ENDOGENOUS INTRACELLULAR GLUTATHIONYL RADICALS ARE GENERATED IN NEUROBLASTOMA-CELLS UNDER HYDROGEN-PEROXIDE OXIDATIVE STRESS

We report the detection of endogenous intracellular glutathionyl (GS(. )) radicals in the intact neuroblastoma cell line NCB-20 under oxidati ve stress. Spin-trapping and electron paramagnetic resonance (EPR) spe ctroscopic methods were used for monitoring the radicals. The cells in cubated with the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) wer e challenged with H2O2 generated by the enzymic reaction of glucose/gl ucose oxidase. These cells exhibit the EPR spectrum of the GS(.) radic al adduct of DMPO (DMPO-(.)SG) without exogenous reduced glutathione ( GSH). The identity of this radical adduct was confirmed by observing h yperfine coupling constants identical to previously reported values in in vitro studies, which utilized known enzymic reactions, such as hor seradish peroxidase and Cu/Zn superoxide dismutase, with GSH and H2O2 as substrates. The formation of the GS(.) radicals required viable cel ls and continuous biosynthesis of GSH. No significant effect on the re sonance amplitude by the addition of a membrane-impermeable paramagnet ic broadening agent indicated that these radicals were located inside the intact cell. N-Acetyl-L-cysteine (NAC)-treated cells produced NAC derived free radicals (NAC(.)) in place of GS(.) radicals, The time co urse studies showed that DMPO-(.)SG formation exhibited a large increa se in its concentration after a lag period, whereas DMPO-NAGC(.) forma tion from NAG-treated cells did not show this sudden increase. These r esults were discussed in terms of the limit of antioxidant enzyme defe nses in cells and the potential role of the GS(.) radical burst in act ivation of the transcription nuclear factor NF-kappa B in response to oxidative stress.