From the Departments of Medicine and
Molecular
Biology and Pharmacology, Washington University School of Medicine, St.
Louis, Missouri 63110 and ¶ Research Service, Veterans Affairs
Medical Center and Department of Internal Medicine, University of Iowa,
Iowa City, Iowa 52241
Received for publication, June 20, 2000, and in revised form, October 27, 2000
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ABSTRACT |
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The existence of interhalogen compounds was
proposed more than a century ago, but no biological roles have been
attributed to these highly oxidizing intermediates. In this study, we
determined whether the peroxidases of white blood cells can generate
the interhalogen gas bromine chloride (BrCl). Myeloperoxidase, the heme
enzyme secreted by activated neutrophils and monocytes, uses H2O2 and Cl Reactive oxidants generated by phagocytic white blood cells are
critical to host defense because they kill invading pathogens (1-5).
However, they are also potentially dangerous because they may damage
tissues at sites of inflammation. The heme enzyme myeloperoxidase, synthesized and secreted by neutrophils and monocytic cells, is an
important source of oxidants. It uses H2O2
generated by the phagocyte NADPH oxidase to produce potent cytotoxins.
At plasma halide concentrations, its major initial product is HOCl (6, 7).
to produce
HOCl, a chlorinating intermediate. In contrast, eosinophil peroxidase
preferentially converts Br
to HOBr. Remarkably, both
myeloperoxidase and eosinophil peroxidase were able to brominate
deoxycytidine, a nucleoside, and uracil, a nucleobase, at plasma
concentrations of Br
(100 µM) and
Cl
(100 mM). The two enzymes used different
reaction pathways, however. When HOCl brominated deoxycytidine, the
reaction required Br
and was inhibited by taurine. In
contrast, bromination by HOBr was independent of Br
and
unaffected by taurine. Moreover, taurine inhibited 5-bromodeoxycytidine production by the
myeloperoxidase-H2O2-Cl
-
Br
system but not by the eosinophil
peroxidase-H2O2-Cl
-Br
system, indicating that bromination by myeloperoxidase involves the
initial production of HOCl. Both HOCl-Br
and the
myeloperoxidase-H2O2-Cl
-Br
system generated a gas that converted cyclohexene into
1-bromo-2-chlorocyclohexane, implicating BrCl in the reaction.
Moreover, human neutrophils used myeloperoxidase,
H2O2, and Br
to brominate
deoxycytidine by a taurine-sensitive pathway, suggesting that
transhalogenation reactions may be physiologically relevant. 5-Bromouracil incorporated into nuclear DNA is a well known mutagen. Our observations therefore raise the possibility that transhalogenation reactions initiated by phagocytes provide one pathway for mutagenesis and cytotoxicity at sites of inflammation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
HOCl can oxidize sulfhydryl groups (8), halogenate and oxygenate
unsaturated lipids (9, 10), and halogenate aromatic compounds (11-13).
Myeloperoxidase-derived chlorinating agents also generate secondary
oxidants such as monochloramines, dichloramines (14-16), and amino
acid-derived aldehydes (17-19).
(Eq. 1)
We previously demonstrated that HOCl generated by myeloperoxidase is in
equilibrium with molecular chlorine (Cl2) through a
reaction that requires chloride (Cl) and H+
(12, 20).
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(Eq. 2) |
Chronic inflammation also increases the risk of cancer, raising the possibility that reactive intermediates generated by neutrophils, monocytes, and macrophages might damage nucleic acids and compromise the integrity of the genome (22-24). Genetic epidemiological studies have revealed that a polymorphism in the myeloperoxidase promoter region alters the risk for various cancers (25-30). These results suggest that myeloperoxidase may play an important role in carcinogenesis, perhaps by generating mutagenic oxidants during the inflammatory response.
A structurally related heme protein, eosinophil peroxidase, is released
by activated eosinophils, which help kill invading parasites. This
peroxidase contributes to the characteristic staining of eosinophils.
At plasma concentrations of halide (100 mM
Cl, 20-100 µM bromide, <1
µM iodide; Refs. 31 and 32), eosinophil peroxidase
preferentially oxidizes bromide (Br
) to produce the
potent brominating agent HOBr (33-35).
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(Eq. 3) |
More than 100 years ago, inorganic chemists proposed the existence of
interhalogens, which are combinations of different halogens (XXn'). Both binary (BrCl, IBr, and ICl) and ternary (ICl3) interhalogens have since been characterized. One
pathway for their formation requires hypohalous acid (HOX) and halide ion (X; Refs. 40 and 41).
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(Eq. 4) |
In the current studies, we show that myeloperoxidase generates reactive
brominating species that oxidize nucleobases by a reaction involving
HOCl, Br, and formation of BrCl, an interhalogen gas. We
also found that human neutrophils used myeloperoxidase,
Cl
, and Br
to brominate deoxycytidine,
suggesting that transhalogenation reactions may be physiologically
relevant. Our observations suggest that transhalogenation reactions
executed by phagocytes may represent one pathway for mutagenesis and
cytotoxicity at sites of inflammation.
