Elsevier

Biochemical Pharmacology

Volume 73, Issue 7, 1 April 2007, Pages 972-980
Biochemical Pharmacology

Production of extracellular superoxide by human lymphoblast cell lines: Comparison of electron spin resonance techniques and cytochrome C reduction assay

https://doi.org/10.1016/j.bcp.2006.12.012Get rights and content

Abstract

Superoxide production by NADPH oxidases plays an important role in the development and progression of cardiovascular disease (CVD). However, measurement of superoxide (O2radical dot), a marker of oxidative stress, remains a challenging task in clinical and translational studies. In this study we analyzed O2radical dot production in cultured human lymphoblast cell lines by three different methods: (a) superoxide dismutase (SOD)-inhibitable cytochrome C reduction, (b) spin trapping of superoxide with 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), and (c) using electron spin resonance (ESR) with the cell-permeable spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH). Lymphocytes were isolated and immortalized by an Epstein–Barr Virus (EBV)-transformation procedure. Superoxide was measured in cultured lymphoblast cell lines at baseline and upon stimulation with phorbol 12-myristate 13-acetate (PMA). Cytochrome C and the spin traps EMPO and DEPMPO detected two to five times less superoxide compared to CMH. Thus, CMH provided the most quantitative measurement of superoxide generation in human lymphoblast cell lines. Superoxide detection with CMH was linear dependent on cell concentration and was inhibited by SOD but not by catalase. Both cell-permeable polyethylene glycol (PEG)–SOD and extracellular Cu,Zn–SOD inhibited O2radical dot detection by 90% in PMA-stimulated cells, suggesting a predominantly extracellular O2radical dot generation in human lymphoblasts. Our study describes a new technique for O2radical dot measurement in cultured human lymphoblasts using ESR and CMH. A highly sensitive in vitro measurement of O2radical dot in human cell lines would allow investigators to study genotype/phenotype interactions in translational studies.

Introduction

Superoxide (O2radical dot) production plays an important role in redox cell signaling and development of pathophysiological conditions, such as hypertension, ischemia-reperfusion injury, inflammation and atherosclerosis [1]. However, detection of O2radical dot is still a challenging problem. One of the most sensitive and definitive methods of O2radical dot detection is electron spin resonance (ESR) [2], [3]. The ESR spin-trapping technique has been used to detect O2radical dot radicals induced by inflammation via neutrophil NADPH oxidase in cellular systems in vitro [4]. However, the commonly used nitrone spin traps have a very low efficacy for trapping of O2radical dot radicals (Fig. 1) [5]. Thus, formation of the radical adduct is limited by slow kinetics of O2radical dot trapping and obstruction by antioxidants. Furthermore, superoxide radical adducts suffer from decomposition to hydroxyl (radical dotOH)-radical adducts by glutathione (GSH) peroxidase [6]. Finally, both O2radical dot and radical dotOH radical adducts can be reduced to ESR silent hydroxylamines by ascorbate, transition metals, or flavin enzymes (Fig. 1) [7].

Recently, cyclic hydroxylamines were found to be effective scavengers of O2radical dot radicals [8], [9]. Hydroxylamine probes 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PPH) and 1-hydroxy-3-carboxy-pyrrolidine (CPH) have been previously used for quantitative detection of extracellular O2radical dot. The advantage of hydroxylamine probes is that they are effective scavengers of O2radical dot and produce a stable nitroxide radical [8]. Previously, we reported the activity of the phagocytic NADPH oxidase in neutrophils from healthy subjects using CPH, and measuring O2radical dot as SOD-inhibitable formation of 3-carboxyproxyl [10].

In the current investigation, we studied superoxide production in cultured lymphoblast cell lines at baseline and upon stimulation with phorbol 12-myristate 13-acetate (PMA) by three methods: (a) superoxide dismutase (SOD)-inhibitable cytochrome C reduction, (b) spin trapping of superoxide with 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), and (c) using ESR with the cell-permeable spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) (Fig. 1) [11]. Reaction of O2radical dot with CMH is much faster (1.2 × 104 M−1 s−1) than with nitrone spin traps, thereby enabling the hydroxylamines to compete with cellular antioxidants and react with both extra- and intracellular O2radical dot.

Our study describes a new technique for O2radical dot measurement in cultured human lymphoblasts using ESR and CMH.

Section snippets

Reagents

Spin traps 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO) and spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) were purchased from Alexis Corporation (San Diego, USA). Polyethylene-glycol-conjugated superoxide dismutase (PEG–SOD), and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma–Aldrich (St. Louis, MO). All other reagents were obtained from Sigma–Aldrich.

Establishment of immortalized cell lines

In collaboration with the

Measurement of O2radical dot in stimulated and unstimulated lymphoblasts using CMH

Incubation of human lymphoblasts with the spin probe CMH resulted in the generation of the ESR signal of 3-methoxycarbonyl-proxyl nitroxide (CMradical dot) (Fig. 2A). Time scan of the low-field component of the ESR signal showed linear accumulation of CMradical dot in unstimulated cells. Stimulation of cells by PMA (10 μM) led to several-fold increase in the slope of CMradical dot kinetics (Fig. 2B).

CMH detects both extra- and intracellular O2radical dot[11], [15]. Extracellular O2radical dot can be quantified by inhibition of the ESR signal by

Discussion

In the present study, we observed that the cell-permeable spin probe CMH provided the most quantitative measurement of O2radical dot generation in human lymphoblast cell lines. As shown in Table 1, cytochrome C and the spin traps EMPO and DEPMPO detected two to four times less O2radical dot compared to CMH. Higher reactivity of CMH with O2radical dot and the stability of the CM-nitroxide (product of CMH and O2radical dot reaction) are likely responsible for higher O2radical dot detection by CMH. In addition, we show that human lymphoblasts

Acknowledgements

This research was supported by National Institutes of Health grants PO-1 HL058000-05 and PO-1 HL075209, the Emory University Research Committee, the Emory University General Clinical Research Center (M01-RR00039), Atlanta, GA, and by National Institutes of Health cardiovascular training grant T-32 HL07745 to PM and SSW.

References (28)

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