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Dihydrorhodamine 123: a fluorescent probe for superoxide generation?

L M Henderson¹, J B Chappell

¹Department of Biochemistry, School of Medical Sciences, University of Bristol, England.

European Journal of Biochemistry

|November 1, 1993

Summary

Dihydrorhodamine 123 (DHR) oxidation by neutrophils primarily indicates hydrogen peroxide (H2O2) presence and intracellular peroxidases, not superoxide (O2-.) generation. DHR is unsuitable for single-cell superoxide detection.

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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.