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EXPERIMENTAL PROCEDURES |
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Materials
Organic solvents, H2O2, sodium hypochlorite, and sodium phosphate were obtained from Fisher. Bis(trimethylsilyl)trifluoroacetamide and silylation grade acetonitrile were from Regis Technologies, Inc. (Morton Grove, IL). All other materials were purchased from Sigma, except where indicated.
Methods
Myeloperoxidase and Eosinophil Peroxidase--
Myeloperoxidase
(A430/A280 > 0.8) was
isolated from HL-60 cells by sequential lectin affinity, ion exchange,
and size exclusion chromatographies (43, 44). Enzyme concentration was
determined spectrophotometrically (430 = 178 mM
1 cm
1; Ref. 45). Porcine
eosinophil peroxidase (Ahis/A280 > 0.9) was provided by ExOxEmis (San Antonio, TX).
Peroxidase Activity Assay-- The purity of myeloperoxidase and eosinophil peroxidase were assessed by peroxidase activity using nondenaturing polyacrylamide slab gel electrophoresis and gel system 8 (46, 47). Glycerol (25% w/v) and cetyltrimethylammonium bromide (0.05% w/v) were included in all buffers. Riboflavin (0.024 mg/ml) was used as the polymerization catalyst, and the stacking gel was omitted. Peroxidase activity was visualized by incubating the gel in 400 µM tetramethylbenzidine, 10 mM sodium citrate, pH 5, 10 mM EDTA, 5 mM NaBr, and 200 µM H2O2.
Preparation of Hypochlorous Acid and Taurine
Chloramine--
Chloride-free NaOCl was prepared by a modification of
previously described methods (16). Reagent NaOCl (100 ml) mixed with ethyl acetate (100 ml) was protonated by dropwise addition of concentrated phosphoric acid (final pH 6) with intermittent shaking. The organic phase containing HOCl was washed twice with water,
and HOCl was re-extracted into the aqueous phase by dropwise addition
of NaOH (final pH
9). Residual ethyl acetate in the aqueous
solution of chloride-free NaOCl was removed by bubbling with nitrogen
gas. The concentration of NaOCl was determined spectrophotometrically (
292 = 350 M
1
cm
1; Ref. 48). Taurine monochloramine was prepared by
addition of HOCl to taurine (1:100; mol/mol). Taurine monochloramine
concentration was determined spectrophotometrically (
252 = 429 M
1 cm
1; Ref. 16)
Preparation of Hypobromous Acid and Taurine
Bromamine--
Bromide-free HOBr was prepared as described (49).
Briefly, silver nitrate solution was added to ~80 mM
bromine water in a molar ratio of 1.5:1. The precipitate was removed by
centrifugation, and 30 ml of the supernatant was distilled under vacuum
using a foil-covered microscale distillation apparatus. The distillate was collected in a foil covered vial at 4 °C. Reagent taurine monobromamine was prepared by addition of reagent HOBr to a 100-fold excess of taurine. HOBr concentration was determined
spectrophotometrically following formation of taurine monobromamine
(288 = 430 M
1
cm
1; Ref. 35).
Oxidation of Uracil and Deoxycytidine--
All reactions were
performed in gas-tight vials and initiated by addition with a gas-tight
syringe of oxidant (H2O2 or
HOCl/OCl) through a septum while vortexing the sample.
Reactions were terminated by addition of L-methionine to a
final concentration of 6 mM. The concentration of
H2O2 was determined spectrophotometrically (
240 = 43.6 M
1
cm
1; Ref. 50). The pH dependence of 5-bromouracil and
5-bromodeoxycytidine formation was performed using reaction mixtures
containing phosphoric acid, monobasic sodium phosphate, and dibasic
sodium phosphate (final concentration, 50 mM). The pH of
the reaction mixture (which did not contain L-methionine)
was determined at the end of the incubation.
Human Neutrophils-- Neutrophils were prepared by density gradient centrifugation (51) and suspended in Dulbecco's phosphate-buffered saline supplemented with 1 mg/ml dextrose, 1 mM deoxycytidine, 100 µM NaBr, and 100 µM DTPA,2 pH 5.9. Differential cell counts revealed that neutrophil preparations contained 96-100% neutrophils and 0-4% eosinophils. Cells (3 ml) were activated with 200 nM phorbol myristate acetate, incubated at 37 °C for 60 min, and maintained in suspension with intermittent inversion. The reaction was terminated by addition of methionine to 6 mM and centrifugation of the cells at 400 × g for 10 min. The supernatant was concentrated to dryness under vacuum, dissolved in 0.4 ml of HPLC solvent A, centrifuged at 14,000 × g for 10 min, and the supernatant was subjected to HPLC analysis.
