Bleomycin versus OH-radical-induced malonaldehydic-product formation in DNA

Purpose: To compare the actions of bleomycin and ionizing radiation on DNA regarding the formation of malonaldehyde-like products. Materials and methods: Calf thymus DNA was treated with iron/bleomycin or gamma-radiation at pH 7. Products Mere analysed by HPLC. The thiobarbituric-acid reactivity of the samples was determined directly or after HPLC by post-column derivatization. ESI mass spectra were taken on-line following HPLC. Results: Malonaldehyde and malonaldehyde-like products as detected by the sensitive 2-thiobarbituric acid (TBA) assay are formed in gamma-irradiated DNA and thymidine solutions as well as upon treatment of DNA with bleomycin/iron. In gamma-irradiated DNA solutions in the presence of oxygen, no base propenals were detected, and the major TEA-active product was malonaldehyde. In the gamma-radiolysis of thymidine, thymine propenal was formed only in traces (not more than 0.07 per cent of the OH-radical yield). Malonaldehyde was practically absent after treatment with bleomycin; three other TEA-active products were seen by HPLC which have been identified as the cytosine thymine and adenine propenals. Guanine propenal was not detected under our conditions. Conclusions: The absence of these base propenals upon gamma-radiolysis implies that although the initiating step of OH-radical and bleomycin action [i.e. H-abstraction at C(4')] may be the same, the bleomycin-iron complex must participate in subsequent steps en route to the base propenals. It is proposed that the bleomycin pathway may involve the interaction of the C(4')-peroxyl radical with the 'spent' bleomycin-iron complex by ligand exchange, under formation of a bleomycin-iron-peroxyl-radical complex, Blm(Fe(4+), (.)OOR), which then decomposes by heterolysis into the alkoxy cation precursor (+)OR of the base propenal and reconstitution of the bleomycin-iron complex Blm(Fe, O)(3+), i.e. gives rise to base propenal formation without the involvement of a C(4')-hydroperoxide.