Bioreductive activation of mitomycin C by DT-diaphorase.
The role of DT-diaphorase (DTD, EC 1.6.99.2) in the bioreductive activation of mitomycin C was examined using purified rat hepatic DTD. The formation of adducts with reduced glutathione (GSH), binding of [3H]mitomycin C to DNA, and mitomycin C-induced DNA interstrand cross-linking were used as indicators of bioactivation. Mitomycin C was metabolized by DTD in a pH-dependent manner with increasing amounts of metabolism observed as the pH was decreased from 7.8 to 5.8. The major metabolite observed during DTD-mediated reduction of mitomycin C was 2,7-diaminomitosene. GSH adduct formation, binding of [3H]mitomycin C and mitomycin C-induced DNA interstrand cross-linking were observed during DTD-mediated metabolism. In agreement with the pH dependence of metabolism, increased bioactivation was observed at lower pH values. Temporal studies and experiments using authentic material showed that 2,7-diaminomitosene could be further metabolized by DTD resulting in the formation of mitosene adducts with GSH. DNA cross-linking during either chemical (sodium borohydride) or enzymatic (DTD) mediated reduction of mitomycin C could be observed at pH 7.4, but it increased as the pH was decreased to 5.8, showing the critical role of pH in the cross-linking process. These data provide unequivocal evidence that the obligate two-electron reductase DTD can bioactivate mitomycin C to reactive species which can form adducts with GSH and DNA and induce DNA cross-linking. The use of mitomycin C may be a viable approach to the therapy of tumors high in DTD activity, particularly when combined with strategies to lower tumor pH.
Siegel
D
D
Molecular Toxicology and Environmental Health Sciences Program, School of Pharmacy, University of Colorado Health Sciences Center, Denver 80262.
Beall
H
H
Senekowitsch
C
C
Kasai
M
M
Arai
H
H
Gibson
N W
NW
Ross
D
D
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D016685:Mitomycin / Q000378:metabolism*; D008937:Mitomycins / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains
D008937:Mitomycins; D009243:NAD; D016685:Mitomycin; C056707:2,7-diaminomitosene; D004247:DNA; D016660:NAD(P)H Dehydrogenase (Quinone); D005978:Glutathione