Inhibition of DNA Cross-linking by Mitomycin C by Peroxidase-mediated Oxidation of Mitomycin C Hydroquinone

Abstract

Mitomycin C requires reductive activation to cross-link DNA and express anticancer activity. Reduction of mitomycin C (40 μM) by sodium borohydride (200 μM) in 20 mM Tris-HCl, 1 mM EDTA at 37 °C, pH 7.4, gives a 50-60% yield of the reactive intermediate mitomycin C hydroquinone. The hydroquinone decays with first order kinetics or pseudo first order kinetics with a t 1/2 of ∼15 s under these conditions. The cross-linking of T7 DNA in this system followed matching kinetics, with the conversion of mitomycin C hydroquinone to leuco-aziridinomitosene appearing to be the rate-determining step. Several peroxidases were found to oxidize mitomycin C hydroquinone to mitomycin C and to block DNA cross-linking to various degrees. Concentrations of the various peroxidases that largely blocked DNA cross-linking, regenerated 10-70% mitomycin C from the reduced material. Thus, significant quantities of products other than mitomycin C were produced by the peroxidase-mediated oxidation of mitomycin C hydroquinone or products derived therefrom. Variations in the sensitivity of cells to mitomycin C have been attributed to differing levels of activating enzymes, export pumps, and DNA repair. Mitomycin C hydroquinone-oxidizing enzymes give rise to a new mechanism by which oxic/hypoxic toxicity differentials and resistance can occur.