Reverse-phase HPLC of Reaction Products-- Uracil and deoxycytidine reaction products were analyzed by reverse-phase HPLC with a C18 column (Beckman Porasil, 5 µm resin, 4.6 × 250 mm) at a flow of 1 ml/min and UV detection at 274 and 295 nm, respectively. Uracil reactions were analyzed by injection of 100 µl of reaction mixture onto the column followed by isocratic elution with 20 mM ammonium formate. For analysis of deoxycytidine reactions, 100 µl of the reaction mixture was injected onto the column and eluted with a gradient of: 95% solvent A (0.1% trifluoroacetic acid, pH 2.5) and 5% solvent B (0.1% trifluoroacetic acid in methanol, pH 2.5) for 4 min, 5-100% solvent B over 20 min, and then 100% solvent B for 10 min. 5-Bromouracil and 5-bromodeoxycytidine yields were quantified by comparison of integrated peak areas to standard curves generated using commercially available compounds. For mass spectrometric analysis, HPLC fractions were collected and concentrated under vacuum. For NMR analysis, 10-fold concentrated reaction mixtures were fractionated on a semi-preparative C18 column (µPorasil; 5 µm resin, 10 × 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 C18 column (µPorasil; 5 µm resin, 4.6 × 250 mm; Beckman) column using 5% methanol at a flow rate of 1 ml/min.
NMR Studies-- Reaction products were isolated by HPLC, solubilized in D2O, and analyzed at 25 °C with a Varian Unity-Plus 500 spectrometer (499.843 MHz for 1H) equipped with a Nalorac indirect detection probe. 1H Chemical shifts were referenced to external sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 in D2O. Spectra were recorded from 8 transients with a 12-s preacquisition delay over a spectral width of 8000 Hz. Pyrimidine resonances of the brominated deoxycytidine (8.23 ppm; singlet, H6) and uracil (7.79 ppm; singlet, H6) reaction products were essentially identical to those of commercially available 5-bromodeoxycytidine and 5-bromouracil. When compared with substrate, the aromatic region of each product spectrum was notable for the lack of proton resonances at C-5, a downfield shift in the proton resonance at C-6, and conversion of the C-6 proton resonance from a doublet to a singlet, both of which are consistent with substitution of a bromine atom at the C-5 position.
Gas Chromatography-Mass Spectrometry (GC/MS)-- After nucleobases were dried under vacuum, residual water was removed by forming an azeotrope with 50 µl of pyridine and again drying the suspension under vacuum. DNA bases were converted to trimethylsilyl derivatives with excess bis(trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane in acetonitrile (3:1 v/v) at 100 °C for 60 min. Aliquots (1 µl) were analyzed in the positive electron ionization mode using full mass scanning on either a Hewlett Packard 5890 Series II gas chromatograph (Santa Clarita, CA) interfaced with a Hewlett Packard 5972 Series Mass Selective Detector or a Varian Star 3400 CX gas chromatograph (Walnut Creek, CA) interfaced with a Finnegan SSQ 7000 mass spectrometer (San Jose, CA). Each gas chromatograph was equipped with a 12-m DB-1 capillary column (inner diameter, 0.2 mm; film thickness, 0.33 µm; J&W Scientific, Folsom, CA). Injector and interface temperatures were 250 and 280 °C, respectively. The initial GC oven temperature was 70 °C for 2 min, followed by a 60 °C/min increase to 180 °C and a final 10 °C/min ramp to 220 °C. Derivatizing agent injections were analyzed between samples to ensure that no residual analyte remained in the injection port.
Cyclohexene addition products (0.2 µl) were analyzed by selected ion monitoring of ions of m/z 195-201 using an initial GC temperature of 50 °C for 2 min, followed by a 20 °C/min increase to 100 °C and a final 60 °C/min ramp to 220 °C.
Electrospray Ionization-Mass Spectrometry-- Aliquots from HPLC fractions were analyzed on a Waters Alliance 2670 HPLC equipped with a C18 column (Porasil, 5 µm resin, 2.1 × 150 mm; Beckman) interfaced with a Finnigan LCQ. Sample (5 µl) was injected at a flow rate of 200 µl/min. Solvent C was 1% acetic acid in 4% methanol, and solvent D was 1% acetic acid in 95% methanol. The sample was eluted from the column by a discontinuous gradient of solvent D. The gradient was: 0% solvent D for 2 min, 0-100% solvent D over 8 min, and then 100% for 10 min. Mass spectrometric analysis was carried out in full mass scanning, zoom scanning, and low energy collisionally activated dissociation modes as described (20). Helium was used as a damping gas and collision activation partner. The temperature of the heated capillary was 220 °C. In the full scan mode (m/z 150-300), each scan consisted of three 300-ms microscans. Full scan mass spectra consisted of 10 signal-averaged scans with subtraction of the carrier solvent background from the same number of scans.
