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 condition s was analyzed by a P-32-postlabeling method. Antibiotic activation by rat liver NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and bovine mi lk xanthine oxidase (EC 1.2.3.2) produced similar results. Five P-32-l abeled MMC adducts were separated by thin layer chromatography and hig h performance liquid chromatography from DNA alkylated at either pH. F our of the radioactive spots separated by thin layer chromatography we re identified as two monofunctional monoadducts [1 ''alpha and 1 ''bet a forms of N2-(2''beta,7''-diaminomitosen-1 ''-yl)-2'-deoxyguanylic ac id], one bifunctional monoadduct ',7''-diaminomitosen-1''alpha-yl)-2'- deoxyguanylic acid], and one cross-linked adduct [N2-(2'' guanyl-N2-yl -mitosen-1''alpha-yl)-2'-deoxyguanylic acid]. One minor radioactive sp ot was not identified. By comparing DNA alkylated at the two pH values , based on equal amounts of P-32 radioactivity, similar amounts of cro ss-links were detected. However, the DNA showed different ratios of th e alpha and beta isomers of the monofunctional monoadduct. Furthermore , the DNA alkylated at pH 6.0 showed more bifunctional monoadducts tha n 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 simil ar to that observed for the five MMC-DNA adducts. The distribution of adducts in DNA alkylated at the same pH was similar irrespective of wh ich enzyme activated MMC or PFM. The pH of the reaction during DNA and MMC interaction was the determining factor for the quantitative distr ibution of the adducts. This pH effect may be important for the cytoto xicity of MMC and PFM in tumor cells that have high levels of reductiv e enzymes with low optimal pH values.