Redox cycling of bleomycin-Fe(III) and DNA degradation by isolated NADH-cytochrome b5 reductase: involvement of cytochrome b5

Isolated and purified microsomal NADH-cytochrome b5 reductase (EC 1.6.2.2) was incubated with bleomycin (BLM) and FeCl3 in the presence of NADH. Only when purified cytochrome b5 was added could an increased NADH consumption be observed indicating redox cycling of the BLM-Fe(III) complex. In the presence of DNA, BLM-Fe(III)-related NADH consumption was accompanied by malondialdehyde (MDA) formation, further evidence for BLM activation yielding oxidative DNA cleavage. BLM, FeCl3, cytochrome b5 and NADH were absolutely necessary to provide these effects. Addition of DNA changed the initial velocity (V0) and the shape of the NADH consumption curves, both probably due to an interaction between DNA and BLM-Fe(III). Furthermore, DNA effectively protected BLM-Fe(III) from autoxidative degradation during redox cycling. BLM-Fe(III)-related, reductase-catalyzed NADH consumption and MDA formation were also dependent on oxygen, showing the involvement of oxygen in the reduction process and in the action of the drug-metal complex in attacking DNA. However, superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) did not affect NADH consumption. Also, superoxide dismutase and catalase were almost without influence on MDA formation, suggesting that no free (or freely accessible) reactive oxygen species occurred during the redox cycle and DNA damage. The results reveal that the BLM-Fe(III) complex undergoes redox cycling by the microsomal NADH-dependent cytochrome b5 reductase-cytochrome b5 system. The significance of this effect for the action of BLM and the involvement of cytochrome b5 is discussed with regard to the presence of these enzymes in the cell nucleus.