Fast track paperSuperoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide
Introduction
One of the most popular assays for detecting superoxide in cells and tissues involves the use of fluorescence-based techniques 1, 2, 3, 4, 5, 6. Generally, the red fluorescence arising from oxidation of hydroethidine (HE) (also dihydroethidium [DHE]) is detected 7, 8, 9, 10, 11, 12, 13, 14 (Fig. 1). This red fluorescence, often referred to as the “ethidium fluorescence,” is inhibited by intracellular superoxide dismutase and other superoxide scavengers 7, 8, 9, 10, 11, 12, 13, 14. HE is synthesized from sodium borohydride reduction of ethidium, a two-electron oxidation product [15]. Most previous fluorescence measurements have been performed using a kinetic mode and typically acquired at a single wavelength corresponding to that of ethidium 16, 17, 18, 19, 20. Alternatively, the red fluorescence due to oxidized HE was visualized in cells and tissues using fluorescence or confocal microscopy. To our knowledge, the question of whether superoxide-dependent oxidation of HE actually generates ethidium as a product has never been considered.
Like many other investigators in this field, we were under the impression that HE is selectively oxidized by superoxide to ethidium 1, 2, 3, 21. Recently, we began a systematic analysis of the chemistry of oxidant-sensitive fluorescent dyes, including dichlorodihydrofluorescein and HE 22, 23. We discovered that superoxide reacts with HE to form a fluorescent product that is distinctly different from ethidium. In the present study, we provide fluorescence, HPLC, and mass spectral evidence in support of this new finding. The potential implications of this finding with respect to intracellular detection, quantitation, and imaging of superoxide using HE as a fluorescent probe, are discussed.
Section snippets
Materials
Hydroethidine (dihydroethidium) was purchased from Molecular Probes Inc. (Eugene, OR, USA). Ethidium bromide, calf thymus, DNA, ferricytochrome c, NADPH, l-arginine, xanthine, calcium chloride, potassium superoxide, and diethylenetriamine pentaacetic acid (DTPA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The potassium superoxide in dimethyl sulfoxide (DMSO) was prepared immediately prior to use [24]. Bovine brain calmodulin was obtained from Calbiochem (San Diego, CA, USA).
Fluorescence spectra of the product formed from superoxide-dependent oxidation of HE
Figure 2A shows the fluorescence spectra for HE (emission maximum = 595 nm) (Table 1), the oxidation product of HE by X/XO (emission maximum = 586 nm) and for ethidium (emission maximum = 605 nm) in phosphate buffer. Superoxide generation was measured to be 8 μM/min. Figure 2B shows the enhancement in the fluorescence intensity of the three compounds in the presence of DNA. DNA did not have any effect on the rate of oxidation of HE in this system (data not shown). Addition of superoxide
Discussion
Our findings demonstrate that superoxide reacts with HE to form a characteristic fluorescent product that is different from E+. In the presence of other reactive oxygen and nitrogen species (e.g., hydrogen peroxide, hydroxyl radical, or peroxynitrite), HE was not oxidized to form the same fluorescent product. The present findings point to a potentially new and improved fluorescence marker for detecting superoxide in cells.
Abbreviations
BH4—6R-5, 6, 7, 8-tetrahydrobiopterin
BMPO—5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide
DEA-NONOate—2-(N, N-diethylamino)-diazenolate-2-oxide
DHE—dihydroethidium
E+—ethidium
HE—hydroethidine
NOS—nitric oxide synthase
X—xanthine
XO—xanthine oxidase
Acknowledgements
This work was supported by grants 1PIHL68769-01, RR01008, NS40494, HL067244, NS39958, and CA77822 from the National Institutes of Health. The authors thank Mr. Hanbing Lu for his help in obtaining the fluorescence photograph of the DNA gel.
References (31)
- et al.
Mitochondrial membrane potential and hydroethidine-monitored superoxide generation in cultured cerebellar granule cells
FEBS Lett
(1997) - et al.
Detection of reactive oxygen species by flow cytometry after spinal cord injury
J. Neurosci. Methods
(2002) - et al.
Proton magnetic resonance studies of ethidium bromide and its sodium borohydride reduced derivative
FEBS Lett
(1972) - et al.
Increased mitochondrial superoxide generation in neurons from trisomy 16 micea model of Down’s syndrome
Free Radic. Biol. Med.
(2000) - et al.
Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical
Free Radic. Biol. Med.
(1998) - et al.
Significant levels of oxidants are generated by isolated cardiomyocytes during ischemia prior to reperfusion
J. Mol. Cell. Cardiol.
(1997) - et al.
Transferrin receptor-dependent iron uptake is responsible for doxorubicin-mediated apoptosis in endothelial cells. Role of oxidant-induced iron signaling in apoptosis
J. Biol. Chem.
(2002) - et al.
Further studies on the formation of oxygen radicals by potassium superoxide in aqueous medium for biochemical investigations
Toxicol. Lett.
(1986) - et al.
Synthesis and biochemical applications of a solid cyclic nitrone spin trapa relatively superior trap for detecting superoxide anions and glutathiyl radicals
Free Radic. Biol. Med.
(2001) - et al.
Detection of superoxide anion using an isotopically labeled nitrone spin trappotential biological applications
FEBS Lett
(2000)
In vitro quantitation of biological superoxide and hydrogen peroxide generation
Methods Enzymol
Reaction of tetrahydrobiopterin with superoxideEPR-kinetic analysis and characterization of the pteridine radical
Free Radic. Biol. Med.
The role of tetrahydrobiopterin in the regulation of neuronal nitric-oxide synthase-generated superoxide
J. Biol. Chem.
Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2′, 7′-dichlorofluorescin
J. Leukoc. Biol.
Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells
J. Leukoc. Biol.
Cited by (674)
Reactive oxygen species drive foraging decisions in Caenorhabditis elegans
2023, Redox BiologyRecent advances in analytical methods of oxidative stress biomarkers induced by environmental pollutant exposure
2023, TrAC - Trends in Analytical ChemistrySemiconducting mineral induced photochemical conversion of PAHs in aquatic environment: Mechanism study and fate prediction
2023, Science of the Total Environment