Formation of reactive sulfite-derived free radicals by the activation of human neutrophils: an ESR study

Abstract

The objective of this study was to determine the effect of (bi)sulfite (hydrated sulfur dioxide) on human neutrophils and the ability of these immune cells to produce reactive free radicals due to (bi)sulfite oxidation. Myeloperoxidase (MPO) is an abundant heme protein in neutrophils that catalyzes the formation of cytotoxic oxidants implicated in asthma and inflammatory disorders. In this study sulfite ((•)SO(3)(-)) and sulfate (SO(4)(•-)) anion radicals are characterized with the ESR spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in the reaction of (bi)sulfite oxidation by human MPO and human neutrophils via sulfite radical chain reaction chemistry. After treatment with (bi)sulfite, phorbol 12-myristate 13-acetate-stimulated neutrophils produced DMPO-sulfite anion radical, -superoxide, and -hydroxyl radical adducts. The last adduct probably resulted, in part, from the conversion of DMPO-sulfate to DMPO-hydroxyl radical adduct via a nucleophilic substitution reaction of the radical adduct. This anion radical (SO(4)(•-)) is highly reactive and, presumably, can oxidize target proteins to protein radicals, thereby initiating protein oxidation. Therefore, we propose that the potential toxicity of (bi)sulfite during pulmonary inflammation or lung-associated diseases such as asthma may be related to free radical formation.

Fig. 1

Reduction of MPO-compound II by sulfite. (A) Pseudo-first-order rate constants for reduction of MPO-compound I by (bi)sulfite. The second-order rate constant is calculated from the slope. The inset shows the time traces of the reaction followed at 430 nm using the sequential mixing mode. Final concentrations were 1.25 μM MPO and12.5 μM H₂O₂, and the concentration of (bi)sulfite for each time trace was (a) 12.5 μM, (b) 62.5 μM, (c) 125 μM, (d) 625 μM, and (e) 1.25 mM. (B) Spectral changes upon addition of 50 μM Na₂SO₃ to MPO-compound II. The resting MPO was recorded first (spectrum a). MPO-compound II was formed by mixing 400 nM ferric MPO with 300 nM homovanillic acid (HVA) and 50 μM H₂O₂ and waiting for 1 min (b). Spectrum c was taken 5 min after the addition of sulfite, and the resting enzyme was reformed after 30 min (spectrum d). (C) Pseudo-first-order rate constants for reduction of MPO-compound II by (bi)sulfite. The inset shows the time traces and fits of the reduction of compound II at pH 7.4 by Na₂SO₃. The concentration of (bi)sulfite for each time trace was (a) 10 μM, (b) 20 μM, (c) 50 μM, and (d) 100 μM.

Fig. 1

Reduction of MPO-compound II by sulfite. (A) Pseudo-first-order rate constants for reduction of MPO-compound I by (bi)sulfite. The second-order rate constant is calculated from the slope. The inset shows the time traces of the reaction followed at 430 nm using the sequential mixing mode. Final concentrations were 1.25 μM MPO and12.5 μM H₂O₂, and the concentration of (bi)sulfite for each time trace was (a) 12.5 μM, (b) 62.5 μM, (c) 125 μM, (d) 625 μM, and (e) 1.25 mM. (B) Spectral changes upon addition of 50 μM Na₂SO₃ to MPO-compound II. The resting MPO was recorded first (spectrum a). MPO-compound II was formed by mixing 400 nM ferric MPO with 300 nM homovanillic acid (HVA) and 50 μM H₂O₂ and waiting for 1 min (b). Spectrum c was taken 5 min after the addition of sulfite, and the resting enzyme was reformed after 30 min (spectrum d). (C) Pseudo-first-order rate constants for reduction of MPO-compound II by (bi)sulfite. The inset shows the time traces and fits of the reduction of compound II at pH 7.4 by Na₂SO₃. The concentration of (bi)sulfite for each time trace was (a) 10 μM, (b) 20 μM, (c) 50 μM, and (d) 100 μM.

Fig. 1

Reduction of MPO-compound II by sulfite. (A) Pseudo-first-order rate constants for reduction of MPO-compound I by (bi)sulfite. The second-order rate constant is calculated from the slope. The inset shows the time traces of the reaction followed at 430 nm using the sequential mixing mode. Final concentrations were 1.25 μM MPO and12.5 μM H₂O₂, and the concentration of (bi)sulfite for each time trace was (a) 12.5 μM, (b) 62.5 μM, (c) 125 μM, (d) 625 μM, and (e) 1.25 mM. (B) Spectral changes upon addition of 50 μM Na₂SO₃ to MPO-compound II. The resting MPO was recorded first (spectrum a). MPO-compound II was formed by mixing 400 nM ferric MPO with 300 nM homovanillic acid (HVA) and 50 μM H₂O₂ and waiting for 1 min (b). Spectrum c was taken 5 min after the addition of sulfite, and the resting enzyme was reformed after 30 min (spectrum d). (C) Pseudo-first-order rate constants for reduction of MPO-compound II by (bi)sulfite. The inset shows the time traces and fits of the reduction of compound II at pH 7.4 by Na₂SO₃. The concentration of (bi)sulfite for each time trace was (a) 10 μM, (b) 20 μM, (c) 50 μM, and (d) 100 μM.

