Products Cited in Publications Worldwide Amplex Red cellular uptake produces radical intermediates by myeloperoxidase and mediates oxidative stress

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

Liu, Huan; Tonoyan, Lusine; Reiz, Béla; Siraki, Arno G

DOI:

Abstract

Amplex Red (AR) is commonly used to detect extracellular hydrogen peroxide (H2O2) and is considered a cellimpermeable 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.

Keywords

Amplex Red ; Myeloperoxidase ; EPR ; Free radicals ; Oxidative stress ; Protein radicals

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ADHP

A501086119171-73-298%

ADHP

438.0/250mg

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