Isolation and identification of the adducts of mitomycin C and porfiromycin with DNA formed in vitro and in vivo

The antitumor antibiotics, mitomycin C (MC) and porfiromycin (PM), are shown to form covalent complexes with DNA in vitro, under reductive activation conditions (both chemical and enzymatic). Three major covalent adducts have been isolated and identified as (1) N2-guanine adduct with MC (structure 4a), (2) N2-guanine adduct with 10-decarbamoyl mitomycin [(10-DMC); structure 16a], and a bisadduct of MC linked to two Gs at their N2-positions (structure 6). The adducts of PM with DNA formed in vitro are analogous (structures 19, 20, and 21). Formation of adducts 6 and 16a in CHO mammalian cells has been shown after exposing them to MC or 10-DMC, whereas formation of crosslink 6 in vivo has been demonstrated after injecting rats with MC. The experiments done in tissue cultures with [1a-3H]-polyfiromycin show [3H]-label in the unmodified A, G, and T thus suggesting the demethylation of PM to MC in cells. The methyl group containing [3H] label was incorporated into nucleosides via de novo purine and thymidylate biosynthesis. A consolidated enzymatic scheme for the hydrolysis of MC-modified DNA has been established and the resistance of such DNA to cleavage by several nucleases has been shown. Thus, only DNase I/SVD/alkaline phosphatase or nuclease P1/SVD/alkaline phosphatase combinations can degrade MC-modified DNA into nucleosides. A modified version of 32P-postlabeling has been developed with in vitro authentic standards and this can be conveniently used in the future to detect MC-modified lesions obtained in vivo. By utilizing the alkaline ethidium bromide fluorescence assay, the crosslinking effect of MC, PM, and 10-DMC has been shown to occur in cells