Mass Spectrometric Analysis of Oxidation Products--
The
electrospray positive ion mass spectra of authentic
5-bromodeoxycytidine and the product from myeloperoxidase, HOCl + Br, and HOBr yielded the same major [M + H]+ ion at m/z 306. All compounds
also exhibited a prominent ion at m/z 308. The
relative abundances of the ions at m/z 306 and 308 reflected that of the natural isotopic abundance of
79Br and 81Br, strongly suggesting that the
deoxycytidine oxidation product was monobrominated. The collisionally
activated dissociation tandem mass spectrum of the
m/z 306 ion generated a product ion at
m/z 190, which is consistent with cleavage of the
N-glycoside bond of 5-bromodeoxycytidine to yield
79Br-substituted cytosine. The collisionally activated
dissociation tandem mass spectrum of the m/z 308 ion likewise generated a product ion at m/z 192, consistent with cleavage of the brominated nucleoside to yield
81Br-substituted cytosine. The electrospray ionization
MS/MS spectrum of the myeloperoxidase product indicates that the
bromine substitution site resides on the cytosine base of deoxycytidine.
Further structural characterization of the modified nucleobase
generated from deoxycytidine and uracil was achieved using GC/MS to
obtain an informative electron ionization mass spectrum and GC
retention time from material isolated by HPLC. This procedure yields
information only about the deoxycytidine nucleobase because the
N-glycoside bond of the nucleoside is hydrolyzed during the derivatization reactions. The mass spectrum of the reaction products exhibited low abundance ions consistent with the molecular ion [M+·] of the bis-trimethylsilyl derivatives
(m/z 333 and 334 for brominated deoxycytidine and
uracil, respectively). The major ion from each analyte was consistent
with loss of CH3· as expected for trimethylsilyl
derivatives. The [M]·+ and
[M-CH3·]+ ions exhibited prominent M+2
isotopes as expected for monobrominated compounds containing the
natural 1:1 abundance of 79Br or 81Br. Ions
observed for [M Br
]+ and [M
CH3·
HBr]+ or [M
CH4
Br
]+ fragments lacked the
prominent M+2 fragments as expected for compounds that lack bromine.
The positive ion mass spectra and GC retention times of the
trimethylsilyl derivatives of the reaction products were essentially
identical to those obtained for commercially available
5-bromodeoxycytidine or 5-bromouracil.
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RESULTS |
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5-Bromodeoxycytidine Is the Major Product when the
Myeloperoxidase-H2O2-Br-Cl
System Oxidizes Deoxycytidine at Plasma Concentrations of
Halide--
We previously demonstrated that the
myeloperoxidase-H2O2-Cl
system
(containing 100 mM Cl
) oxidizes deoxycytidine
to 5-chlorodeoxycytidine (20). When we supplemented this system with a
plasma concentration of Br
(100 µM), HPLC
analysis detected a peak of material that migrated with a retention
time distinct from that of 5-chlorodeoxycytidine (Fig.
1A). The new oxidation product
was isolated by HPLC and identified as 5-bromodeoxycytidine on the
basis of its HPLC retention time, ultraviolet absorption spectrum, GC
retention time and positive ion mass spectrum, electrospray ionization
tandem mass spectrum, and 1H NMR spectrum (see
"Methods").
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Under these reaction conditions, 5-bromodeoxycytidine was also the
major product when eosinophil peroxidase or lactoperoxidase oxidized
deoxycytidine. It was therefore important to determine whether our
myeloperoxidase preparation was contaminated by other peroxidases, even
though we isolated the enzyme from HL-60 cells, a promyelocytic cell
line that is not known to express other peroxidases. Myeloperoxidase
was apparently pure as assessed by its heme spectrum and by denaturing
polyacrylamide gel electrophoresis. Moreover, it yielded a single band
of peroxidase activity that migrated with a retention time distinct
from that of eosinophil peroxidase on nondenaturing gel
electrophoresis. These observations indicate that the reactive
intermediates that brominated deoxycytidine at plasma concentrations of
Cl and Br
resulted from the action of myeloperoxidase.
5-Bromouracil Is the Major Product When the
Myeloperoxidase-H2O2-Br-Cl
System Oxidizes Uracil at Plasma Concentrations of Halide--
To
determine whether the enzyme can halogenate other pyrimidines, we
incubated uracil with the
myeloperoxidase-H2O2-Cl
system.
In the absence of added Br
, a new peak of material
(retention time, 13.7 min) was detectable in the reaction mixture by
HPLC analysis (Fig. 1B). In the presence of 100 µM Br
, we observed a second peak (retention
time, 18 min). We isolated both peaks of material by HPLC and
determined their structures by ultraviolet absorption spectroscopy, GC,
and positive ion mass spectrometry, electrospray ionization tandem mass
spectrometry, and 1H NMR spectroscopy (see "Methods").
