Ability of hypochlorous acid and N -chloramines to chlorinate DNA and its constituents

Article abstract of DOI:10.1021/tx100188b

Myeloperoxidase is a heme enzyme released by activated phagocytes that is responsible for the generation of the strong oxidant hypochlorous acid (HOCl). Although HOCl has potent bactericidal properties and plays an important role in the human immune system, this oxidant also causes damage to tissues, particularly under inflammatory conditions. There is a strong link between chronic inflammation and the incidence of many cancers, which may be associated with the ability of HOCl and related oxidants such as N-chloramines to damage DNA. However, in contrast to HOCl, little is known about the reactivity of N-chloramines with DNA and its constituents. In this study, we examine the ability of HOCl and various N-chloramines to form chlorinated base products on nucleosides, nucleotides, DNA, and in cellular systems. Experiments were performed with N-chloramines formed on Nα-acetyl-histidine (His-C), Nα-acetyl-lysine (Lys-C), glycine (Gly-C), taurine (Tau-C), and ammonia (Mono-C). Treatment of DNA and related materials with HOCl and His-C resulted in the formation of 5-chloro-2'-deoxycytidine (5CldC), 8-chloro-2'-deoxyadenosine (8CldA) and 8-chloro-2'-deoxyguanosine (8CldG). With the nucleosides, 8CldG was the favored product in each case, and HOCl was the most efficient chlorinating agent. 5Cl(d)C was the most abundant product on exposure of the nucleotides and DNA to HOCl and His-C, with only low levels of chlorinated products observed with Lys-C, Gly-C, Tau-C, and Mono-C. 5CldC was also formed on exposure of smooth muscle cells to either HOCl or His-C. Cellular RNA was also a target for HOCl and His-C, with evidence for the formation of 5-chloro-cytidine (5ClC). This study shows that HOCl and the model N-chloramine, His-C, are able to chlorinate cellular genetic material, which may play a role in the development of various inflammatory cancers.

Full text of DOI:10.1021/tx100188b

Chem. Res. Toxicol. 2010, 23, 1293–1302
1293
Ability of Hypochlorous Acid and N-Chloramines to Chlorinate
DNA and Its Constituents
Naomi R. Stanley, David I. Pattison, and Clare L. Hawkins*
The Heart Research Institute, 7 Eliza Street, Newtown, Sydney, NSW 2042, Australia
ReceiVed May 27, 2010
Myeloperoxidase is a heme enzyme released by activated phagocytes that is responsible for the
generation of the strong oxidant hypochlorous acid (HOCl). Although HOCl has potent bactericidal
properties and plays an important role in the human immune system, this oxidant also causes damage to
tissues, particularly under inflammatory conditions. There is a strong link between chronic inflammation
and the incidence of many cancers, which may be associated with the ability of HOCl and related oxidants
such as N-chloramines to damage DNA. However, in contrast to HOCl, little is known about the reactivity
of N-chloramines with DNA and its constituents. In this study, we examine the ability of HOCl and
various N-chloramines to form chlorinated base products on nucleosides, nucleotides, DNA, and in cellular
systems. Experiments were performed with N-chloramines formed on NR-acetyl-histidine (His-C), NR-
acetyl-lysine (Lys-C), glycine (Gly-C), taurine (Tau-C), and ammonia (Mono-C). Treatment of DNA
and related materials with HOCl and His-C resulted in the formation of 5-chloro-2′-deoxycytidine (5CldC),
8
8
-chloro-2′-deoxyadenosine (8CldA) and 8-chloro-2′-deoxyguanosine (8CldG). With the nucleosides,
CldG was the favored product in each case, and HOCl was the most efficient chlorinating agent. 5Cl(d)C
was the most abundant product on exposure of the nucleotides and DNA to HOCl and His-C, with only
low levels of chlorinated products observed with Lys-C, Gly-C, Tau-C, and Mono-C. 5CldC was also
formed on exposure of smooth muscle cells to either HOCl or His-C. Cellular RNA was also a target for
HOCl and His-C, with evidence for the formation of 5-chloro-cytidine (5ClC). This study shows that
HOCl and the model N-chloramine, His-C, are able to chlorinate cellular genetic material, which may
play a role in the development of various inflammatory cancers.
Introduction
(reviewed in refs 6, 12, and 13). Proteins are important major
targets for HOCl, owing to their abundance and high
reactivity with this oxidant (14). Protein-derived N-chloram-
ines are formed readily on reaction of HOCl with Lys and
His side-chains (12). These species are reactive and can
mediate further protein damage (e.g., ref 15) and induce the
oxidation of other biological molecules, including lipids (16)
and DNA (17).
