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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

Iron-induced formation of reactive oxygen species. In the presence of water and dissolved oxygen, ferrous iron will form both H2O2 and hydroxyl radicals (eqs 1 to 3).
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Figure 2: Iron-induced formation of reactive oxygen species. In the presence of water and dissolved oxygen, ferrous iron will form both H2O2 and hydroxyl radicals (eqs 1 to 3).

Mentions: In an aqueous solution containing dissolved oxygen, the addition of soluble ferrous salts or ferrous iron-containing minerals such as pyrite results in the formation of both hydrogen peroxide and hydroxyl radicals (eqs 1 and 2). All of the probes were incubated with multiple concentrations of ferrous iron in 25 mM phosphate buffer, pH 7.4 (Fig. 2). The results show differences in each probe's sensitivity to hydroxyl radicals generated in ferrous iron solutions. When less than 50 μM ferrous iron salts were added to the probes, the fluorescence intensities of all of the probes were observed to increase. The apparent concentration of detected hydroxyl radicals declined when greater concentrations of ferrous iron were added to tempo-9-ac. Since the calibration curve generated for proxyl fluorescamine has a very shallow slope, small changes in the fluorescence intensity of this probe equate to very large apparent changes in hydroxyl radical concentration, reflected in the vertical axis for proxyl fluorescamine on the right side of Figure 2. An additional complication in detection of hydroxyl radicals generated by ferrous salts and minerals using amplex ultrared as a probe arises from the capacity of the dye to be oxidized by H2O2 to the fluorescent resorufin in the presence of HRP. It is evident that H2O2 is indeed formed in addition to hydroxyl radicals upon addition of ferrous iron salts or minerals because addition of HRP to the incubation mixtures containing the iron samples and amplex ultrared results in a significant increase in fluorescence.


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

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

Iron-induced formation of reactive oxygen species. In the presence of water and dissolved oxygen, ferrous iron will form both H2O2 and hydroxyl radicals (eqs 1 to 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Iron-induced formation of reactive oxygen species. In the presence of water and dissolved oxygen, ferrous iron will form both H2O2 and hydroxyl radicals (eqs 1 to 3).
Mentions: In an aqueous solution containing dissolved oxygen, the addition of soluble ferrous salts or ferrous iron-containing minerals such as pyrite results in the formation of both hydrogen peroxide and hydroxyl radicals (eqs 1 and 2). All of the probes were incubated with multiple concentrations of ferrous iron in 25 mM phosphate buffer, pH 7.4 (Fig. 2). The results show differences in each probe's sensitivity to hydroxyl radicals generated in ferrous iron solutions. When less than 50 μM ferrous iron salts were added to the probes, the fluorescence intensities of all of the probes were observed to increase. The apparent concentration of detected hydroxyl radicals declined when greater concentrations of ferrous iron were added to tempo-9-ac. Since the calibration curve generated for proxyl fluorescamine has a very shallow slope, small changes in the fluorescence intensity of this probe equate to very large apparent changes in hydroxyl radical concentration, reflected in the vertical axis for proxyl fluorescamine on the right side of Figure 2. An additional complication in detection of hydroxyl radicals generated by ferrous salts and minerals using amplex ultrared as a probe arises from the capacity of the dye to be oxidized by H2O2 to the fluorescent resorufin in the presence of HRP. It is evident that H2O2 is indeed formed in addition to hydroxyl radicals upon addition of ferrous iron salts or minerals because addition of HRP to the incubation mixtures containing the iron samples and amplex ultrared results in a significant increase in fluorescence.

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