DETECTION OF MITOMYCIN C-DNA ADDUCTS IN-VIVO BY P-32 POSTLABELING - TIME COARSE FOR FORMATION AND REMOVAL OF ADDUCTS AND BIOCHEMICAL MODULATION

Mitomycin C (MMC) is a DNA cross-linking agent that has been used in c ancer chemotherapy for over 20 years, yet little is known either quali tatively or quantitatively about MMC-induced DNA adduct formation and repair in vivo. As an initial means of investigating this, we used a r ecently developed P-32-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 cou ld he detected in the liver which were tentatively identified as the ( CpG) N(2)G-MMC-N(2)G interstrand cross-link, the bifunctionally activa ted MMC-N(2)G monoadduct, and two isomers (alpha and beta) of the mono functionally activated MMC-N(2)G monoadduct. The (GpG) N(2)G-MMC-N(2)G 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 f our detectable adducts increased substantially within the first 2 h af ter MMC treatment, reached maximal levels by 6 h, and decreased progre ssively thereafter through 24 h, although low levels of certain adduct s persisted beyond 24 h, Lung and kidney had comparable levels of tota l MMC adducts, which were approximately 60% those of the liver, and th ere were no significant differences in the proportion of specific addu cts among the three tissues. The interstrand cross-link represented si milar to 13-14% of the total MMC adducts, which is approximately 5-fol d greater than the proportion of CpG sites in the genome. In addition, the interstrand cross-link was selectively decreased after 16 h relat ive to the three monoadducts, suggesting preferential repair, The effe ct 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, ethacr ynic acid, or N-acetylcysteine pretreatments, was examined to characte rize the possible pathways of MMC metabolism and adduct formation in v ivo. Surprisingly, none of these pretreatments had a significant effec t on individual or total adducts with the exception of dexamethasone, which caused an almost 2-fold proportional increase in all four adduct s in the liver.