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

To compare the actions of bleomycin and ionizing radiation on DNA regarding the formation of malonaldehyde-like products.

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.

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.

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.