Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay

Abstract

. Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay. Am J Physiol Cell Physiol 287: C895-C902, 2004. First published August 11, 2004 10.1152 10. /ajpcell.00028.2004 was demonstrated that superoxide oxidizes dihydroethidium to a specific fluorescent product (oxyethidium) that differs from ethidium by the presence of an additional oxygen atom in its molecular structure (Zhao H, Kalivendi S, Zhang H, Joseph J, Nithipatikom K, Vásquez-Vivar J, and Kalyanaraman B. Free Radic Biol Med 34: 1359 -1368. We have adapted this new HPLC-based assay to quantify this product as a tool to estimate intracellular superoxide in intact tissues. Ethidium and oxyethidium were separated using a C-18 column and quantified using fluorescence detection. Initial cell-free experiments with potassium superoxide and xanthine oxidase confirmed the formation of oxyethidium from dihydroethidium. The formation of oxyethidium was inhibited by superoxide dismutase but not catalase and did not occur upon the addition of H2O2, peroxynitrite, or hypochlorous acid. In bovine aortic endothelial cells (BAEC) and murine aortas, the redox cycling drug menadione increased the formation of oxyethidium from dihydroethidium ninefold (0.4 nmol/mg in control vs. 3.6 nmol/mg with 20 M menadione), and polyethylene glycolconjugated superoxide dismutase (PEG-SOD) significantly inhibited this effect. Treatment of BAEC with angiotensin II caused a twofold increase in oxyethidium formation, and this effect also was reduced by PEG-SOD (0.5 nmol/mg). In addition, in the aortas of mice with angiotensin II-induced hypertension and DOCA-salt hypertension, the formation of oxyethidium was increased in a manner corresponding to superoxide production estimated on the basis of cytochrome c reduction. Detection of oxyethidium using HPLC represents a new, convenient, quantitative method for the detection of superoxide in intact cells and tissues.oxyethidium; hypertension; menadione; angiotensin II; endothelium IT HAS BECOME EVIDENT that mammalian cells produce reactive oxygen species (ROS) and that these can serve as signaling molecules that modulate events such as enzyme phosphorylation, cell growth, hypertrophy, and programmed cell death. When produced in excessive amounts, ROS can contribute to cellular dysfunction (9). Overproduction of ROS has been implicated in diverse diseases such as cancer, hypertension, atherosclerosis, Alzheimer's disease, lung injury, and aging (20).The production of ROS is mediated by a variety of mammalian enzymes that are capable of reducing molecular oxygen. While occasional enzymes such as glucose oxidase and xanthine oxidase are capable of performing a two-electron reduction of oxygen to form hydrogen peroxide, the most common scenario is a one-electron reduction, leading to formation of superoxide (O 2 Ϫ ⅐). O 2 Ϫ ⅐ can in turn serve as a progenitor for other ROS such as hydrogen peroxide, peroxynitrite, and the hydroxyl radical. In vascular cells, increased ...

Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: Identification of homo- and heterodimers

“…Except for a few studies [2,5] the formation of the characteristic marker product of superoxide/HE reaction, 2-OH-E + , was not investigated. As compared to 2-hydroxyethidium, ethidium was always detected in much higher amounts in these studies [2,5]. Thus, the "red fluorescence" generated from HE in cells is mostly due to ethidium in nearly all of these studies.…”

“…Although subtle differences in the spectroscopic properties between E + and 2-OH-E + were used to selectively detect 2-OH-E + and E + in cells under well-defined conditions [4], the fluorescence techniques were largely deemed to be unsuitable for superoxide measurements in cells [2,5]. Using the HPLC-fluorescence method, it was previously shown that the increase in intracellular "red fluorescence" arising from HE could not be solely attributed to 2-OH-E + , as E + was also formed intracellularly from HE during oxidation [2,5]. However, the mechanism of E + formation in cells remained unclear, although E + could be formed from photosensitized oxidation of HE in superoxide-dependent manner [6].…”

Omega-3 fatty acids differentially modulate enzymatic anti-oxidant systems in skeletal muscle cells

“…To evaluate the effects of omega-3 polyunsaturated fatty acids on skeletal muscle cells, we determined superoxide production using the dihydroethidium (DHE) oxidation method (Fink et al 2004;Zhao et al 2003). After 24 h of treatment with 100 μM EPA or DHA freshly prepared before each assay (described above), cells were incubated with 1 μM DHE in DPBS containing 5 mM glucose, for 30 min, at 37°C.…”

Estudio de la estabilidad genética asociada a los procesos de crioconservación de ápices de menta

“…Algunos de estos productos secundarios son el etileno y etanol (relativamente inocuos) y otros pueden ser aldehídos como el malondialdehído (MDA) o hidroxialquenales como el hidroxi-2-nonenal (HNE) que son citotóxicos, alteran la función celular y pueden ser mutagénicos (Dumet y Benson, 2000;. El MDA reacciona con las bases del ADN, especialmente guanina, provocando lesiones mutagénicas y el HNE inhibe el crecimiento celular y es genotóxico (Dizdaroglu, 1992;Cadet et al 1999 (Fink et al 2004) y el diacetato 4,5-diaminofluorescina (DAF-2DA), para detectar la presencia de NO Kojima et al 1998). …”

“…•-y NO, respectivamente, dentro de los tejidos observándolos bajo Microscopio Láser Confocal espectral (CLSM; Rodríguez-Serrano et al 2006;Fink et al 2004;Kojima et al 1998). DCF-DA es un compuesto no fluorescente que es capaz de atravesar las membranas biológicas, y una vez dentro de la célula los grupos acetato son hidrolizados por esterasas intracelulares, convirtiéndose así en un compuesto fluorescente capaz de reaccionar con el H2O2 (Brul et al 1997).…”