Chlorination of Guanosine and Other Nucleosides by Hypochlorous Acid and Myeloperoxidase of Activated Human Neutrophils

Abstract

Activated human neutrophils secrete myeloperoxidase, which generates HOCl from H 2 O 2 and Cl ؊ . We have found that various (2-deoxy)nucleosides react with HOCl to form chlorinated (2-deoxy)nucleosides, including novel 8-chloro(2-deoxy)guanosine, 5-chloro(2-deoxy)cytidine, and 8-chloro(2-deoxy)adenosine formed in yields of 1.6, 1.6, and 0.2%, respectively, when 0.5 mM nucleoside reacted with 0.5 mM HOCl at pH 7.4. The relative chlorination, oxidation, and nitration activities of HOCl, myeloperoxidase, and activated human neutrophils in the presence and absence of nitrite were studied by analyzing 8-chloro-, 8-oxo-7,8-dihydro-, and 8-nitroguanosine, respectively, using guanosine as a probe. 8-Chloroguanosine was always more easily formed than 8-oxo-7,8-dihydro-or 8-nitro-guanosine. Using electrospray ionization tandem mass spectrometry, we show that several chlorinated nucleosides including 8-chloro(2-deoxy)guanosine are formed following exposure of isolated DNA or RNA to HOCl. Micromolar concentrations of tertiary amines such as nicotine and trimethylamine dramatically enhanced chlorination of free (2-deoxy)nucleosides and nucleosides in RNA by HOCl. As the G؊463A polymorphism of the MPO gene, which strongly reduces myeloproxidase mRNA expression, is associated with a reduced risk of lung cancer, chlorination damage of DNA /RNA and nucleosides by myeloperoxidase and its enhancement by nicotine may be important in the pathophysiology of human diseases associated with tobacco habits.

Myeloperoxidase Generates 5-Chlorouracil in Human Atherosclerotic Tissue

“…1B), which is consistent with the observation that GM-CSF causes myeloperoxidase expression to continue when human monocytes differentiate (11). In accordance with previous studies demonstrating that nicotine (a tertiary amine) promotes the HOCl-dependent oxidation of the deoxynucleosides of adenine, guanine, and cytidine (29,55,56), nicotine dramatically enhanced (ϳ9-fold) the yield of 5-chlorouracil generated by activated macrophages. Cotinine, a nicotine metabolite and tertiary amine, did not promote uracil chlorination.…”

“…Previous work has shown that tertiary amines such as nicotine, trimethylamine, and quinine enhance chlorination of various biological substrates by HOCl (29,30,55,56). Cigarette smoke, a major risk factor for accelerated atherosclerosis and sudden death, contains high concentrations of nicotine, and in the current study this tertiary amine markedly enhanced uracil chlorination by macrophages, the myeloperoxidase-H 2 O 2 -Cl Ϫ system, and N-chloramines.…”

“…Because N-chloramines readily react with primary amines in transchlorination reactions (36), this finding suggests that N-chlorotaurine formed by chlorine transfer from N ⑀ -chloro-N ␣ -acetyllysine also chlorinates uracil. (29,30,55,56), and quinuclidine derivatives activate NH 2 Cl-induced oxidation of NADH (56). To determine whether tertiary amines similarly promote uracil chlorination, we supplemented the N-chloramine reaction mixture with various concentrations of nicotine.…”

Assessment of Myeloperoxidase Activity by the Conversion of Hydroethidine to 2-Chloroethidium

“…However, from our experiments with NETQ, we cannot exclude initial chlorination of the tertiary amine during the conversion of HE to 2-Cl-E ϩ . Indeed, tertiary chloramines have been proposed as intermediates in HOCl-mediated chlorination of guanosine (45), although in the case of HE a tertiary chloramine may deactivate the ring for subsequent chlorination at the C2 position to the primary amine.…”

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

“…Free amino and thiol groups of amino acids and peptides constitute important targets for HOCl, yielding unstable chloramines and sulfenyl chloride intermediates, respectively (14)(15)(16). Chloramine intermediates are also detected in the reaction of HOCl with exocyclic (RNH 2 ) and heterocyclic (RNHR) amine functions in DNA bases (14,17). HOCl reacts with aromatic rings, such as in tyrosine, yielding 3-chlorotyrosine and 3,5-dichlorotyrosine (5,18,19).…”