IDENTIFICATION OF FREE-RADICALS PRODUCED IN RAT ERYTHROCYTES EXPOSED TO HEMOLYTIC CONCENTRATIONS OF PHENYLHYDROXYLAMINE

Previous studies have shown that incubation of rat red blood cells in vitro with phenylhydroxylamine (50-300 mu M) induces rapid splenic seq uestration of the red cells on reintroduction to isologous rats. EPR a nd the spin trapping agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), w ere utilized to determine if free radical species could be identified under these experimental conditions. Hemolytic concentrations of pheny lhydroxylamine, in the presence of DMPO and 5-20% lysed or intact rat erythrocyte suspensions, gave rise to a four-line (1:2:2:1) EPR spectr um. No signal was obtained if phenylhydroxylamine, DMPO, or red cells was omitted. Comparison of the phenylhydroxylamine-induced signal with authentic hydroxyl radical- and GSH thiyl radical-DMPO standard adduc t signals identified the phenylhydroxylamine-induced species as a GSH thiyl free radical. Removal of GSH from a red cell lysate abolished th e GSH thiyl radical signal without the appearance of any other signal, while addition of exogenous GSH resulted in its return. When erythroc ytes were exposed to concentrations of phenylhydroxylamine greater tha n or equal to 200 mu M, a time-dependent transition of the GSH thiyl r adical signal to a hemoglobin thiyl radical signal was observed. The d ata are consistent with the postulate that thiyl radical species, gene rated from the interaction of phenylhydroxylamine and oxyhemoglobin, p lay a key role in the development of hemolytic injury to the rat red c ell.