Ascorbate both activates and inactivates bleomycin by free radical generation

Abstract

ABSTRACT:The chemotherapeutic agent bleomycin (BLM) is activated by reducing agents to break isolated DNA. Paradoxically, these same reducing agents protect cellular DNA from BLM damage. To resolve this paradox, we have examined the reaction of FelIIBLM with DNA in the presence of ascorbate. As expected, ascorbate augments FeII'BLM-induced DNA damage. However, when ascorbate is added to FelIIBLM prior to exposure to DNA, a redox-inactive BLM is produced in a reaction that generates the ascorbyl radical. This reaction occurs in both ascorbate-supplemented buffer and unsupplemented plasma. In buffered solution, this reaction was found to be stoichiometric; for each mole of BLM present, 6.9 mol of ascorbate was oxidized and 4.7 mol of oxygen was consumed. Iron was found to serve only as a catalyst for the reaction. These data suggest that both activation of BLM and the generation of redox-inactive BLM occur via the same reaction and that BLM-induced DNA damage depends upon BLM reaching DNA prior to its interaction with reducing agents.

Oxidative cell wall damage mediated by bleomycin-Fe(II) in Saccharomyces cerevisiae

“…The cells were then washed and assayed for rate of spheroplast formation. the reaction with DNA, the ascorbate did not activate a BLM complex with Fe(III) (4,40). ␤-Mercaptoethanol, another reductant, also inhibited BLM-Fe(II)-O 2 -mediated cell wall damage (data not shown).…”

“…We tested two classes of effectors for their effect on BLM-Fe(II)-O 2 -mediated cell wall damage. Reducing agents such as ascorbate protect against BLM in cellular cytotoxicity assays (4,35). In the presence of DNA, ascorbate can initiate BLM-induced strand scission in the presence of Fe(II) or Fe(III), whereas in the absence of DNA, ascorbate reacts with BLM to produce the ascorbyl radical and a redox-inactive BLM incapable of nicking DNA.…”

Vitamin C as a Modulator of the Response to Cancer Therapy

“…At the same time, because cellular reducing factors such as AA can scavenge free radicals and other oxidative species, it has been assumed that such reducing agents would protect cells by suppressing the oxidative processes initiated by bleomycin. Buettner et al demonstrated that both phenomena depend upon whether bleomycin reaching DNA before (DNA damage effect) or after (free radicals scavenging) its interaction with reducing agents [77]. The authors demonstrated that in the presence of DNA, AA initiated bleomycin-induced strand breaks whereas in the absence of DNA, AA reacted with bleomycin to produce the ascorbyl radical and a redox-inactive bleomycin that was incapable of nicking DNA.…”

Vitamin C increases DNA breaks and suppresses DNA damage-independent activation of ATM by bleomycin

“…The main cellular thiol glutathione (GSH) was noted as a weak activator of DNA nicking by BLM-iron [5]. Ascorbate (Asc), the reduced form of vitamin C, is known as an excellent reducer of Fe(III) and its addition to BLM-Fe(III) reactions stimulated DNA cleavage [14,15]. Although BLM has been extensively used as a tool for the induction of DSBs in cultured cells, how different cellular reducers contribute to the activation of BLM is poorly understood.…”