Glutathione propagates oxidative stress triggered by Myeloperoxidase in HL-60 cells - Evidence for glutathionyl radical-induced peroxidation of phospholipids and cytotoxicity

(G)lutathione acts as a universal scavenger of free radicals at the expense of the formation of the glutathionyl radicals (GS(circle)). Here we demonstrated that GS(circle) radicals specifically interact with a reporter molecule, paramagnetic and non-fluorescent 4-((9-acridinecarbonyl)amino)-2,2,6,6-tetramethylpiperidine-1-oxyl (Ac-Tempo), and convert it into a non-paramagnetic fluorescent product, identified as 4-((9-acridinecarbonyl) amino)-2,2,6,6-tetramethylpiperidine (Ac-piperidine). Horseradish peroxidase-, myeloperoxidase-, and cyclooxygenase-catalyzed oxidation of phenol in the presence of H2O2 and GSH caused the generation of phenoxyl radicals and GS(circle) radicals, of which only the latter reacted with Ac-Tempo. Oxidation of several other phenolic compounds (e.g. etoposide and tyrosine) was accompanied by the formation of GS(circle) radicals along with a characteristic fluorescence response from Ac-Tempo. In myeloperoxidase-rich HL-60 cells treated with H2O2 and phenol, fluorescence microscopic imaging of Ac-Tempo revealed the production of GS(circle) radicals. A thiol-blocking reagent, N-ethylmaleimide, as well as myeloperoxidase inhibitors (succinyl acetone and azide), blocked formation of fluorescent acridine-piperidine. H2O2/phenol-induced peroxidation of major classes of phospholipids in HL-60 cells was completely inhibited by Ac-Tempo, indicating that GS(circle) radicals were responsible for phospholipid peroxidation. Thus, GSH, commonly viewed as a universal free radical scavenger and major intracellular antioxidant, acts as a pro-oxidant during myeloperoxidase-catalyzed metabolism of phenol in HL-60 cells.