We identified the early and late eluting materials as 5-chlorouracil
and 5-bromouracil, respectively.
Production of 5-bromouracil by myeloperoxidase was dependent on the
concentration of Br in the reaction mixture (Fig.
2). Increasing [Br
] from
0 to 100 µM in the presence of 100 mM
Cl
reduced uracil chlorination but increased uracil
bromination. The apparent Km values for
Cl
and Br
binding by myeloperoxidase are
175 and 2 mM (46), respectively, suggesting that the enzyme
did not directly oxidize Br
to generate 5-bromouracil.
The relative yields of the halogenated pyrimidines also differed; under
optimal conditions, the yield of 5-bromouracil was 5-fold greater than
that of 5-chlorouracil.
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We used HPLC to investigate the reaction requirements for generation of
5-bromodeoxycytidine by the myeloperoxidase and eosinophil peroxidase-H2O2-Cl-Br
systems (Table I). Both systems required
enzyme and H2O2 and were blocked by catalase, a
scavenger of H2O2. The product yield of the
reaction was nearly quantitative relative to
H2O2. Omitting Br
did not
completely prevent bromination, perhaps because the NaCl used as the
salt contained up to 0.01% NaBr (up to a final [Br
] of
10 µM). Two heme-enzyme inhibitors, cyanide and
aminotriazole, also inhibited product formation. Both myeloperoxidase
and eosinophil peroxidase brominated deoxycytidine in the absence of
added Cl
, which is consistent with the ability of
myeloperoxidase to convert Br
into HOBr (46). These
results demonstrate that bromination of deoxycytidine by
myeloperoxidase or eosinophil peroxidase requires active enzyme,
Br
, and H2O2.
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The two enzymes differed in one important respect, however. Bromination by myeloperoxidase but not by eosinophil peroxidase was inhibited by taurine (Table I), which can react with HOCl or HOBr to form chloramines or bromamines (14, 35). This observation suggests that the enzymes brominate pyrimidines by different reaction pathways.
We used HPLC to establish the optimal reaction conditions for
bromination of uracil (Fig. 3) and
deoxycytidine (Fig. 4) by the
myeloperoxidase-H2O2-Cl-Br
system. Bromination of both substrates was proportional to
[H2O2] from 0 to 100 µM. The
yields of both products increased with rising [Br
] over
a physiologically relevant range. The pH dependence demonstrated a
distinct optimum of 4.5-5.0 for 5-bromodeoxycytidine generation (Fig.
4C) and a broader range of 4.0-6.0 for 5-bromouracil
generation (Fig. 3B). Both reactions were largely complete
after 60 min.
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Reagent HOCl Generates 5-Bromouracil and 5-Bromodeoxycytidine by a
Reaction That Requires Br--
Previous studies have
indicated that myeloperoxidase preferentially oxidizes Cl
to HOCl at plasma concentrations of Br
and
Cl
. In contrast, eosinophil peroxidase selectively
oxidizes Br
to an HOBr-like species. To determine whether
HOCl could be an intermediate when myeloperoxidize generates
5-bromouracil (Fig. 5) or
5-bromodeoxycytidine (Fig. 6), we
compared the Br
, oxidant, and pH dependences of product
production by reagent HOBr and HOCl. When we used HOBr, product yield
was independent of [Br
] (Figs. 5A and
6A). In striking contrast, HOCl required Br
to
brominate deoxycytidine.
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Production of 5-bromouracil and 5-bromodeoxycytidine increased linearly
with [HOBr] (Figs. 5B and 6B). In contrast, the
yield of brominated products reached a plateau at around 75 µM of HOCl, a concentration similar to that of
Br (100 µM) in the reaction mixture. The pH
dependences of product generation by HOCl and HOBr were similar under
acidic and neutral conditions. However, the yields of 5-bromouracil
differed under alkaline conditions: at pH 8, HOBr but not HOCl
generated large amounts of product (Fig. 5C). The decline in
uracil bromination by HOCl at pH 8 was accompanied by an increase in
uracil chlorination (data not shown). These results indicate that HOCl
readily oxidizes Br
to generate a brominating agent at
physiologically plausible concentrations of halide.
Hypochlorous Acid and Myeloperoxidase Generate the Interhalogen Gas
BrCl at Physiologic Concentrations of Bromide and
Chloride--
Previous studies indicate that reagent HOCl oxidizes
Br to BrCl (40, 41).
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(Eq. 5) |
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Mass spectrometric analysis of cyclohexene exposed to the gas
sparged from the HOCl reaction mixture revealed a major peak of
material with the mass-to-charge-ratio (Fig.
7A) and isotopic pattern expected for the molecular ion of 1-bromo-2-chlorocyclohexane. This material exhibited a retention time midway between that of authentic 1,2-dichlorocyclohexane and that of 1,2-dibromocyclohexane. It was not detectable when we omitted HOCl from the reaction mixture (Fig. 7A). When we replaced
HOCl-Cl-Br
with the
myeloperoxidase-H2O2-Cl
-Br
system, we also observed a peak of material with the retention time and
characteristic isotopic pattern of 1-bromo-2-chlorocyclohexane (Fig.
