DNA alkylation by enzyme-activated mitomycin C.
After anaerobic reductive activation by either NADPH cytochrome P-450 reductase (EC 1.6.2.4) or xanthine oxidase (EC 1.2.3.2), mitomycin C readily alkylated DNA. When the mitomycin C-alkylated DNA is digested by DNase, snake venom phosphodiasterase, and alkaline phosphatase, only partial release of the monofunctionally linked mitomycin C nucleotide adduct occurs. Cross-linked adducts are not released into dinucleotides but resist nuclease digestion and remain in oligonucleotides and insoluble precipitates. Kinetic analyses show that the nuclease-resistant fraction which is indicative of DNA cross-linking by mitomycin C takes place quite readily. This nuclease-resistant fraction is particularly significant when the amount of total bound mitomycin C is less than 15 mumol/mmol of DNA. The cross-linked mitomycin C product accounts for more than half of the total alkylation under all pH conditions tested. Our data suggest that particular DNA sites are available for DNA cross-linking by mitomycin C, and these sites are probably the preferred and immediate alkylating targets. Furthermore, DNA cross-links by mitomycin C are not the secondary product of monofunctional adducts. Activity of both flavoenzymes is pH dependent, hence, mitomycin C activation and the rate of DNA alkylation are pH dependent. At elevated mitomycin C alkylation of DNA, the highest amount of cross-linking occurs at neutral pH. High pressure liquid chromatographic separation of the nuclease-digested DNA detected one major and two less prominent mitomycin C adducts. These were verified to be mononucleotide mitosene types by UV spectra showing maximum absorbance at 312 and 250 nm. The major adduct was purified and identified as O6-(2'-deoxyguanosyl)-2,7-diaminomitosene by NMR, indicating that the O6 position of guanine is a preferred site in DNA for at least monofunctional linkage formation.
D000477:Alkylating Agents; D000818:Animals; D001711:Biotransformation; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004247:DNA; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016685:Mitomycin; D008937:Mitomycins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D013057:Spectrum Analysis; D014969:Xanthine Oxidase / Q000378:metabolism
D000477:Alkylating Agents; D008937:Mitomycins; D016685:Mitomycin; D004247:DNA; D014969:Xanthine Oxidase; D009251:NADPH-Ferrihemoprotein Reductase