1
Myeloperoxidase (MPO ) is released by activated phagocytes
during inflammation, as part of the innate immune system. In
the presence of physiological concentrations of chloride ions
100-150 mM) (1) and hydrogen peroxide (H
generates the potent antibacterial agent hypochlorous acid
HOCl), whose formation accounts for 20-70% of the H
liberated during inflammation (2). At sites of inflammation in
ViVo, the concentration of HOCl has been estimated to reach
5-50 mM (3), while kinetic modeling studies of the phago-
some suggest that concentrations as high as 100 mM are feasible
4). The high concentrations of HOCl observed under inflam-
(
2 2
O ), MPO
(
2 2
O
Less is known about the consequences of the reaction of
HOCl with RNA and DNA. The reactivity of HOCl with
nucleosides is primarily determined by the location of the NH
groups, with the heterocyclic (ring) NH groups generally more
2
(
matory conditions are postulated to result in damage to host
tissue and contribute to disease progression (reviewed in refs 5
and 6). MPO has been linked to many inflammatory diseases,
such as systemic vasculitides (7), neurogenerative disease (8),
cancer (9), kidney disease (10), and atherosclerosis (11).
reactive than the exocyclic (free) NH
2
groups (e.g., k
2
) 2.1 ×
4
-1 -1
-1 -1
10 M s , cf. 2.4 M
s
for the reaction with N-1 and NH
2
at C-2 on GMP, respectively) (18). The majority of HOCl
consumed in the reaction with DNA results in the formation of
unstable, nucleoside-derived, N-chloramines (17, 19). These
intermediates decompose readily, and may be involved in the
formation of stable chlorinated products (19-21). A range of
stable products are produced from the reaction of HOCl with
DNA, including thymine glycol, 5-hydroxy-cytosine, 5-hydroxy-
uracil, and hypoxanthine (22, 23). Similarly, there is evidence
for the formation of a number of chlorinated bases including
5-chlorouracil (5ClU) (22-25), 8-chloro(2′-deoxy)adenosine
HOCl is a highly reactive compound that reacts readily
with biomolecules, including proteins, lipids, and DNA
*
To whom correspondence should be addressed. Tel: +61-2-8208-8900.
Fax: +61-2-9565-5584. E-mail: hawkinsc@hri.org.au.
1
Abbreviations: BCASMC, bovine coronary aortic smooth muscle cells;
8
Cl(d)A, 8-chloro(2′-deoxy)adenosine; 5Cl(d)C, 5-chloro(2′-deoxy)cytidine;
8
Cl(d)G, 8-chloro(2′-deoxy)guanosine; 5Cl(d)U, 5-chloro(deoxy)uridine;
(
(
8Cl(d)A) (20, 23, 26, 27), 5-chloro(2′-deoxy)cytidine (5Cl(d)C)
20, 23, 24, 27-29), and 8-chloro(2′-deoxy)guanosine (8Cl(d)G)
dA, 2′-deoxyadenosine; dC, 2′-deoxycytidine; dG, 2′-deoxyguanosine; dT,
2
′-deoxythymidine; dU, 2′-deoxyuridine; DMEM, Dulbecco’s modified
Eagle’s medium; DTNB, 5,5′-dithio-2-nitrobenzoic acid; Gly-C, glycine
chloramine; HBSS, Hank’s buffered salt solution; His-C, NR-acetyl-histidine
chloramine; Lys-C, NR-acetyl-lysine chloramine; Mono-C, monochloramine;
MPO, myeloperoxidase; SRM, selective reaction monitoring; Tau-C, taurine
chloramine; TNB, 5-thio-2-nitrobenzoic acid.
(20, 27) on reaction of HOCl with DNA. These products are
specific for MPO-derived chlorinating oxidants and, in some
cases, have been used as biomarkers for HOCl-induced damage
(e.g., refs 30-32).
1
0.1021/tx100188b  2010 American Chemical Society
Published on Web 07/01/2010

1
294 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Stanley et al.
The presence of increased levels of chlorinated nucleosides
molecular-weight cutoff filters (Millipore) for 5 min at 16000g to
remove the DNA before quantifying the remaining oxidant using
TNB.
Quantification of Chlorinated Products on Nucleosides and
Nucleotides. The nucleosides deoxyadenosine (dA), deoxycytidine
in the inflammatory exudate of humans (30) and rats (31), and
elevated levels of 5ClU in atherosclerotic plaques compared to
healthy arteries (32) suggest that the chlorination of nucleic acids
may be relevant under pathological conditions. Indeed, 8CldG
has recently been reported to be a biomarker of early inflam-
mation, with elevated levels of this chlorinated nucleoside
detected in the urine of lipopolysaccaride-treated rats and
diabetic patients (33). However, the mechanism of MPO
oxidant-induced DNA chlorination in ViVo is not well under-
stood. It is likely that direct reaction of HOCl with cellular DNA
may be a minor pathway, owing to the extracellular production
of HOCl and its high reactivity with biological substrates. Under
physiological conditions, N-chloramines are formed readily on
(
dC), and deoxyguanosine (dG), and equimolar mixtures of dA,
dC, dG, and deoxythymidine (dT) (500 µM) were incubated with
equal volumes of HOCl (250 µM) or N-chloramines at 20 °C before
quenching the reaction with Met (10 mM), after the required
incubation time. The nucleotides ATP, CTP, and GTP, and an
equimolar mixture of ATP, CTP, GTP, and TTP (500 µM) were
treated as described above and the reaction quenched with 2.5 mM
Met. The nucleotide samples were subsequently dephosphorylated
for 1 h at 37 °C with alkaline phosphatase (25 U) in the presence
of phosphatase buffer (80 mM Tris-HOCl and 160 µM EDTA, pH
8
). Nucleotide samples were filtered through 10 kDa molecular-
3
ammonia (NH ), taurine, free amino acids, as well as peptides
weight cutoff filters for 5 min at 16000g to remove alkaline phos-
phatase prior to analysis.
and proteins (2, 34, 35). In Vitro, various N-chloramines convert
uracil to 5ClU (32). The role of taurine chloramine (Tau-C) in
the chlorination of uracil is unclear, as it can both prevent (30)
and promote (32) 5ClU formation. There is a lack of data
regarding the ability of N-chloramines to mediate the chlorina-
tion of RNA and DNA, though tertiary amines, including
nicotine and trimethylamine, enhance the chlorination of nucleo-
sides and RNA by HOCl (20).
Quantification of base chlorination was carried out on a Shimadzu
HPLC system using a Beckman Coulter Ultrasphere 5 µm ODS
column (4.6 mm ×25 cm) fitted with a Pelliguard C-18 guard
column (Supelco) at 30 °C. Samples were separated using a gradient
elution system at a flow rate of 1 mL min- with 5 mM ammonium
formate (Buffer A) and 5 mM ammonium formate in 50% (v/v)
acetonitrile (Buffer B) as follows: 100% Buffer A for 10 min,
followed by a linear increase of Buffer B to 20% over 30 min,
50% over the next 10 min, and 100% over a further 5 min. After
isocratic 100% Buffer B for 3 min, Buffer A was returned to 100%
over 3 min, followed by a 4 min re-equilibration. A modified
gradient was used for nucleotide samples: 100% Buffer A for 5
min, followed by a linear increase of Buffer B to 30% over 10
min, 50% over the next 20 min, and 100% over a further 5 min.
After isocratic 100% Buffer B for 5 min, Buffer A was returned to
1
In this study, we investigated the ability of five biologically
relevant N-chloramines to chlorinate nucleosides, nucleotides,
isolated DNA, and cellular RNA and DNA. Experiments were
performed with N-chloramines generated on Gly (Gly-C), to
model the N-terminal amino group, and NR-acetyl-Lys (Lys-
C) and NR-acetyl-His (His-C) to model N-chloramines formed
on protein side chains. Mono-C is known to be reactive with
cells and is also believed to be formed under physiological
conditions (4). Tau-C was also studied as taurine is present in
high concentrations in neutrophils (36), and its role in mediating
nucleoside chlorination is unclear.
1
00% over 5 min, followed by a 5 min re-equilibration. Bases were
quantified by UV absorbance at 260 nm.
Chlorinated Base Production on Isolated DNA. Fifty micro-
grams of DNA (200 µL) was treated with an equal volume of HOCl
or N-chloramines (25 µM-1 mM) at 20 °C for 5 min and 4 h (HOCl
and His-C only) and 1 week (all). The samples were hydrolyzed
and dephosphorylated as described previously (41), prior to LC-
MS analysis.
