Hypochlorite and superoxide radicals can act synergistically to induce fragmentation of hyaluronan and chondroitin sulphates

Activated phagocytes release the haem enzyme MPO (myeloperoxidase) and also generate superoxide radicals (O₂^•−), and hence H₂O₂, via an oxidative burst. Reaction of MPO with H₂O₂ in the presence of chloride ions generates HOCl (the physiological mixture of hypochlorous acid and its anion present at pH 7.4). Exposure of glycosaminoglycans to a MPO–H₂O₂–Cl⁻ system or reagent HOCl generates long-lived chloramides [R-NCl-C(O)-R′] derived from the glycosamine N-acetyl functions. Decomposition of these species by transition metal ions gives polymer-derived amidyl (nitrogen-centred) radicals [R-N^•-C(O)-R′], polymer-derived carbon-centred radicals and site-specific strand scission. In the present study, we have shown that exposure of glycosaminoglycan chloramides to O₂^•− also promotes chloramide decomposition and glycosaminoglycan fragmentation. These processes are inhibited by superoxide dismutase, metal ion chelators and the metal ion-binding protein BSA, consistent with chloramide decomposition and polymer fragmentation occurring via O₂^•−-dependent one-electron reduction, possibly catalysed by trace metal ions. Polymer fragmentation induced by O₂^•− [generated by the superoxide thermal source 1, di-(4-carboxybenzyl)hyponitrite] was demonstrated to be entirely chloramide dependent as no fragmentation occurred with the native polymers or when the chloramides were quenched by prior treatment with methionine. EPR spin-trapping experiments using 5,5-dimethyl1-pyrroline-N-oxide and 2-methyl-2-nitrosopropane have provided evidence for both O₂^•− and polymer-derived carbon-centred radicals as intermediates. The results obtained are consistent with a mechanism involving one-electron reduction of the chloramides to yield polymer-derived amidyl radicals, which subsequently undergo intramolecular hydrogen atom abstraction reactions to give carbon-centred radicals. The latter undergo fragmentation reactions in a site-specific manner. This synergistic damage to glycosaminoglycans induced by HOCl and O₂^•− may be of significance at sites of inflammation where both oxidants are generated concurrently.