Effect of pH on DNA alkylation by enzyme-activated mitomycin C and porfiromycin.
DNA adduct formation by enzyme-activated antibiotics, mitomycin C (MMC) or porfiromycin (PFM), at pH 7.6 or pH 6.0 under anaerobic conditions was analyzed by a 32P-postlabeling method. Antibiotic activation by rat liver NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and bovine milk xanthine oxidase (EC 1.2.3.2) produced similar results. Five 32P-labeled MMC adducts were separated by thin layer chromatography and high performance liquid chromatography from DNA alkylated at either pH. Four of the radioactive spots separated by thin layer chromatography were identified as two monofunctional monoadducts [1" alpha and 1" beta forms of N2-(2" beta,7"-diaminomitosen-1"-yl)-2'-deoxyguanylic acid], one bifunctional monoadduct [N2-(10"-decarbamoyl-2",7"-diaminomitosen-1" alpha-yl)-2'-deoxyguanylic acid], and one cross-linked adduct [N2-(2" beta,7"-diamino-10"-deoxyguanyl-N2-yl-mitosen- 1" alpha-yl)-2'-deoxyguanylic acid]. One minor radioactive spot was not identified. By comparing DNA alkylated at the two pH values, based on equal amounts of 32P radioactivity, similar amounts of cross-links were detected. However, the DNA showed different ratios of the alpha and beta isomers of the monofunctional monoadduct. Furthermore, the DNA alkylated at pH 6.0 showed more bifunctional monoadducts than did the DNA alkylated at pH 7.6. Analysis of alkylated DNA by enzyme-activated PFM showed a similar spectrum of DNA adduct formation. The effect of pH on the distribution of the five PFM-DNA adducts was similar to that observed for the five MMC-DNA adducts. The distribution of adducts in DNA alkylated at the same pH was similar irrespective of which enzyme activated MMC or PFM. The pH of the reaction during DNA and MMC interaction was the determining factor for the quantitative distribution of the adducts. This pH effect may be important for the cytotoxicity of MMC and PFM in tumor cells that have high levels of reductive enzymes with low optimal pH values.
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Division of Developmental Therapeutics, University of Maryland Cancer Center, Baltimore 21201.
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D000478:Alkylation; D000818:Animals; D001711:Biotransformation; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004247:DNA / Q000378:metabolism*; D018736:DNA Adducts; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology; D016685:Mitomycin / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011160:Porfiromycin / Q000378:metabolism*; D051381:Rats; D014969:Xanthine Oxidase / Q000378:metabolism
D018736:DNA Adducts; C056417:mitomycin C-DNA adduct; C081800:porfiromycin-DNA adduct; D016685:Mitomycin; D004247:DNA; D014969:Xanthine Oxidase; D009251:NADPH-Ferrihemoprotein Reductase; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; D011160:Porfiromycin