Amplex Red cellular uptake produces radical intermediates by myeloperoxidase and mediates oxidative stress

Amplex Red (AR) is commonly used to detect extracellular hydrogen peroxide (H₂O₂) and is considered a cell-impermeable compound. However, it would appear capable of entering cells based on its phenoxazine substructure and the report of its mitochondrial membrane permeability. Additionally, myeloperoxidase (MPO) oxidation of AR produces a fluorescent compound, resorufin, which has been reported, though the mechanism is not well-studied. EPR spin trapping using glutathione (GSH) revealed that AR metabolism produced AR radicals and glutathionyl radicals (GS^•). An intermediate metabolite, 3,7-dihydroxyphenoxazine, was observed by liquid chromatography-mass spectrometry (LC-MS), which supported AR radical disproportionation first and subsequently N-oxidation. Besides, in the presence of GSH, the formation of resorufin decreased significantly evidencing the reactivity of radical intermediates. Three types of AR-GS adduct were found using LC-MS and the resorufin GS-adduct was the dominant one. Regarding intracellular findings in HL-60 cells (that highly express MPO), LC-MS and fluorescence analysis showed AR penetrated the cell membrane and was oxidized by cellular MPO. Interestingly, we demonstrated that the oxidation of AR in HL-60 cells showed a significant time dependence; PF-1355, an MPO inhibitor, inhibited the oxidation of AR by MPO. Cell viability (ATP) revealed that 200 μM AR significantly decreased viability in HL-60 cells in 6 h. We also found that AR-mediated decreased total GSH and increased protein-radical formation. These findings revealed that AR is cell-permeable, and AR radicals induce cellular oxidative distress and lead to the formation of protein radicals, which correlate with the MPO-mediated mechanism of cytotoxicity.