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

Abstract

Superoxide production by NADPH oxidases plays an important role in the development and progression of cardiovascular disease (CVD). However, measurement of superoxide (O(2)(-)), a marker of oxidative stress, remains a challenging task in clinical and translational studies. In this study we analyzed O(2)(-) 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 O(2)(-) detection by 90% in PMA-stimulated cells, suggesting a predominantly extracellular O(2)(-) generation in human lymphoblasts. Our study describes a new technique for O(2)(-) measurement in cultured human lymphoblasts using ESR and CMH. A highly sensitive in vitro measurement of O(2)(-) in human cell lines would allow investigators to study genotype/phenotype interactions in translational studies.

Figure 1

ESR detection of O₂^•−. Spin trapping of O₂^•− is limited by slow reaction of O₂^•− with spin traps (EMPO) in the presence of antioxidants (SOD, ascorbate), biodegradation (glutathione peroxidase, GPx) and bioreduction of the radical adducts. Fast reaction of O₂^•− with spin probe CMH permits detection of cellular O₂^•−. CMH produces stable CM nitroxide, which can be quantified by ESR.

Figure 2

Quantification of O₂^•− in human lymphoblasts using cell-permeable CMH. (A) Typical ESR spectrum of CM-nitroxide. Arrow indicates low field component of ESR spectrum which was used to monitor accumulation of CM-nitroxide. (B) Kinetics of CM-nitroxide accumulation in the probes with cells (2.5 x 10³/μl), Cu,Zn-SOD (50 U/ml) and PMA (10 μM). Cu,Zn-SOD significantly inhibits CM accumulation both in resting and PMA-stimulated cells.

Figure 3

Inhibition of O₂^•− detection in human lymphoblasts treated by PEG-SOD or Cu-Zn-SOD. Superoxide is measured in unstimulated (A) or PMA-stimulated cells (B) in the presence of cell-permeable PEG-SOD or extracellular Cu,Zn-SOD (50 U/ml). Both PEG-SOD and Cu,Zn-SOD inhibit O₂^•− detection to similar levels.

Figure 4

Superoxide detection in human lymphoblasts treated with PEG-catalase. (A) Typical ESR spectrum of CM-nitroxide. (B) Accumulation of CM-nitroxide in the cellular probes (2.5 x 10³/μl) containing PEG-catalase (100 U/ml), Cu,Zn-SOD (50 U/ml) and PMA (10 μM). (C) Production of O₂^•− calculated as a difference between CM-accumulation in the cellular sample and buffer. PEG-catalase does not affect basal O₂^•− production but attenuates stimulation of cells by PMA.

Figure 4

Superoxide detection in human lymphoblasts treated with PEG-catalase. (A) Typical ESR spectrum of CM-nitroxide. (B) Accumulation of CM-nitroxide in the cellular probes (2.5 x 10³/μl) containing PEG-catalase (100 U/ml), Cu,Zn-SOD (50 U/ml) and PMA (10 μM). (C) Production of O₂^•− calculated as a difference between CM-accumulation in the cellular sample and buffer. PEG-catalase does not affect basal O₂^•− production but attenuates stimulation of cells by PMA.

Figure 5

Dependence of O₂^•− production on the dose and duration of PMA treatment. (A) Suspension of human lymphoblasts (2.5 x 10³/μl) were treated for 5 minutes with different doses of PMA (0–15 μM) before addition of CMH (n=3). (B) Time course of cell activation was tested in cells incubated with 10 μM PMA for various time periods before addition of CMH (n=3).

Figure 6

Dependence of ESR signal on concentrations of cells and CMH. Accumulation of CM-nitroxide was linearly proportional to concentrations of cells (A). Superoxide measurements were performed at different CMH concentrations (B). Amount of O₂^•− was not increased at CMH concentrations above 1mM. Graphs show average of three independent measurements.

Figure 7

Detection of O₂^•− by DEPMPO and CMH in human lymphoblasts. Typical ESR spectra of 50 mM DEPMPO (A) or 1 mM CMH (B) with xanthine oxidase O₂^•− generating system (0.1 mM xanthine + 10 U/ml xanthine oxidase); in the absence of cells; in the presence of 2.5 x 10³/μl cells; cells plus Cu,Zn-SOD (50 U/ml); cells plus PMA (10 μM); cells plus PMA (10 μM) and Cu,Zn-SOD (50 U/ml). Accumulations of the DEPMPO radical adducts (C) or CM-nitroxide (D) in the probes with resting and PMA-stimulated cells in the presence or absence of Cu,Zn-SOD (50 U/ml). Figure shows typical ESR data representative of multiple measurements (n=3).

Figure 8

Detection of O₂^•− by EMPO and CMH in lymphoblasts. Typical ESR spectra of 60 mM EMPO (A) or CMH (B) in the presence or absence of cells (2.5 x 10³/μl); cells plus Cu,Zn-SOD (50 U/ml); cells plus PMA (10 μM); cells plus PMA (10 μM) and Cu,Zn-SOD (50 U/ml). Accumulations of the EMPO radical adducts (C) or CM-nitroxide (D) in the probes with resting and PMA-stimulated cells in the presence or absence of Cu,Zn-SOD (50 U/ml). Figure shows typical ESR data representative of multiple measurements (n=3).