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Comparison of fluorescence-based techniques for the quantification of particle-induced hydroxyl radicals.

Cohn CA, Simon SR, Schoonen MA - Part Fibre Toxicol (2008)

Bottom Line: Here, three fluorescein derivatives [aminophenyl fluorescamine (APF), amplex ultrared, and dichlorofluorescein (DCFH)] and two radical species, proxyl fluorescamine and tempo-9-ac have been compared for their usefulness to measure hydroxyl radicals generated in two different systems: a solution containing ferrous iron and a suspension of pyrite particles.Proxyl fluorescamine and tempo-9-ac do not react with hydroxyl radicals directly, which reduces their sensitivity.Since both DCFH and amplex ultrared will react with reactive oxygen species other than hydroxyl radicals and another highly reactive species, peroxynitite, they lack specificity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Environmental Molecular Science, Stony Brook University, Stony Brook, USA. coreycohn@hotmail.com.

ABSTRACT

Background: Reactive oxygen species including hydroxyl radicals can cause oxidative stress and mutations. Inhaled particulate matter can trigger formation of hydroxyl radicals, which have been implicated as one of the causes of particulate-induced lung disease. The extreme reactivity of hydroxyl radicals presents challenges to their detection and quantification. Here, three fluorescein derivatives [aminophenyl fluorescamine (APF), amplex ultrared, and dichlorofluorescein (DCFH)] and two radical species, proxyl fluorescamine and tempo-9-ac have been compared for their usefulness to measure hydroxyl radicals generated in two different systems: a solution containing ferrous iron and a suspension of pyrite particles.

Results: APF, amplex ultrared, and DCFH react similarly to the presence of hydroxyl radicals. Proxyl fluorescamine and tempo-9-ac do not react with hydroxyl radicals directly, which reduces their sensitivity. Since both DCFH and amplex ultrared will react with reactive oxygen species other than hydroxyl radicals and another highly reactive species, peroxynitite, they lack specificity.

Conclusion: The most useful probe evaluated here for hydroxyl radicals formed from cell-free particle suspensions is APF due to its sensitivity and selectivity.

No MeSH data available.


Related in: MedlinePlus

The effect of H2O2 concentration on inducing fluorescence in the probes. In the presence of 0.2 μM HRP, 50 mM phosphate pH 7.4 buffer and H2O2, oxidation of the probes results in an increase in fluorescence. On the left side of the vertical axis are the fluorescence intensities when no HRP nor H2O2 are added to the probe and pH buffer. The fluorometer was calibrated so that pure water resulted in a zero fluorescence and the reaction involving the probe with HRP, pH buffer, and 100 nM H2O2 resulted in a fluorescence reading of 1000. The fluorescence intensity results presented in the next two figures are calibrated using two points: the fluorescence intensity of the probe without H2O2 or HRP added, and the fluorescence intensity at 1000 nM H2O2. The measurements were repeated four times and standard deviation error bars have been added. Many of the error bars, however, are within the size of the points.
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Figure 1: The effect of H2O2 concentration on inducing fluorescence in the probes. In the presence of 0.2 μM HRP, 50 mM phosphate pH 7.4 buffer and H2O2, oxidation of the probes results in an increase in fluorescence. On the left side of the vertical axis are the fluorescence intensities when no HRP nor H2O2 are added to the probe and pH buffer. The fluorometer was calibrated so that pure water resulted in a zero fluorescence and the reaction involving the probe with HRP, pH buffer, and 100 nM H2O2 resulted in a fluorescence reading of 1000. The fluorescence intensity results presented in the next two figures are calibrated using two points: the fluorescence intensity of the probe without H2O2 or HRP added, and the fluorescence intensity at 1000 nM H2O2. The measurements were repeated four times and standard deviation error bars have been added. Many of the error bars, however, are within the size of the points.

Mentions: In order to determine hydroxyl radical generation from hydrogen peroxide in the presence of HRP or from ferrous iron solutions and particulate suspensions, calibration curves were first produced. In the presence of HRP, the fluorescence intensity of all of the probes increases with increasing hydrogen peroxide concentration (Fig. 1). The gain on the fluorimeter was adjusted in this series of experiments to display a relative fluoresence intensity of 1000 in the presence of 1000 nM H2O2 along with 0.2 μM HRP and each probe at its concentration as listed above in Methods. The baseline of the instrument was adjusted to display a relative intensity of zero in the presence of water alone. It is evident from Figure 1 that in the absence of H2O2, all of the probes except for proxyl fluorescamine and tempo-9-ac show relative fluorescence intensities that are less than 5% of the signals they generate in the presence of 1000 nM H2O2 and HRP. With addition of HRP alone, in the absence of exogenously added H2O2, APF, DCFH, and amplex ultrared show small increases in fluorescence intensity which may reflect spontaneous formation of H2O2 but these signals are still less than 20% of the signals generated in the presence of 1000 nM exogenous H2O2 and HRP. The two fluorogenic free radical scavengers, tempo-9-ac and proxyl fluorescamine, however, clearly undergo significantly smaller increases in fluorescence with increasing H2O2 concentration than the other probes. We hypothesize that these smaller increases reflect the indirect mechanisms by which hydroxyl radicals modify the two spin traps (through phenoxy- and thiyl radicals in the case of tempo-9-ac and through methyl radicals in the case of proxyl fluorescamine), resulting in suboptimal conversion of the probes to the spin-neutral species.


