Sandra J. Jordan

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.

Generation of nitro radical anions of some 5-nitrofurans, and 2- and 5-nitroimidazoles by rat hepatocytes.

Nitrofurantoin, nifurtimox, nifuroxime, nitrofurazone, misonidazole, benznidazole, ronidazole, and ornidazole were reduced to their respective nitro radical anions by intact rat hepatocytes at pH 7.4. The nitrofurantoin radical anion and other nitro anion radicals generated inside these cells were detected with ESR spectroscopy. Broadening of the signals from nitrofurantoin anion radicals was accomplished with paramagnetic transition metals, implying that the radicals were outside the cell in the medium. Rat hepatocytes are well suited for in situ electron spin resonance investigations of free radical metabolites and represent a model for the as yet unobtained direct detection of free radical metabolites in liver.

Fatty acid radical formation in rats administered oxidized fatty acids: In vivo spin trapping investigation

We report in vivo evidence for fatty acid-derived free radical metabolite formation in bile of rats dosed with spin traps and oxidized polyunsaturated fatty acids (PUFA). When rats were dosed with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and oxidized PUFA, the DMPO thiyl radical adduct was formed due to a reaction between oxidized PUFA and/or its metabolites with biliary glutathione. In vitro experiments were performed to determine the conditions necessary for the elimination of radical adduct formation by ex vivo reactions. Fatty acid-derived radical adducts of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) were detected in vivo in bile samples collected into a mixture of iodoacetamide, desferrioxamine, and glutathione peroxidase. Upon the administration of oxidized 13C-algal fatty acids and 4-POBN, the EPR spectrum of the radical adducts present in the bile exhibited hyperfine couplings due to 13C. Our data demonstrate that the carbon-centered radical adducts observed in in vivo experiments are unequivocally derived from oxidized PUFA. This in vivo evidence for PUFA-derived free radical formation supports the proposal that processes involving free radicals may be the molecular basis for the previously described cytotoxicity of dietary oxidized PUFA.

Nitric oxide formation during light-induced decomposition of phenyl N-tert-butylnitrone

Phenyl N-tert-butylnitrone (PBN) is a spin trap commonly employed in free radical research. PBN has been shown to have adverse and beneficial effects on various biological systems. We report here evidence that photolysis (or even ambient light) decomposes PBN to nitric oxide in aqueous solutions. Non-heme and heme proteins have been employed to form nitrosyl complexes, which were detected using EPR spectroscopy. Concomitantly, nitrite formation was detected after light-induced decomposition of PBN. In addition, we found that tert-nitrosobutane and decomposed PBN caused an activation of guanylate cyclase. We propose a mechanism where PBN is decomposed by light to tert-nitrosobutane. The latter compound is, in turn, decomposed to nitric oxide. This study suggests the possibility that PBN or PBN radical adducts may be sources of nitric oxide in biological environments. When using PBN as a spin trap in biological samples, not only is the trapping of reactive free radicals operative, but nitric oxide produced from PBN decomposition may play an important role in altering biological functions.