Influence of the antioxidant N-acetylcysteine and its metabolites on damage induced by bleomycin in PM2 bacteriophage DNA

Bleomycin is considered to be a useful model compound for studying environmental carcinogenesis, due to its broad spectrum of DNA damaging properties. In addition, bleomycin is a useful antitumor drug because of its cytotoxic properties. To investigate the influence of the antioxidant N-acetylcysteine and its metabolites glutathione and cysteine on bleomycin-induced DNA damage and more importantly to gain insight into the biological relevance of such damage. PM2 DNA was exposed to Cu²⁺-bleomycin in the presence and absence of the thiols N-acetylcysteine, glutathione and cysteine. It was found that the presence of these thiols led to a considerable enhancement of bleomycin-induced single- and double-strand breaks and a concomitant decrease in the biological activity of PM2 DNA in a dose-dependent way. A similar observation was made when ascorbic acid was used. Bleomycin showed no DNA damaging activity when PM2 DNA was pretreated with the strong Fe ion chelator desferal and its activity was strongly inhibited by the addition of CU²⁺ ions or under hypoxic (N₂) conditions. Cu²⁺-bleomycin under our conditions is not active by itself, but most probably after binding to DNA exchanges CU²⁺ for Fe³⁺ bound to DNA. Fe³⁺-bleomycin is then reduced to Fe²⁺-bleomycin, a process potentiated by the added antioxidants, and subsequently activated by O₂. The contribution to biological inactivation of bleomycin alone or in the presence of ascorbic acid is only ~15%. The contribution to lethality in the presence of thiols is higher. These results indicate that ascorbic acid only enhances the DNA damaging properties of bleomycin, whereas the thiol compounds in addition influence the type of DNA damage. The remainder of the biological inactivation is probably caused by double damage, such as single-strand breaks with closely opposed alkali-labile sites or base damage.