Fig. 2

Formation of radical adducts in reaction between sulfite (Na₂SO₃) and myeloperoxidase (MPO)/hydrogen peroxide (H₂O₂) as a function of DMPO concentrations and time. (A) Reactions including Na₂SO₃ (1 mM) and MPO (1 μM) were initiated with H₂O₂ (100 μM) in 100 mM phosphate buffer (pH 7.4) in the presence of various concentrations of DMPO. After initiation with H₂O₂, the mixture was immediately placed into the flat cell. The concentration of the spin trap for each spectrum was (a) 100 mM, (b) 50 mM, (c) 20 mM, and (d) 6 mM. (B) Spectrum a is the same as spectrum d in Panel (A). Spectrum b is the composite simulation of 43% DMPO/^•OSO₃⁻, 33% DMPO/^•OH, and 24% DMPO/^•SO₃⁻. Spectrum c is the computer simulation of DMPO/^•OSO₃⁻ radical adduct (a^N = 13.7 G, a^H_β = 10.1 G, a^H_γ1 = 1.42 G, and a^H_γ2 = 0.75 G). Spectrum d is the simulation of DMPO/^•OH (a^N = 14.9 G, a^H_β = 14.9 G). Spectrum e is the simulation of DMPO/^•SO₃⁻ (a^N = 14.6 G, a^H_β = 16.2 G). (C) A reaction mixture containing Na₂SO₃ (1 mM), DMPO (6 mM), and MPO (1 μM) was initiated with H₂O₂ (100 μM) in 100 mM phosphate buffer (pH 7.4) and immediately placed into the flat cell (spectrum a). Spectra b, c, and d were detected after 3, 6, and 10 min, respectively. Spectrum e was detected immediately after the initiation, but in the presence of 100 mM DMSO. Spectrum f was detected immediately after the initiation, but in the presence of 100 mM HCOONa (the appearance of DMPO/^•CO₂⁻ radical adduct is marked with asterisks; a^N = 15.8 G and a^H_β = 18.7 G).

Fig. 3

Formation of radical adducts in reaction between sulfite (Na₂SO₃) and human neutrophils upon PMA activation in the presence of DMPO. (Spectrum a) Reaction mixture containing 1 × 10⁷ cells/ml, Na₂SO₃ (1 mM), and DMPO (100 mM) in PBS, pH 7.4. After cell activation with PMA (500 ng/ml) followed by incubation at 37°C for 3 min, the mixture was placed into the flat cell. The dotted spectrum is the composite computer simulation of 26% DMPO/^•OOH (a^N = 14.1 G, a^H_β = 11.2 G, and a^H_γ = 1.24 G), 42% DMPO/^•OH (a^N = 14.9 G and a^H_β = 14.9 G), and 32% DMPO/^•SO₃⁻ (a^N = 14.7 G and a^H_β = 16.0 G) radical adducts. (Spectrum b) Same as in spectrum a without Na₂SO₃. (Spectrum c) Same as in spectrum a, except PMA was not added. (Spectrum d) Same as in spectrum a without Na₂SO₃ and PMA. (Spectrum e) Same as inspectrum a without neutrophils.

Fig. 4

Effect of halides and pseudohalides on the formation of DMPO/^•SO₃⁻ radical. A reaction mixture containing 1 × 10⁷ cells/ml, Na₂SO₃ (1 mM), and DMPO (100 mM) in PBS, pH 7.4. Na₂SO₃ (1 mM) was used for the remaining experiments. After cell activation with PMA (500 ng/ml), the mixture was placed into the flat cell. The spectrum was attenuated in the presence of 100 mM NaCl (spectrum b), 100 μM NaBr (spectrum c), 100 μM NaSCN (spectrum d), and in the presence of the mixture of NaCl, NaBr, and NaSCN with the indicated concentrations (spectrum e).

Fig. 5

Effect of inhibitors and radical scavengers on the formation of DMPO/^•SO₃⁻ radical. Reaction mixture containing 1 × 10⁷ cells/ml, Na₂SO₃ (1 mM), and DMPO (100 mM) in PBS, pH 7.4. Azide and ABAH were preincubated for 15 min before the addition of DMPO. After cell activation with PMA (500 ng/ml), the mixture was placed into the flat cell. Na₂SO₃ (1 mM) was used for the remaining experiments. The spectrum was attenuated in the presence of NaN₃ (500 μM) (spectrum b), ABAH (500 μM) (spectrum c), catalase (150 μg/ml) (spectrum d), and Cu, Zn-SOD (50 μM) (spectrum e).

Fig. 6

Effect of Gd-DTPA as a line-broadening agent on the DMPO/^•SO₃⁻ radical adduct formation. The ESR spectrum of the DMPO/^•SO₃⁻ generated in a system of neutrophils (1 × 10⁷ cells/ml), Na₂SO₃ (1 mM), and DMPO (100 mM) in PBS, pH 7.4. After cell activation with PMA (500 ng/ml), the mixture was placed into the flat cell (spectrum a). (Spectrum b) Same as in spectrum a, but in the presence of 20 mM Gd-DTPA added prior to cell activation. (Spectrum c) Same as in spectrum b, but without neutrophils. (Spectrum d) Same as in spectrum a, but in the presence of 20 mM La-DTPA added prior to cell activation.

Scheme 1

Scheme 2