Bromination of deoxycytidine by eosinophil peroxidase: A mechanism for mutagenesis by oxidative damage of nucleotide precursors Jeffrey P. Henderson*, Jaeman Byun*, Michelle V. Williams*, Michael L. McCormick†, William C. Parks‡, Lisa A. Ridnour§, and Jay W. Heinecke*¶ʈ *Departments of Medicine, ‡Pediatrics, §Radiology, and ¶Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110; and †Research Service, Veterans Administration Medical Center and Department of Internal Medicine, University of Iowa, Iowa City, IA 52241 Edited by Bernard M. Babior, The Scripps Research Institute, La Jolla, CA, and approved December 19, 2000 (received for review April 3, 2000) Oxidants generated by eosinophils during chronic inflammation eggs of which trigger an intense eosinophilic granulomatous may lead to mutagenesis in adjacent epithelial cells. Eosinophil reaction. Many lines of evidence implicate Schistosoma, which is peroxidase, a heme enzyme released by eosinophils, generates distributed widely in Africa, the Middle East, and Asia in the hypobromous acid that damages tissue in inflammatory condi- genesis of bladder cancer, liver cancer, and colon cancer (21). tions. We show that human eosinophils use eosinophil peroxidase Thus, oxidants generated by eosinophils during a chronic in- to produce 5-bromodeoxycytidine. Flow cytometric, immunohisto- flammatory response may lead to mutagenesis in adjacent chemical, and mass spectrometric analyses all demonstrated that epithelial cells. 5-bromodeoxycytidine generated by eosinophil peroxidase was Oxidative damage of DNA has been implicated in carcino- taken up by cultured cells and incorporated into genomic DNA as genesis (2, 3, 6, 22, 23). In vitro studies have identified numerous 5-bromodeoxyuridine. Although previous studies have focused on modified nucleic acids as products of phagocytic oxidative reactions. For example, activated phagocytes generate the mu- oxidation of chromosomal DNA, our observations suggest another tagenic nucleobase 8-oxoguanine by pathways that may involve mechanism for oxidative damage of DNA. In this scenario, perox- hydroxyl radical or singlet oxygen (24). Nitric oxide, or its idase-catalyzed halogenation of nucleotide precursors yields prod- degradation products, deaminate nucleobases in vitro and en- ucts that subsequently can be incorporated into DNA. Because the hance mutagenesis in cultured cells (25, 26). Human neutrophils thymine analog 5-BrUra mispairs with guanine in DNA, generation produce 8-nitrodeoxyguanosine by a pathway involving myelo- of brominated pyrimidines by eosinophils might constitute a mech- peroxidase, nitrite, and H2O2 (27). Moreover, we showed re- anism for cytotoxicity and mutagenesis at sites of inflammation. cently that myeloperoxidase will also convert deoxycytidine (dC) to 5-chlorodeoxycytidine (28). nder normal conditions, eosinophils are a minor component Most studies have focused on oxidative damage of chromo- Uof circulating phagocytic white blood cells. However, they somal DNA, but we reasoned that potentially mutagenic adducts become much more abundant in blood and tissues in a variety of might be generated in the abundant cellular pools of ribo- and inflammatory disorders, including chronic allergic conditions, deoxyribonucleoside precursors. In the current studies, we used deoxynucleosides to determine whether the brominating inter- helminthic infections, and cancer (1). Moreover, eosinophils are mediates generated by eosinophil peroxidase can modify nucleic implicated in tissue damage associated with asthma, dermatitis, acid bases. Through MS and NMR spectroscopy, we identified vasculitis, and the hypereosinophilia syndromes. These granu- 5-bromodeoxycytidine (BrdC) as a product at physiologically locytes also may play important roles in host defenses against plausible BrϪ and ClϪ concentrations. Activated eosinophils microbial parasites and tumor cells. One potential mechanism of generated BrdC in a reaction that was inhibited by catalase, eosinophil-mediated damage and defense involves their produc- implicating a peroxidase in the cellular pathway. Cultured cells CELL BIOLOGY tion of oxidizing intermediates. incorporated the BrdC into genomic DNA as BrdUrd, a well Chronic inflammation is associated with an increased risk of established mutagen. Our observations indicate that brominat- cancer, raising the possibility that reactive intermediates gener- ing intermediates generated by eosinophil peroxidase represent ated by eosinophils, neutrophils, monocytes, and macrophages one potential pathway for modifying nucleic acids at sites of might damage nucleic acids and compromise the integrity of the inflammation in vivo. They also suggest a mechanism for the genome (2–6). Activated eosinophils generate such intermedi- oxidative damage of DNA by phagocytes. In this scenario, ates by first producing superoxide using a membrane-associated reactive species halogenate nucleotides and nucleotide precur- NADPH oxidase (7, 8). Superoxide then dismutates to hydrogen sors that subsequently are incorporated into DNA, in which they can exert cytotoxic and mutagenic effects. peroxide (H2O2), which is the oxidizing substrate for eosinophil peroxidase, a granule enzyme. Reactive intermediates generated Experimental Procedures by eosinophil peroxidase may represent one pathway for tissue damage by eosinophils (9–14). At plasma concentrations of Peroxidase Activity Assay. Porcine eosinophil peroxidase (ExOx- halide ion (ClϪ Ϸ 100 mM, BrϪ 20–100 ␮M, IϪ Ͻ1 ␮M; refs. 15 Emis, Little Rock, AR) yielded a single band of active material as assessed by nondenaturing PAGE (29, 30). and 16), the major product of eosinophil peroxidase is thought to be hypobromous acid (HOBr; refs. 9, 11, and 13). Ϫ ϩ ϩ ϩ 3 ϩ This paper was submitted directly (Track II) to the PNAS office. Br H2O2 H HOBr H2O [1] Abbreviations: dC, deoxycytidine; BrdC, 5-bromodeoxycytidine. The pseudohalide thiocyanate also has been proposed to be a ʈTo whom reprint requests should be addressed at: Division of Atherosclerosis, Nutrition major substrate (17). Recent studies provide compelling evi- and Lipid Research, Campus Box 8046, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail: [email protected]. dence that HOBr generated by activated eosinophils is one The publication costs of this article were defrayed in part by page charge payment. This pathway for tissue damage in asthma (18). article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. A striking example in which inflammation-mediated DNA §1734 solely to indicate this fact. damage may lead to a carcinogenic insult is schistosomiasis (19, Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.041146998. 20). This disease is caused by the blood fluke Schistosoma, the Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.041146998 PNAS ͉ February 13, 2001 ͉ vol. 98 ͉ no. 4 ͉ 1631–1636 Downloaded by guest on September 27, 2021 Hypobromous Acid. Bromide-free HOBr was prepared by addition of silver nitrate to Ϸ80 mM bromine water (1.5:1, mol͞mol) and vacuum distillation (31). Reagent taurine monobromamine was prepared by adding HOBr to a 100-fold mole excess of taurine. Oxidation of dC. Reactions were performed in gas-tight vials and initiated by adding oxidant from a gas-tight syringe through the septum while vortexing the sample. Reactions were terminated by adding L-methionine to a final concentration of 6 mM. HOBr ␧ ϭ Ϫ1⅐ Ϫ1 ␧ and taurine bromamine ( 240 43.6 M cm ), and H2O2 ( 288 ϭ 430 MϪ1⅐cmϪ1) concentrations were determined spectropho- tometrically (13, 32). The pH dependence of product formation was determined as described (28). Human Eosinophils. Human polymorphonuclear cells were pre- pared from blood by density-gradient centrifugation (33). Neu- Fig. 1. Reverse-phase HPLC analyses of dC oxidized by the eosinophil perox- trophils were removed by passage over beads coupled to anti- Ϫ Ͼ idase-H2O2-Br system. dC (1 mM) was incubated with 3 nM eosinophil per- CD16 antibody (R & D Systems). Cells ( 95% eosinophils and oxidase (EPO), 100 ␮MH2O2, and 100 ␮M NaBr in buffer A (50 mM sodium Ͻ5% lymphocytes) were suspended in PBS (100 mM NaCl͞10 phosphate͞100 mM sodium chloride͞100 ␮M diethylenetriaminepentaacetic ͞ Ϫ mM sodium phosphate supplemented with 2 mM dextrose 1.4 acid, pH 4.5) for 60 min at 37°C (EPO͞H2O2͞Br ). Where indicated, eosinophil ͞ ͞ ͞ ␮ ͞ ␮ ͞ Ϫ mM CaCl2 1.4 mM MgCl2 1mMdC100 M NaBr 100 M peroxidase was omitted from the reaction mixture (H2O2 Br ). Also shown is diethylenetriaminepentaacetic acid), incubated at 37°C for 60 a chromatogram of 20 ␮M authentic BrdC. Reactions were initiated by adding min, and maintained in suspension with intermittent inversion. H2O2 and terminated with 6 mM L-methionine. The reaction was terminated by addition of 6 mM methionine and removal of cells by centrifugation. antibromodeoxyuridine monoclonal antibody (Becton-Dickin- son Immunocytometry Systems) and FITC-conjugated goat Reverse-Phase HPLC. Reaction mixtures were analyzed by reverse- ␮ ϫ anti-mouse IgG (Sigma). Flow-cytometric analysis was carried phase HPLC with a C18 column (Porasil, 5- m resin, 4.6 250 mm; Beckman–Altex) at a flow of 1 ml͞min with UV detection at 295 out on a Becton-Dickinson FACS 440 equipped with an I-90–5 nm (28). For NMR analysis, concentrated reaction mixtures were UV laser (Coherent Radiation, Palo Alto, CA). Data from at least 20,000 nuclei were collected. For immunohistochemistry, fractionated