Metabolism of mitomycin C by DT-diaphorase: role in mitomycin C-induced DNA damage and cytotoxicity in human colon carcinoma cells.
The role of DT-diaphorase in bioreductive activation of mitomycin C was examined using HT-29 and BE human carcinoma cells which have high and low levels of DT-diaphorase activity, respectively. HT-29 cells were more sensitive to mitomycin C-induced cytotoxicity than the DT-diaphorase-deficient BE cell line. Mitomycin C induced DNA interstrand cross-linking in HT-29 cells but not in BE cells. Both mitomycin C-induced cytotoxicity and induction of DNA interstrand cross-links could be inhibited by pretreatment of HT-29 cells with dicoumarol. Metabolism of mitomycin C by HT-29 cell cytosol was pH dependent and increased as the pH was lowered to 5.8, the lowest pH tested. Metabolism of mitomycin C by HT-29 cytosol was inhibited by prior boiling of cytosol or by the inclusion of dicoumarol. Little metabolism was detected in BE cytosols. When purified rat hepatic DT-diaphorase was used, metabolism of mitomycin C increased as the pH was decreased and could be detected at pH 5.8, 6.4, 7.0, 7.4, but not at 7.8. Metabolism of mitomycin C was NADH dependent and inhibited by dicoumarol or by prior boiling of enzyme. An approximate 1:1 stoichiometry between NADH and mitomycin C removal was demonstrated and no oxygen consumption could be detected. Metabolism of mitomycin C by purified HT-29 DT-diaphorase was also dicoumarol inhibitable and pH dependent. The major metabolite formed during metabolism of mitomycin C by HT-29 cytosol, purified HT-29, and rat hepatic DT-diaphorase was characterized as 2,7-diaminomitosene. These data suggest that two-electron reduction of mitomycin C by DT-diaphorase may be an important determinant of mitomycin C-induced genotoxicity and cytotoxicity.
Molecular Toxicology and Environmental Health Sciences Program, School of Pharmacy, University of Colorado, Boulder 80309.
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Siegel
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Gibson
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Preusch
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Ross
D000970:Antineoplastic Agents / Q000009:adverse effects / Q000378:metabolism*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism*; D003114:Colony-Forming Units Assay; D004249:DNA Damage / Q000187:drug effects*; D001728:Dicumarol / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D016685:Mitomycin; D008937:Mitomycins / Q000009:adverse effects / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases / Q000502:physiology*
D000970:Antineoplastic Agents; D008937:Mitomycins; D009243:NAD; D016685:Mitomycin; D001728:Dicumarol; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases