In vivo thiyl free radical formation from hemoglobin following administration of hydroperoxides

Although free radical formation due to the reaction between red blood cells and organic hydroperoxides in vitro has been well documented, the analogous in vivo ESR spectroscopic evidence for free radical formation has yet to be reported. We successfully employed ESR to detect the formation of the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)/hemoglobin thiyl free radical adduct in the blood of rats dosed with DMPO and tert-butyl hydroperoxide, cumene hydroperoxide, ethyl hydrogen peroxide, 2-butanone hydroperoxide, 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid, or hydrogen peroxide. We found that pretreating the rats with either buthionine sulfoximine or diethylmaleate prior to dosing with tert-butyl hydroperoxide decreased the concentration of nonprotein thiols within the red blood cells and significantly enhanced the DMPO/hemoglobin thiyl radical adduct concentration. Finally, we found that pretreating rats with the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea prior to dosing with tert-butyl hydroperoxide enhanced the DMPO/hemoglobin thiyl radical adduct concentration and induced the greatest decrease in nonprotein thiol concentration within the red blood cells.