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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

Pyrite-induced formation of reactive oxygen species. Varying amounts of pyrite particles were incubated with the probes for 24 hrs followed by filtration and fluorescence measurements.
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Figure 3: Pyrite-induced formation of reactive oxygen species. Varying amounts of pyrite particles were incubated with the probes for 24 hrs followed by filtration and fluorescence measurements.

Mentions: One of our goals of having an ROS detection method is for its implementation for the detection of particulate-generated hydroxyl radicals. From previous research, we know that aqueous suspensions of pyrite particles will form hydroxyl radicals [21]. In this study, all of the probes were incubated with several different concentrations of pyrite particles and then evaluated for the generation of hydroxyl radicals (Fig. 3). The results show that for all of the probes except amplex ultrared (in the presence of HRP) and tempo-9-ac, fluorescence intensity generated by the probes increases proportionally to the total pyrite surface area exposed to the aqueous solvent. In other words, the probes record an increase in the formation of hydroxyl radicals with an increase in available surface area. The increased fluorescence from tempo-9-ac in the presence of pyrite particles shows a similar iron dose dependence to that observed in the presence of soluble ferrous salts: the fluorescence increases with small amounts of ferrous iron or pyrite and then decreases when more of the iron-containing species is added. The results from amplex ultrared are similar but when no HRP is added, the fluorescence increases nearly linearly. As in the studies with soluble ferrous iron salts described above, the sensitivity of proxyl fluorescamine to hydroxyl radicals from pyrite suspensions is so low that the concentrations of hydroxyl radicals formed cannot be detected with precision, and the apparent values, shown on the right vertical axis, are clearly outside the range of those detected by the other probes.


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

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

Pyrite-induced formation of reactive oxygen species. Varying amounts of pyrite particles were incubated with the probes for 24 hrs followed by filtration and fluorescence measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Pyrite-induced formation of reactive oxygen species. Varying amounts of pyrite particles were incubated with the probes for 24 hrs followed by filtration and fluorescence measurements.
Mentions: One of our goals of having an ROS detection method is for its implementation for the detection of particulate-generated hydroxyl radicals. From previous research, we know that aqueous suspensions of pyrite particles will form hydroxyl radicals [21]. In this study, all of the probes were incubated with several different concentrations of pyrite particles and then evaluated for the generation of hydroxyl radicals (Fig. 3). The results show that for all of the probes except amplex ultrared (in the presence of HRP) and tempo-9-ac, fluorescence intensity generated by the probes increases proportionally to the total pyrite surface area exposed to the aqueous solvent. In other words, the probes record an increase in the formation of hydroxyl radicals with an increase in available surface area. The increased fluorescence from tempo-9-ac in the presence of pyrite particles shows a similar iron dose dependence to that observed in the presence of soluble ferrous salts: the fluorescence increases with small amounts of ferrous iron or pyrite and then decreases when more of the iron-containing species is added. The results from amplex ultrared are similar but when no HRP is added, the fluorescence increases nearly linearly. As in the studies with soluble ferrous iron salts described above, the sensitivity of proxyl fluorescamine to hydroxyl radicals from pyrite suspensions is so low that the concentrations of hydroxyl radicals formed cannot be detected with precision, and the apparent values, shown on the right vertical axis, are clearly outside the range of those detected by the other probes.

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