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-hyd roxylamine metabolites. Addition of dapsone hydroxylamine (DDS-NOH) to red cell suspensions causes damage such that when reintroduced into t he circulation of isologous rats, the injured cells are rapidly remove d by the spleen. Hemolytic activity is associated with the extensive f ormation of disulfide-linked hemoglobin adducts on red cell membrane s keletal proteins. To determine if free radicals could be involved in t his 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 subjec ted to EPR analysis. Addition of DDS-NOH (25-50 mu M) to a red cell su spension 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 sign al to be replaced by a cysteamine thiyl radical adduct signal. DDS-NOH -treated red cells were also found to contain ferrylhemoglobin, indica ting the presence of hydrogen peroxide. Furthermore, DDS-NOH was found to stimulate salicylate hydroxylation in red cell suspensions, confir ming the presence of oxygen radicals. These data support the hypothesi s that oxygen radicals are involved in the mechanism underlying dapson e-induced hemolytic anemia. (C) 1997 Elsevier Science Inc.