The objective of the present study is 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 the present study sulfite (•SO3−) and sulfate (SO4•−) 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, PMA-stimulated neutrophils produced DMPO-sulfite anion radical, -superoxide, and -hydroxyl radical adducts. The latter 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 (SO4•−) 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.

Previous studies focused on catalyzed oxidation of (bi)sulfite, leading to the formation of reactive sulfur trioxide (•SO3−), peroxymonosulfate (−O3SOO•) and sulfate (SO4•−) anion radicals, which can damage target proteins and oxidize them to protein radicals. It is known that these very reactive sulfur- and oxygen-centered radicals can be formed by oxidation of (bi)sulfite by peroxidases. Myeloperoxidase (MPO), an abundant heme protein secreted from activated neutrophils that play a central role in host defense mechanisms, allergic reactions and asthma, is a likely candidate for initiating the respiratory damage caused by sulfur dioxide. The objective of the present study is to examine the oxidative damage caused by (bi)sulfite-derived free radicals in human neutrophils through formation of protein radicals. We used immuno-spin trapping and confocal microscopy to study the protein oxidations driven by sulfite-derived radicals. We found that the presence of sulfite can cause MPO-catalyzed oxidation of MPO to a protein radical in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils. We trapped the MPO-derived radicals in situ using the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and detected them immunologically as nitrone adducts in cells. Our present study demonstrates that myeloperoxidase initiates (bi)sulfite oxidation leading to MPO radical damage possibly leading to (bi)sulfite-exacerbated allergic reactions.

The spontaneous addition of air oxygen to a dienolic compound, yielding a cyclic peroxide, was followed by spin trapping (ST) combined with EPR spectroscopy and mass spectrometry (MS). Using two different nitrones, the ST/EPR study allowed the detection of the spin adduct of a radical intermediate, and the radical centre in the addend was identified after similar experiments performed with two different (13)C-labelled analogues of the substrate. The media were also submitted to electrospray ionisation, in both positive and negative modes, for structural characterisation of the spin adducts by tandem mass spectrometry. This allowed the structure of the hydroxylamine derivatives of the nitroxides formed to be identified. Following these results, a mechanism pathway was proposed for this autoxidation.