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

Abstract

Purpose: To compare the actions of bleomycin and ionizing radiation on DNA regarding the formation of malonaldehydelike products. Materials and methods: Calf thymus DNA was treated with iron/bleomycin or gamma -radiation at pH 7. Products were 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 TBA-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 TBA-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(Fe4, 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.