Free radicals induced by adriamycin-sensitive and adriamycin-resistant cells: a spin-trapping study

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Abstract
The radicals generated by adriamycin-sensitive (CHO-AB) and adriamycin-resistant (CHO-C5) Chinese hamster ovary cells as well as by adriamycin-sensitive and -resistant human breast cancer cells (MCF7- WT and MCF7-ADR) have been studied with spin-trapping and ESR spectroscopy. During anoxic exposure to adriamycin (ADR) both pairs of cell lines produced the broad ESR singlet characteristic of ADR semiquinone (AQ.). By use of tris(oxalato)chromate (CrOx) as an extracellular line-broadening agent, the distribution of AQ. between the intra- and extracellular compartments was studied. For cell densities of (1-3) .times. 107 cells/mL, CrOx eliminated most, though not all, of the ESR signal, indicating that the AQ. radicals freely diffuse and partition between the intra- and extracellular compartments proportionally to their respective volumes. Similar behavior was exhibited by all four cell lines studied. Upon introduction of oxygen to anoxic cells in the presence of the spin trap 5,5-dimethylpyrroline N-oxide (DMPO) the AQ. signal was replaced by that of the DMPO-OH spin adduct. Metal chelators such as desferroxamine had no effect on DMPO-OH or AQ. formation. Superoxide dismutase, not catalase, totally eliminated the ESR signal, indicating that DMPO-OH produced by ADR-treated cells originates from superoxide rather than from .OH produced from H2O2. In the presence of CrOx, the DMPO-OH signal was not distinguishable from the background noise, thus excluding any contribution to the signal by intracellular spin adducts. Without excluding a possible role for intracellular O2.- and .OH radicals, the present results show that AQ. radicals partition predominantly outside the cell and react there with oxygen to yield extracellular superoxide. Since ADR-resistant and -sensitive cell lines generated comparable levels of the radicals, there is little support for the assumption that ESR-observable oxygen-derived radicals play a role in ADR antitumor activity.

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