7B). Production of 1-bromo-2-chlorocyclohexane by
myeloperoxidase required enzyme, H2O2,
Cl
, and Br
. These observations indicate
that the myeloperoxidase pathway can produce BrCl by generating HOCl,
which reacts with Br
to form the interhalogen gas.
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Physiologic Concentrations of Chloride Stimulate the Bromination of
Deoxycytidine by Reagent HOCl--
BrCl is hydrolyzed to HOBr and
Cl in reaction mixtures containing
H2O.
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(Eq. 6) |
|
Bromamines and Chloramines Produce 5-Bromodeoxycytidine--
HOCl
can react with amines to form N-chloro compounds, which then
could react with Br to generate brominating species (52).
Alternatively, chloramine formation might compete with
Br
, inhibiting the formation of brominating species by
HOCl. Our observation that taurine inhibits bromination by HOCl and
Br
(Table I) is consistent with the latter possibility.
To distinguish between these two possibilities, we determined whether
different haloamines can produce 5-bromodeoxycytidine (Fig.
9). Both N-chlorotaurine and
N-bromotaurine generated high concentrations of
5-bromodeoxycytidine below pH 5 in the presence of 100 µM
Br
and 100 mM Cl
. Over the pH
range 5.5-7, however, only N-bromotaurine generated significant levels of 5-bromodeoxycytidine.
|
Unlike N-chlorotaurine, N-chlorodeoxycytidine
failed to convert cytosine to 5-bromocytosine except under strongly
acidic conditions (<pH 3), and the yield was low even at pH 2 (Fig.
9). The chloramine of deoxycytidine therefore is unlikely to be an
intermediate when myeloperoxidase brominates deoxycytidine. These
observations indicate that primary haloamines can brominate pyrimidines
in the presence of Br. They also suggest that
Br
oxidation by HOCl is more rapid than nucleoside
chloramine formation.
Activated Human Neutrophils Generate 5-Bromodeoxycytidine and
5-Bromouracil at Plasma Halide Concentrations--
To determine
whether oxidants generated by human neutrophils can brominate
nucleobases, we stimulated the cells with phorbol myristate acetate in
physiological salt solution supplemented with 100 µM NaBr
and 1 mM deoxycytidine (Table
II) or uracil (data not shown). HPLC and
mass spectrometric analysis detected substantial quantities of
5-bromodeoxycytidine and 5-bromouracil in the medium of the activated
cells. Bromination required stimulation of the cells with phorbol ester
and was inhibited by catalase and heme poisons, implicating
H2O2 and a heme protein in the reaction (Table II). Omitting supplemental Br significantly reduced but
did not eliminate 5-bromodeoxycytidine production, most likely because
the medium was contaminated with Br
. Superoxide dismutase
failed to affect the reaction.
|
Generation of 5-bromodeoxycytidine (Fig.
10, upper panel) by
activated neutrophils was strongly affected by the pH of the culture medium. Acidification to the range observed in inflamed tissue and the
phagolysosome significantly enhanced the cellular production of
5-bromodeoxycytidine. The progress curve of the reaction was essentially complete 30 min after the cells were activated with phorbol
ester (Fig. 10, lower panel).
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Human Neutrophils Generate Brominating Intermediates by a Pathway
Involving HOCl--
In vitro studies demonstrated that
N-bromotaurine, but not N-chlorotaurine,
brominates deoxycytidine at pH 5.9 in the presence of 100 µM Br and 100 mM
Cl
(Fig. 9). This observation suggests that reagent HOBr,
but not HOCl, should brominate deoxycytidine in the presence of taurine under these conditions. Indeed, this reaction occurred when
mM concentrations of taurine were included in the reaction
mixture (Fig. 11A). In
contrast, taurine inhibited bromination by HOCl under the same
conditions, probably because it consumed the oxidant to form
N-chlorotaurine, which is unreactive at pH 5.9. Taurine was
also inhibitory (Fig. 11A) when the
myeloperoxidase-H2O2 system replaced HOCl. In
contrast, it had little affect on bromination by the eosinophil
peroxidase-H2O2 system. It is noteworthy that, in the absence of taurine, product yields (relative to oxidant) of
5-bromodeoxycytidine were similar with reagent HOCl, reagent HOBr,
myeloperoxidase, and eosinophil peroxidase. These observations indicate
that reactive species generated by HOCl or HOBr brominate deoxycytidine
with similar efficiency under these conditions. They also demonstrate
that use of taurine, a potent scavenger of hypohalous acids, can
distinguish between bromination reactions mediated by HOBr and
HOCl.
|
We used taurine to determine whether human neutrophils use HOCl or HOBr
to brominate deoxycytidine (Fig. 11B). We activated the
cells with phorbol ester, incubated them for 60 min at pH 5.9 in buffer
containing 100 µM NaBr, 100 mM NaCl, and 1 mM deoxycytidine, and used HPLC to determine whether
5-bromodeoxycytidine was produced. Taurine markedly inhibited the
brominating ability of neutrophils (Fig. 11B), with an
IC50 similar to that of the
myeloperoxidase-H2O2-Cl-Br
system (Fig. 11A). These observations strongly support the
hypothesis that the major pathway by which human neutrophils generate
reactive brominating species involves HOCl production followed by
oxidation of Br
.
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DISCUSSION |
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Our observations demonstrate that the myeloperoxidase system of
human neutrophils generates brominating oxidants that convert uracil
into 5-bromouracil and transform deoxycytidine into 5-bromodeoxyuridine (Scheme 2). Importantly, these
pyrimidines become halogenated at plasma concentrations of
Br (100 µM) and Cl
(100 mM), suggesting that this pathway may be physiologically relevant.
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Hypochlorous acid is thought to be the major product when
myeloperoxidase is exposed to plasma halide concentrations. In
contrast, eosinophil peroxidase preferentially converts
Br to HOBr. It was therefore important to determine
whether myeloperoxidase was oxidizing Cl
or
Br
in our experiments. We obtained several lines of
evidence to support the proposal that myeloperoxidase initially
generates HOCl, which then is converted into reactive brominating
species. First, reagent HOCl brominated uracil and deoxycytidine as
effectively as HOBr. The HOCl-mediated reaction required
Br
and was optimal under acidic conditions. Second,
taurine, which rapidly reacts with HOCl to form
N-chlorotaurine, inhibited bromination of deoxycytidine by
HOCl-Br
or the
myeloperoxidase-H2O2-Cl
-Br
system. Importantly, kinetic studies indicate that chlorination of
taurine by myeloperoxidase may involve an enzymatic intermediate, suggesting that taurine scavenges chlorinating species before they can
diffuse out of the active site of the enzyme (14). In contrast, taurine
had little effect on bromination by HOBr or the eosinophil
peroxidase-H2O2-Cl
-Br
system. Taurine thus selectively inhibits bromination mediated by HOCl
under these experimental conditions. Finally, human neutrophils converted deoxycytidine to 5-bromodeoxycytidine by a reaction requiring
H2O2 and Br
. Bromination was
optimal under acidic conditions and was inhibited by taurine and heme
poisons, implicating HOCl and myeloperoxidase in the reaction pathway.
Collectively, these observations indicate that activated human
phagocytes use myeloperoxidase to brominate pyrimidines at plasma
concentrations of halide by reactions that initially require HOCl.
A key question is the mechanism by which HOCl then generates reactive brominating species. Based on the chemistry of the interhalogen compounds (40, 41), we propose that BrCl is one potential intermediate in the pathway. BrCl is a stronger brominating oxidant than Br2 or HOBr (42), suggesting that it halogenates pyrimidines. Alternatively, bromination may be mediated by HOBr or Br2, which are in equilibrium with BrCl.
To directly detect BrCl, we sparged a reaction mixture containing
HOCl-Br or the
myeloperoxidase-H2O2-Cl
-Br
system with nitrogen gas that we subsequently passed through cyclohexene. Mass spectrometric analysis of the resulting cyclohexene solution detected an ion with the expected mass-to-charge ratio, GC
retention time, and isotopic abundance of 1-bromo-2-cyclohexane. Cyclohexene is an aprotic, nonpolar solvent that should not contain halide ions under these conditions. Thus, bromination of cyclohexene cannot involve a backsided nucleophilic attack of a bromonium ion
intermediate by free Cl
in solvent. Instead, it is likely
to involve the concerted attack on the double bond by
[Br+-Cl
] derived from molecular BrCl
(Scheme 1). Detecting 1-bromo-2-cyclohexane therefore provides strong
evidence that HOCl and myeloperoxidase generate the interhalogen gas BrCl.
Our demonstration that [Cl] increased the yield of
5-bromodeoxycytidine generation by reagent HOCl and Br
is
consistent with this proposal. Chloride ion is likely to stimulate bromination by reacting with HOBr to form BrCl (Equation 6). These observations further support the hypothesis that HOBr and HOCl are in
equilibrium with BrCl under acidic conditions in the transhalogenation pathway (Scheme 3).
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Another important question is whether the acidic conditions that are optimal for myeloperoxidase to generate brominating intermediates are likely to exist in vivo. We suggest that the acidic conditions that result from infection or tissue hypoxia at sites of inflammation may represent such an environment (54, 55). Also, cultured activated macrophages form phagocytic compartments in which the pH falls below 4 (56). Moreover, strongly acidic environments exist in the phagolysosome and stomach, where myeloperoxidase-derived HOCl (or chloramines) might generate brominated pyrimidines. Human neutrophils use a Cl2-like species to generate 3-chlorotyrosine from tyrosine in vitro, and we have shown that 3-chlorotyrosine levels are markedly elevated in human atherosclerosis, a chronic inflammatory condition (13, 21). Therefore, inflammation may generate acidic environments in which myeloperoxidase can produce brominating oxidants.
Chronic inflammation is an important risk factor for cancer, and
oxidants generated by phagocytic cells mutate bacteria and transform
cultured mammalian cells (57, 58). Moreover, recent genetic
epidemiological studies have found a relationship between cancer risk
and a polymorphism in the promoter region of the myeloperoxidase gene.
People with a polymorphism that increases myeloperoxidase expression
were at increased risk for promyelocytic leukemia (26, 27). In
contrast, those with polymorphisms that lower myeloperoxidase expression were at decreased risk for lung and laryngeal cancers (25,
28-30). These observations suggest that nucleobase halogenation might
provide one mechanism for mutagenesis and cytotoxicity in vivo. Indeed, 5-bromouracil, one product of the
myeloperoxidase-H2O2-Cl-Br
system, is incorporated into nuclear DNA as 5-bromodeoxyuridine, a
known mutagen (59-63). Moreover, myeloperoxidase converts
deoxycytidine to 5-bromodeoxycytidine, and we have shown that
5-bromodeoxycytidine generated by eosinophil peroxidase is incorporated
into the genomic DNA of cultured mammalian cells as
5-bromodeoxyuridine.1
Mutagenesis by oxidants is generally thought to occur via direct damage to DNA. However, our results suggest an additional mechanism: halogenation of nucleobases within a precursor pool. In this scenario, activated phagocytes generate halogenating intermediates, which react with nucleotides or nucleotide precursors in the extracellular and intracellular milieu. Consequently, potentially cytotoxic and mutagenic deoxynucleotide derivatives become incorporated into the genomes of daughter cells. If they find their way into tumor suppressor genes, genes for DNA repair, or potential oncogenes, they might increase the risk for cancer. A similar incorporational mechanism of mutagenesis has been suggested for the MutT system in bacteria, which cleanses the deoxynucleotide pool of 8-oxodGTP (64). When MutT is genetically inactivated, the spontaneous mutation rate increases 100-10,000-fold (65-67).
We found that two primary haloamines, N-bromotaurine and N-chlorotaurine, also brominate deoxycytidine at plasma concentrations of halide. However, the reaction with N-chlorotaurine required more acidic conditions than bromination by HOBr or HOCl; bromination by N-bromotaurine and N-chlorotaurine were half-maximal at pH 5.8 and 4.5, respectively. Also, the pyrimidine ring of cytosine contains an exocyclic amino group and two nitrogens, raising the possibility that N-chlorocytosine is an intermediate in the bromination reaction (20, 68). However, we found that N-chlorodeoxycytidine was unable to generate 5-bromodeoxycytidine under our standard experimental conditions. These observations indicate that primary chloramines and bromamines, but not N-chlorocytosine, can be intermediates in the bromination of deoxycytidine. Haloamines are relatively stable compounds that can diffuse long distances and cross plasma membranes. Because hypohalous acids rapidly react with primary amino groups and extracellular fluids contain high concentrations of low molecular weight amines, it is possible that haloamines represent one mechanism for brominating nucleobases under acidic conditions.
Chronic inflammation is a risk factor for cancer, and reactive
chlorinating, brominating, and nitrating intermediates generated by
myeloperoxidase damage nucleobases in vitro (20, 69). These observations suggest that phagocytes may constitute a physiologically important pathway for oxidative damage to DNA. This hypothesis would be
strongly supported by detection of brominated or chlorinated pyrimidines in vivo, with important implications for the
pathogenesis of tissue injury and perhaps mutagenesis during inflammation.
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ACKNOWLEDGEMENTS |
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We thank Dr. A. d'Avignon (Department of Chemistry, Washington University in St. Louis) for assistance with NMR studies. Mass spectrometry experiments were performed at the Washington University School of Medicine Mass Spectrometry Resource.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AG12293, AG19309, and RR00954, the Veterans Affairs Merit Review Program (to M. L. M.), and the Monsanto-Searle/Washington University Biomedical Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a Biophysics Training Grant from the National Institutes of Health.
** To whom correspondence should be addressed: Div. of Atherosclerosis, Nutrition and Lipid Research, Box 8046, 660 S. Euclid Ave., St. Louis, MO 63110.Fax: 314-362-0811; E-mail: heinecke@im.wustl.edu.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M005379200
1 J. P. Henderson and J. W. Heinecke, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: DTPA, diethylenetriaminepentaacetic acid; GC, gas chromatography; MS, mass spectrometry; HPLC, high pressure liquid chromatography.
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