Molecular Chlorine Generated by the Myeloperoxidase-Hydrogen Peroxide-Chloride System of Phagocytes Produces 5-Chlorocytosine in Bacterial RNA

Abstract

Myeloperoxidase, a heme enzyme secreted by activated phagocytes, uses H 2 O 2 and Cl؊ to generate the chlorinating intermediate hypochlorous acid (HOCl). This potent cytotoxic oxidant plays a critical role in host defenses against invading pathogens. In this study, we explore the possibility that myeloperoxidase-derived HOCl might oxidize nucleic acids. When we exposed 2-deoxycytidine to the myeloperoxidase-H 2 O 2 -Cl ؊ system, we obtained a single major product that was identified as 5-chloro-2-deoxycytidine using mass spectrometry, high performance liquid chromatography, UV-visible spectroscopy, and NMR spectroscopy. 5-Chloro-2-deoxycytidine production by myeloperoxidase required H 2 O 2 and Cl and H؉ , in the generation of 5-chloro-2-deoxycytidine. Activated human neutrophils were able to generate 5-chloro-2-deoxycytidine. Cellular chlorination was blocked by catalase and heme poisons, consistent with a myeloperoxidase-catalyzed reaction. The myeloperoxidase-H 2 O 2 -Cl ؊ system generated similar levels of 5-chlorocytosine in RNA and DNA in vitro. In striking contrast, only cell-associated RNA acquired detectable levels of 5-chlorocytosine when intact Escherichia coli was exposed to the myeloperoxidase system. This observation suggests that oxidizing intermediates generated by myeloperoxidase selectively target intracellular RNA for chlorination. Collectively, these results indicate that Cl 2 derived from HOCl generates 5-chloro-2-deoxycytidine during the myeloperoxidase-catalyzed oxidation of 2-deoxycytidine. Phagocytic generation of Cl 2 therefore may constitute one mechanism for oxidizing nucleic acids at sites of inflammation.

Production of Brominating Intermediates by Myeloperoxidase

“…For NMR analysis, 10-fold concentrated reaction mixtures were fractionated on a semipreparative C 18 column (Porasil; 5 m resin, 10 ϫ 250 mm; Beckman) at a flow rate of 2.5 ml/min with an isocratic gradient consisting of 90% 20 mM ammonium formate, pH 6.3, and 10% methanol. N-Chlorodeoxycytidine (retention time, 10 min) was prepared as described (20) and isolated with an analytical C 18 column (Porasil; 5 m resin, 4.6 ϫ 250 mm; Beckman) column using 5% methanol at a flow rate of 1 ml/min.…”

“…Cl 2 generated by this pathway has been implicated in the production of 3-chlorotyrosine and 5-chlorodeoxycytidine by activated neutrophils (12,20). Elevated levels of protein-bound 3-chlorotyrosine and myeloperoxidase are found in human atherosclerotic tissue, strongly suggesting that oxidative reactions involving HOCl damage proteins in this chronic inflammatory disorder (13,21).…”

“…We previously demonstrated that HOCl generated by myeloperoxidase is in equilibrium with molecular chlorine (Cl 2 ) through a reaction that requires chloride (Cl Ϫ ) and H ϩ (12,20).…”

Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease

“…A prominent target of HOCl is tyrosine, which leads to the formation of the stable aromatic residue, 3-chlorotyrosine (Cl-Tyr) (21,22). MPO also produces chlorinating species that react with DNA to form chlorinated adducts such as 5-chloro-2′-deoxycytidine (5-Cl-dC) (23), the presence of which was identified in colon tissue of H. hepaticus-infected Rag2 −/− mice (10). This modification of DNA may provide a mechanistic link between neutrophil activity and colitis-associated carcinoma (10,24,25).…”

Linoleic acid hydroperoxide reacts with hypochlorous acid, generating peroxyl radical intermediates and singlet molecular oxygen

“…3) (11). In vitro studies suggest that Cl 2 might be the chlorinating agent that mediates the formation of chlorinated products during phagocytosis (11)(12)(13).…”