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Detection of Mitomycin C-DNA Adducts in Vivo by ³²P-Postlabeling: Time Course for Formation and Removal of Adducts and Biochemical Modulation¹

Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755-3564 [A. J. W., J. W. H.]; Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755-3835 [A. J. W., J. W. H.]; Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire 03756-0001 [J. W. H.]; and Department of Experimental Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263 [A. E. M.]

Mitomycin C (MMC) is a DNA cross-linking agent that has been used in cancer chemotherapy for over 20 years, yet little is known either qualitatively or quantitatively about MMC-induced DNA adduct formation and repair in vivo. As an initial means of investigating this, we used a recently developed ³²P-postlabeling assay to examine the formation and loss of MMC-DNA adducts in the tissues of a simple in vivo model test system, the chick embryo, following treatment with a chemotherapeutic dose of MMC. As early as 15 min after MMC treatment, four adducts could be detected in the liver which were tentatively identified as the (CpG) N²G-MMC-N²G interstrand cross-link, the bifunctionally activated MMC-N²G monoadduct, and two isomers ( and ß) of the monofunctionally activated MMC-N²G monoadduct. The (GpG) N²G-MMC-N²G intrastrand cross-link appears to be a poor substrate for nuclease P1 and/or T4 kinase and was not evaluable by this assay. Levels of all four detectable adducts increased substantially within the first 2 h after MMC treatment, reached maximal levels by 6 h, and decreased progressively thereafter through 24 h, although low levels of certain adducts persisted beyond 24 h. Lung and kidney had comparable levels of total MMC adducts, which were approximately 60% those of the liver, and there were no significant differences in the proportion of specific adducts among the three tissues. The interstrand cross-link represented 13–14% of the total MMC adducts, which is approximately 5-fold greater than the proportion of CpG sites in the genome. In addition, the interstrand cross-link was selectively decreased after 16 h relative to the three monoadducts, suggesting preferential repair. The effect of modulating different components of the Phase I and Phase II drug metabolism on MMC adduct formation, using either glutethimide, 3,4,3',4'-tetrachlorobiphenyl, dexamethasone, buthionine sulfoximine, ethacrynic acid, or N-acetylcysteine pretreatments, was examined to characterize the possible pathways of MMC metabolism and adduct formation in vivo. Surprisingly, none of these pretreatments had a significant effect on individual or total adducts with the exception of dexamethasone, which caused an almost 2-fold proportional increase in all four adducts in the liver.

¹ This work was supported by Grant CA49002 from the National Cancer Institute, NIH (to J. W. H.), grants from the Norris Cotton Cancer Center and the Hitchcock Foundation, and Grant CH-526 (to A. E. M.) from the American Cancer Society. J. W. H. was also partially supported by the Norris Cotton Cancer Center, and A. J. W. was partially supported by the Dartmouth College Chemistry Department. Support for the Dartmouth College Molecular Biology Core Facility was provided by the Norris Cotton Cancer Center Core Grant CA23108.

² To whom requests for reprints should be addressed, at Department of Pharmacology and Toxicology, Dartmouth Medical School, 7650 Remsen, Hanover, NH 03755-3835. Phone: (603) 650-1316; Fax: (603) 650-1129; E-mail: josh.hamilton@dartmouth.edu.

Received 7/16/97. Accepted 11/25/97.

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