The decay of
nitroxide spin label electron paramagnetic resonance (EPR) absorption intensity was used to investigate the
doxorubicin-mediated intracellular generation of
free radicals. The effects of 50-500 micrograms/ml
doxorubicin on human
tumor cells (MCF-7,
breast cancer cells, and HL-60, promyelocytic
leukemia, cells) were studied by measuring
2,2,6,6-tetramethylpiperidine-1-oxyl (
TEMPO) absorption intensity decay (TAID) at
a TEMPO concentration of 10 microM.
Doxorubicin accelerated the TAID in both cell lines with a detection limit of 50 micrograms/ml for MCF-7 cells and 500 micrograms/ml
doxorubicin for HL-60 cells. Preincubation of cells with the
iron chelating agent,
deferoxamine (5 mM), partially prevented the effects of
doxorubicin on the TAID.
Catalase and
copper,
zinc-
superoxide dismutase (Cu,Zn-SOD) had no influence on the effects of
doxorubicin on the TAID in intact cells. However, Cu,Zn-SOD completely abolished the effects of
doxorubicin on the TAID in a MCF-7 cell-free system. Our findings suggest that
doxorubicin mediates the intracellular generation of O2.- and that
iron is involved in this process.