Role of copper in the oxygen radical-mediated toxicity of the thiol-containing radioprotector dithiothreitol in mammalian cells.

The thiol radioprotector dithiothreitol (DTT) causes biphasic toxicity in V79 cells: exposure to DTT causes loss of clonogenic cell survival at intermediate concentrations of the drug, but is not toxic at lower (< 0.05 mM) or higher (> 2.0 mM) concentrations. Toxicity depends on the medium in which cells are exposed to DTT. Cell killing is less when cells are exposed to DTT in phosphate-buffered saline (PBS) than when in complete, serum-containing medium, and there is no cell killing at any drug concentration when cells are in serum-free medium. When cells are exposed to DTT in PBS containing 10% fetal calf serum (FCS) or 10% dialyzed FCS, cell killing is increased compared to the response in PBS alone, suggesting that some component(s) of serum is involved in DTT toxicity. Addition of micromolar quantities of copper as either free Cu2+ or ceruloplasmin to serum-free medium increases the toxicity of DTT, but addition of free iron or transferrin has no effect. H2O2 is produced during DTT oxidation and appears to be involved in the toxicity of DTT, because toxicity can be prevented by catalase. H2O2 is also toxic to V79 cells in a medium-dependent fashion, but the toxicity is not influenced by addition of copper, ceruloplasmin, iron or transferrin. The rate of DTT oxidation is also medium-dependent and is increased by copper or ceruloplasmin, but not by iron or transferrin. The data are consistent with the hypothesis that the toxicity of DTT is caused by the copper-catalyzed oxidation of DTT, forming H2O2 which, in turn, produces .OH, the ultimate toxic agent, via the Fenton reaction.