Quantification of hydroxyl radical generation with spin trapping ESR technique in batch system

Abstract Spin trapping, a sensitive and specific means of detecting free radicals, is optimally performed on cell suspensions. This makes it unsuitable for the study of adherent endothelial cell monolayers because disrupting the monolayer to induce a cell suspension could introduce confounding factors. This problem was eliminated through the use of endothelial cells that were grown to confluence on microcarrier beads. Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the nature of free radical species generated by suspensions of microcarrier bead adherent porcine pulmonary endothelial cells under various forms of oxidant stress was examined. Exposure of these endothelial cells to paraquat resulted in the spin trapping of superoxide (.O2-). Endothelial cell incubation in the presence of either bolus or continuous fluxes of hydrogen peroxide (H2O2) yielded spin trap evidence of hydroxyl radical formation, which was preventable by pretreating the cells with deferoxamine. Chromium oxalate which eliminates extracellular electron paramagnetic resonance spectrometry (EPR) signals, prevented the detection of DMPO spin adducts generated by paraquat but not H2O2-treated endothelial cells. When endothelial cells were coincubated with PMA-stimulated monocytes evidence of both .O2- and hydroxyl radical production was detected, whereas with PMA- stimulated neutrophils only .O2- production could be confirmed. Neutrophil elastase, cathepsin G, and the combination of PMA and A23187 have previously been suggested to induce endothelial cell oxy-radical generation. However, exposure of endothelial cells to each of these agents did not yield DMPO spin adducts or cyanide-insensitive endothelial cell O2 consumption. These data indicate that endothelial cell exposure: to paraquat induces extracellular .O2- formation; to H2O2 leads to intracellular hydroxyl radical production; and to elastase, cathepsin G, or A23187/PMA does not appear to cause oxy- radical generation.

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Insight into the nature and site of oxygen-centered free radical generation by endothelial cell monolayers using a novel spin trapping technique

Blood ◽

10.1182/blood.v79.3.699.bloodjournal793699 ◽

1992 ◽

Vol 79 (3) ◽

pp. 699-707

Author(s):

BE Britigan ◽

TL Roeder ◽

DM Shasby

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Free Radical ◽

Hydroxyl Radical ◽

Spin Trap ◽

Spin Trapping ◽

Cathepsin G ◽

Cell Monolayers ◽

Radical Production ◽

Radical Generation

Spin trapping, a sensitive and specific means of detecting free radicals, is optimally performed on cell suspensions. This makes it unsuitable for the study of adherent endothelial cell monolayers because disrupting the monolayer to induce a cell suspension could introduce confounding factors. This problem was eliminated through the use of endothelial cells that were grown to confluence on microcarrier beads. Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the nature of free radical species generated by suspensions of microcarrier bead adherent porcine pulmonary endothelial cells under various forms of oxidant stress was examined. Exposure of these endothelial cells to paraquat resulted in the spin trapping of superoxide (.O2-). Endothelial cell incubation in the presence of either bolus or continuous fluxes of hydrogen peroxide (H2O2) yielded spin trap evidence of hydroxyl radical formation, which was preventable by pretreating the cells with deferoxamine. Chromium oxalate which eliminates extracellular electron paramagnetic resonance spectrometry (EPR) signals, prevented the detection of DMPO spin adducts generated by paraquat but not H2O2-treated endothelial cells. When endothelial cells were coincubated with PMA-stimulated monocytes evidence of both .O2- and hydroxyl radical production was detected, whereas with PMA- stimulated neutrophils only .O2- production could be confirmed. Neutrophil elastase, cathepsin G, and the combination of PMA and A23187 have previously been suggested to induce endothelial cell oxy-radical generation. However, exposure of endothelial cells to each of these agents did not yield DMPO spin adducts or cyanide-insensitive endothelial cell O2 consumption. These data indicate that endothelial cell exposure: to paraquat induces extracellular .O2- formation; to H2O2 leads to intracellular hydroxyl radical production; and to elastase, cathepsin G, or A23187/PMA does not appear to cause oxy- radical generation.

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Direct Evidence for In Vivo Hydroxyl Radical Generation in Blood of Mice After Acute Chromium(VI) Intake : Electron Spin Resonance Spin-Trapping Investigation

Biological Trace Element Research ◽

10.1385/bter:76:1:75 ◽

2000 ◽

Vol 76 (1) ◽

pp. 75-84 ◽

Cited By ~ 14

Author(s):

Yasuji Hojo ◽

Ayako Okado ◽

Sadahiro Kawazoe ◽

Tamio Mizutani

Keyword(s):

Electron Spin Resonance ◽

Hydroxyl Radical ◽

Electron Spin ◽

Direct Evidence ◽

Spin Trapping ◽

Chromium Vi ◽

Spin Resonance ◽

Radical Generation ◽

Resonance Spin

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Direct evidence for in vivo hydroxyl-radical generation in experimental iron overload: an ESR spin-trapping investigation.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.88.19.8440 ◽

1991 ◽

Vol 88 (19) ◽

pp. 8440-8444 ◽

Cited By ~ 139

Author(s):

M. J. Burkitt ◽

R. P. Mason

Keyword(s):

Iron Overload ◽

Hydroxyl Radical ◽

Direct Evidence ◽

Spin Trapping ◽

Esr Spin Trapping ◽

Radical Generation

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Enhancement of hydroxyl radical generation by phenols and their reaction intermediates during ozonation

Water Science & Technology ◽

10.2166/wst.1998.0247 ◽

1998 ◽

Vol 38 (6) ◽

pp. 147-154 ◽

Cited By ~ 2

Author(s):

Hideo Utsumi ◽

Sang-Kuk Han ◽

Kazuhiro Ichikawa

Keyword(s):

Hydroxyl Radical ◽

Hydroxyl Radicals ◽

Spin Trapping ◽

Active Species ◽

Generation Rate ◽

Peak Height Ratio ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Phenol Derivatives ◽

Semiquinone Radicals

Generation of hydroxyl radicals, one of the major active species in ozonation of water was directly observed with a spin-trapping/electron spin resonance (ESR) technique using 5,5-dimethyl-1-pyrrolineN-oxide (DMPO) as a spin-trapping reagent. Hydroxyl radical were trapped with DMPO as a stable radical, DMPO-OH. Eighty μM of ozone produced 1.08 X 10-6M of DMPO-OH, indicating that 1.4% of •OH is trapped with DMPO. Generation rate of DMPO-OH was determined by ESR/stopped-flow measurement. Phenol derivatives increased the amount and generation rate of DMPO-OH, indicating that phenol derivatives enhance •OH generation during ozonation of water. Ozonation of 2,3-, 2,5-, 2,6-dichlorophenol gave an ESR spectra of triplet lines whose peak height ratio were 1:2:1. ESR parameters of the triplet lines agreed with those of the corresponding dichloro-psemiquinone radical. Ozonation of 2,4,5- and 2,4,6-trichlorophenol gave the same spectra as those of 2,5- and 2,6-dichlorophenol, respectively, indicating that a chlorine group in p-position is substituted with a hydroxy group during ozonation. Amounts of the radical increased in an ozone-concentration dependent manner and were inhibited by addition of hydroxyl radical scavengers. These results suggest that p-semiquinone radicals are generated from the chlorophenols by hydroxyl radicals during ozonation. The p-semiquinone radicals were at least partly responsible for enhancements of DMPO-OH generation.

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