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Effect of pH on DNA alkylation by enzyme-activated mitomycin C and porfiromycin
1Division of Developmental Therapeutics, University of Maryland Cancer Center, Baltimore 21201.
Abstract:
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.
Insights
Enzyme-activated antibiotics mitomycin C and porfiromycin form DNA adducts. Reaction pH significantly influences the distribution of these adducts, impacting drug cytotoxicity in tumor cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Mitomycin C (MMC) and porfiromycin (PFM) are enzyme-activated antibiotics.
- These drugs form DNA adducts, crucial for their cytotoxic effects.
- Understanding adduct formation is key to optimizing cancer therapy.
Purpose of the Study:
- To analyze DNA adduct formation by enzyme-activated MMC and PFM.
- To investigate the influence of pH on the types and distribution of DNA adducts.
- To correlate adduct formation with potential drug cytotoxicity.
Main Methods:
- Utilized a 32P-postlabeling method to detect and quantify DNA adducts.
- Employed rat liver NADPH-cytochrome P-450 reductase and bovine milk xanthine oxidase for antibiotic activation.
- Separated and identified adducts using thin layer chromatography and high-performance liquid chromatography.
Main Results:
- Identified five distinct MMC-DNA adducts, including monofunctional, bifunctional, and cross-linked adducts.
- Found that pH 6.0 led to a higher proportion of bifunctional monoadducts compared to pH 7.6.
- Observed similar adduct profiles for PFM, with pH being the primary determinant of adduct distribution.
- The choice of activating enzyme did not significantly alter adduct distribution at a given pH.
Conclusions:
- The pH of the reaction environment is a critical factor in determining the quantitative distribution of DNA adducts formed by MMC and PFM.
- This pH-dependent adduct formation may significantly influence the cytotoxicity of these drugs, particularly in tumor cells with reductive enzyme activity at lower pH optima.