Comparison of fluorescence-based techniques for the quantification of particle-induced hydroxyl radicals.

Cohn CA, Simon SR, Schoonen MA - Part Fibre Toxicol (2008)

The effect of H2O2 concentration on inducing fluorescence in the probes. In the presence of 0.2 μM HRP, 50 mM phosphate pH 7.4 buffer and H2O2, oxidation of the probes results in an increase in fluorescence. On the left side of the vertical axis are the fluorescence intensities when no HRP nor H2O2 are added to the probe and pH buffer. The fluorometer was calibrated so that pure water resulted in a zero fluorescence and the reaction involving the probe with HRP, pH buffer, and 100 nM H2O2 resulted in a fluorescence reading of 1000. The fluorescence intensity results presented in the next two figures are calibrated using two points: the fluorescence intensity of the probe without H2O2 or HRP added, and the fluorescence intensity at 1000 nM H2O2. The measurements were repeated four times and standard deviation error bars have been added. Many of the error bars, however, are within the size of the points.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2289838&req=5

Figure 1: The effect of H2O2 concentration on inducing fluorescence in the probes. In the presence of 0.2 μM HRP, 50 mM phosphate pH 7.4 buffer and H2O2, oxidation of the probes results in an increase in fluorescence. On the left side of the vertical axis are the fluorescence intensities when no HRP nor H2O2 are added to the probe and pH buffer. The fluorometer was calibrated so that pure water resulted in a zero fluorescence and the reaction involving the probe with HRP, pH buffer, and 100 nM H2O2 resulted in a fluorescence reading of 1000. The fluorescence intensity results presented in the next two figures are calibrated using two points: the fluorescence intensity of the probe without H2O2 or HRP added, and the fluorescence intensity at 1000 nM H2O2. The measurements were repeated four times and standard deviation error bars have been added. Many of the error bars, however, are within the size of the points.
Mentions: In order to determine hydroxyl radical generation from hydrogen peroxide in the presence of HRP or from ferrous iron solutions and particulate suspensions, calibration curves were first produced. In the presence of HRP, the fluorescence intensity of all of the probes increases with increasing hydrogen peroxide concentration (Fig. 1). The gain on the fluorimeter was adjusted in this series of experiments to display a relative fluoresence intensity of 1000 in the presence of 1000 nM H2O2 along with 0.2 μM HRP and each probe at its concentration as listed above in Methods. The baseline of the instrument was adjusted to display a relative intensity of zero in the presence of water alone. It is evident from Figure 1 that in the absence of H2O2, all of the probes except for proxyl fluorescamine and tempo-9-ac show relative fluorescence intensities that are less than 5% of the signals they generate in the presence of 1000 nM H2O2 and HRP. With addition of HRP alone, in the absence of exogenously added H2O2, APF, DCFH, and amplex ultrared show small increases in fluorescence intensity which may reflect spontaneous formation of H2O2 but these signals are still less than 20% of the signals generated in the presence of 1000 nM exogenous H2O2 and HRP. The two fluorogenic free radical scavengers, tempo-9-ac and proxyl fluorescamine, however, clearly undergo significantly smaller increases in fluorescence with increasing H2O2 concentration than the other probes. We hypothesize that these smaller increases reflect the indirect mechanisms by which hydroxyl radicals modify the two spin traps (through phenoxy- and thiyl radicals in the case of tempo-9-ac and through methyl radicals in the case of proxyl fluorescamine), resulting in suboptimal conversion of the probes to the spin-neutral species.

Bottom Line: Here, three fluorescein derivatives [aminophenyl fluorescamine (APF), amplex ultrared, and dichlorofluorescein (DCFH)] and two radical species, proxyl fluorescamine and tempo-9-ac have been compared for their usefulness to measure hydroxyl radicals generated in two different systems: a solution containing ferrous iron and a suspension of pyrite particles.Proxyl fluorescamine and tempo-9-ac do not react with hydroxyl radicals directly, which reduces their sensitivity.Since both DCFH and amplex ultrared will react with reactive oxygen species other than hydroxyl radicals and another highly reactive species, peroxynitite, they lack specificity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Environmental Molecular Science, Stony Brook University, Stony Brook, USA. coreycohn@hotmail.com.

ABSTRACT

Background: Reactive oxygen species including hydroxyl radicals can cause oxidative stress and mutations. Inhaled particulate matter can trigger formation of hydroxyl radicals, which have been implicated as one of the causes of particulate-induced lung disease. The extreme reactivity of hydroxyl radicals presents challenges to their detection and quantification. Here, three fluorescein derivatives [aminophenyl fluorescamine (APF), amplex ultrared, and dichlorofluorescein (DCFH)] and two radical species, proxyl fluorescamine and tempo-9-ac have been compared for their usefulness to measure hydroxyl radicals generated in two different systems: a solution containing ferrous iron and a suspension of pyrite particles.

Results: APF, amplex ultrared, and DCFH react similarly to the presence of hydroxyl radicals. Proxyl fluorescamine and tempo-9-ac do not react with hydroxyl radicals directly, which reduces their sensitivity. Since both DCFH and amplex ultrared will react with reactive oxygen species other than hydroxyl radicals and another highly reactive species, peroxynitite, they lack specificity.

Conclusion: The most useful probe evaluated here for hydroxyl radicals formed from cell-free particle suspensions is APF due to its sensitivity and selectivity.

No MeSH data available.


Related in: MedlinePlus