Mitochondrial DNA damage by bleomycin.

We have modified a specific and sensitive method of detecting different forms of mitochondrial DNA (mtDNA) and utilized it to study bleomycin (BLM)-induced mtDNA damage. Intact, nicked circular, and linear forms of mtDNA were separated by gel electrophoresis, detected by Southern blot hybridization, and characterized by size markers and alkali treatment. DNA from BLM-treated mitochondria from liver, lung, and L1210 tumors were all equally sensitive to damage by BLM. The extent of BLM-induced mtDNA damage was dependent on experimental conditions. In vitro incubation of isolated mitochondria with BLM showed that 100 microM BLM caused complete conversion of intact to nicked and linear forms. Scission of mtDNA was more extensive if mitochondria were lysed in the presence of BLM than after several washings to remove the drug from the incubation media. Isolated mtDNA was extremely sensitive to BLM such that 10 nM BLM caused loss of intact form. The extent of mtDNA damage by BLM was decreased by the addition of EDTA.