Superoxide radical detection in cells, tissues, organisms (animals, plants, insects, microorganisms) and soils

Abstract

A simple protocol is presented for the assessment of superoxide radical in organisms (animal/plant tissues, microorganisms, cell cultures, biological/culture fluids) and soils, through the quantification of 2-hydroxyethidium (2-OH-E+), its specific reaction product with hydroethidine (HE). It is an alternative to the quantification of 2-OH-E+ by HPLC (restricted to cell cultures), offering the advantage of the in vivo assessment of superoxide radical in a wide range of experimental systems. The protocol includes alkaline-acetone extraction of the sample, purification by microcolumn cation exchange and hydrophobic chromatographies, and fluorescence detection of the isolated 2-OH-E+/HE-oxidation products mixture before and after consumption of 2-OH-E+ by a horseradish peroxidase/hydrogen peroxide system. The protocol is sensitive at <1 pmol 2-OH-E+ per mg protein (extended to the femto level when using large samples) in biological systems, and in soils at 9 pmol superoxide radical per gram of soil. The protocol includes a cytochrome c-based subprotocol for superoxide radical detection in soils at 770 pmol g(-1) soil. For processing ten samples and depending on the experimental material used (soil or biological), the approximate procedure time would be 2-7 h.

Evidence for photochemical production of reactive oxygen species in desert soils

“…À /peroxides and detection of Me tr . We extracted soil metal superoxides as O 2 Á À from desiccated Atacama and Mojave Desert topsoils by acetonitrile and quantified fluorometrically by a modified hydroethidine (HE)-based assay 23,24 , and the identity of O 2 Á À and validity of the HEbased assay were verified by a second assay 25,26 , a modification of the superoxide dismutase (SOD)-inhibited equimolar reduction of cyt. c by O 2 Á À (Methods, Supplementary Methods).…”

Bioreductively Activated Reactive Oxygen Species (ROS) Generators as MRSA Inhibitors

“…Again, we found that 1f was capable of generating H 2 O 2 in the presence of DT-D. 24 Next, the possibility of 1f generating intracellular O 2 −• in bacteria was examined using a DHE assay. 21,22 The bacterial control showed unreacted DHE (Figure 1d), and in the presence of 1f, the formation of 2-OH-E + in a concentrationdependent manner was observed, suggestive of O 2 −• production intracellularly (Figure 1d). Superoxide accumulation intracellularly leads to the production of H 2 O 2 through dismutation.…”

“…19,20 A HPLCbased dihydroethidium (DHE) assay was used to independently assess O 2 −• production (Figure 1b). 21,22 Here, the conversion of DHE to 2-hydroxyethidium (2-OH-E + ) is indicative of O 2 −• generation. 23 During incubation of 1f in the presence of DT-D, the formation of 2-OH-E + was observed, thus, confirming the intermediacy of O 2 −• (Figure 1b).…”

On the use of fluorescence lifetime imaging and dihydroethidium to detect superoxide in intact animals and ex vivo tissues: A reassessment

“…In other studies, chromatographic techniques were used to detect 2-OH-E + in mouse brain tissue (6, 13). There also exist other reports wherein fluorometric approaches have been used to detect 2-OH-E + in brain extracts (14–16).…”