Formation of free radicals and protein mixed disulfides in rat red cells exposed to dapsone hydroxylamine

The hemolytic activity of dapsone is well known to reside in its N- hydroxylamine metabolites. Addition of dapsone hydroxylamine (DDS-NOH) to red cell suspensions causes damage such that when reintroduced into the circulation of isologous rats, the injured cells are rapidly removed by the spleen. Hemolytic activity is associated with the extensive formation of disulfide-linked hemoglobin adducts on red cell membrane skeletal proteins. To determine if free radicals could be involved in this process, rat red cells were incubated with DDS-NOH in the presence of the spin trap, 5,5'- dimethyl-1-pyrroline-N-oxide (DMPO) and subjected to EPR analysis. Addition of DDS-NOH (25-50 μM) to a red cell suspension gave rise to a four-line (1:2:2:1) EPR spectrum with coupling constants identical to those of a DMPO- hydroxyl radical adduct (DMPO-OH) standard. No other radicals were detected; however, preincubation of red cells with cysteamine caused the DDS-NOH- generated DMPO-OH signal to be replaced by a cysteamine thiyl radical adduct signal. DDS-NOH-treated red cells were also found to contain ferrylhemoglobin, indicating the presence of hydrogen peroxide. Furthermore, DDS-NOH was found to stimulate salicylate hydroxylation in red cell suspensions, confirming the presence of oxygen radicals. These data support the hypothesis that oxygen radicals are involved in the mechanism underlying dapsone-induced hemolytic anemia.