of mitomycin C with a Km of 23 microM at pH 6.6. At pH 7.6, the rate of enzymatic reduction of mitomycin C was 61% of that at pH 6.6. NADH:cytochrome b5 reductase catalyzed the activation of mitomycin C to alkylating metabolites under both hypoxic and aerobic conditions, with alkylation being 1.5 times greater in hypoxia. Dicumarol at 100 microM inhibited the NADH:cytochrome b5 reductase-catalyzed reduction of mitomycin C by 24% and by 57% at 300 microM. The degree of inhibition of the enzyme by dicumarol was the same at both pH 6.6 and 7.6. NADH:cytochrome b5 reductase exhibited a small but measurable NADH-oxidase activity, which was unaffected by 300 microM dicumarol. These findings demonstrate that (a) NADH:cytochrome b5 reductase can metabolically activate mitomycin C and (b) dicumarol is capable of inhibiting this enzymatic activity.
Hodnick
W F
WF
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
Sartorelli
A C
AC
D000332:Aerobiosis; D000478:Alkylation; D000818:Animals; D001711:Biotransformation; D015687:Cell Hypoxia; D002460:Cell Line; D003579:Cytochrome Reductases / Q000302:isolation & purification / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D004912:Erythrocytes / Q000201:enzymology; D007700:Kinetics; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism; D051379:Mice; D016685:Mitomycin / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011817:Rabbits; D014407:Tumor Cells, Cultured
D009243:NAD; D016685:Mitomycin; D009249:NADP; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase