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

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.

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.

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).

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.