Dihydrorhodamine 123: a fluorescent probe for superoxide generation?

Area of Science:

• Cellular biology
• Biochemistry
• Immunology

Background:

• Confocal microscopy and fluorescent activated cell scanning enable single-cell response studies.
• Superoxide oxidizes non-fluorescent dihydrorhodamine 123 (DHR) to fluorescent rhodamine 123.

Purpose of the Study:

• To investigate the specificity of DHR as a probe for superoxide generation in human neutrophils.
• To determine whether DHR oxidation accurately reflects superoxide production at the single-cell level.

Main Methods:

• Stimulation of human neutrophils with phorbol 12-myristate 13-acetate.
• Inhibition studies using diphenylene iodonium, azide, and superoxide dismutase.
• Assessment of DHR oxidation in the presence and absence of enzymes and extracellular catalase.
• Observation of rhodamine 123 localization within cells.

Main Results:

• DHR oxidation was inhibited by diphenylene iodonium and azide, but not superoxide dismutase.
• Hydrogen peroxide (H2O2), not superoxide (O2-.), slowly oxidized DHR in the absence of enzymes, with peroxidases greatly enhancing the rate.
• Stimulated cellular fluorescence was eliminated by extracellular catalase, indicating extracellular H2O2 involvement.
• DHR failed to distinguish between superoxide-producing and non-producing HL60 cells in mixed populations.

Conclusions:

• Dihydrorhodamine 123 (DHR) oxidation is primarily an indicator of intracellular peroxidases and hydrogen peroxide (H2O2), not superoxide (O2-.) generation.
• DHR is not a suitable probe for single-cell detection of superoxide production.
• Only cells containing peroxidases will exhibit fluorescence with DHR under these conditions.