Experimental Procedures
Quantification of Chlorinated Products by LC-MS. Detection
of chlorinated bases was performed in the positive ion mode with
a Thermo Finnigan LCQ Deca XP Max ion trap mass spectrometer
coupled to a Thermo Finnigan Surveyor HPLC system (Thermo
Electron Corporation, Australia). Bases were separated on a reverse
phase C-18 Alltima HP column (5 µM, 2.1 mm × 25 cm) equipped
with a Pelliguard C-18 guard column, using 5 mM ammonium
formate (Buffer A) and 5 mM ammonium formate in 50% (v/v)
Materials. Water was filtered through a four-stage Milli-Q
system (Millipore-Waters, Lane Cove, NSW, Australia). DNA (calf
thymus, > 98% purity), free nucleosides and nucleotides, alkaline
phosphatase, and ammonium formate were obtained from Sigma-
Aldrich (St. Louis, MO). DNA solutions were prepared by stirring
gently overnight at 4 °C in the presence of Chelex resin (Bio-Rad,
-1
Hercules, CA; 0.2 g mL ) to remove contaminating trace metal
ions. Chlorinated nucleosides (8ClA; 8CldA; 5ClC; 5CldC; 8ClG;
-1
acetonitrile (Buffer B) at a flow rate of 200 µL min . Samples
were eluted by the following gradient: 100% Buffer A for 10 min,
followed by a linear increase of Buffer B to 20% over 30 min,
8CldG) were obtained from BioLog Life Sciences Institute (Bremen,
Germany). HOCl solutions were prepared immediately before use
by dilution of a concentrated stock solution [ca. 0.5 M in 0.1 M
NaOH (BDH, Poole, U.K.)] into water. HOCl concentrations were
5
0% over a further 10 min and 100% over the next 5 min. This
was maintained for 5 min before returning to 100% A over 5 min
with re-equilibration for 5 min. Parent bases were detected using
an in-line UV detector (Perkin-Elmer -785A) at 260 nm. The
chlorinated bases were subsequently detected by LC-MS-MS with
the following conditions: capillary voltage, 4500 V; capillary
-
determined from the absorbance of OCl at 292 nm at pH 12 using
a molar extinction coefficient of 350 M- cm (37). All other
1
-1
chemicals were of analytical reagent grade.
N-Chloramine Formation and Quantification. Mono-C, Gly-
C, Lys-C, and Tau-C were generated by the reaction of HOCl with
a 5-fold molar excess of the substrates ammonium sulfate, glycine,
NR-acetyl-Lys and taurine, respectively (38). His-C was generated
using a 2-fold molar excess of NR-acetyl-histidine, as previously
described (38). Under these conditions, complete conversion of
HOCl to the respective N-chloramine is observed (38). N-Chloram-
ine concentrations were determined by the reaction with 5-thio-2-
nitrobenzoic acid (TNB) at 412 nm as described previously (39)
temperature, 275 °C; capillary and sweep gases (both N ), 42 and
2
24 units, respectively; and collision gas, helium.
Tandem MS (MS-MS) experiments were performed to detect
chlorinated RNA and DNA products. The normalized collision
energy was set at 30% for all bases. For DNA samples, the MS
was programmed to undergo four scan events throughout the run,
such that every fourth data point in the chromatogram was (a) a
full scan MS, m/z 100-400; (b) SRM of m/z 263 ((2) f 146 ((1)
using an extinction coefficient of 13 600 M- cm (40).
1
-1
+
[5CldC + H] to monitor 5CldC; (c) SRM of m/z 302 ((1) f 186
((1) [8CldG + H]+ to monitor 8CldG; and (d) SRM of m/z 287
Oxidant Consumption on Reaction with DNA. Calf thymus
+
DNA (50 µg in 200 µL H
2
O) was incubated with an equal volume
of HOCl or N-chloramine (500 µM) at 20 °C. The final pH of the
((2) f 170 ((1) [8CldA + H] to monitor 8CldA, as described
previously (27). For RNA samples, every fourth data point in the
chromatogram was (a) a full scan MS, m/z 100-400; (b) SRM of
reaction mixture was 7-8. Samples were filtered through 3 kDa

DNA Chlorination by HOCl and N-Chloramines
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1295
+
m/z 279 ((2) f 146 ((1) [5ClC + H] to monitor 5ClC; (c) SRM
+
of m/z 319 ((2) f 186 ((1) [8ClG + H] to monitor 8ClG; and
+
(
8
d) SRM of m/z 302 ((1) f 170 ((1) [8ClA + H] to monitor
ClA (27).
Cell Culture. A7r5 (rat aortic smooth muscle; ATCC # 1444)
cells were cultured in Dulbecco’s modified Eagle’s medium
DMEM) supplemented with 10% (v/v) heat inactivated fetal bovine
(
-
1
serum, 4 mM glutamine, 3 g L D-glucose, 1 mM sodium pyruvate,
00 U mL- penicillin, and 0.1 mg mL streptomycin (referred to
as complete DMEM). Bovine coronary aortic smooth muscle cells
BCASMC) were cultured in BCASMC growth medium (Cell
Applications). Both cell types were cultured in an atmosphere of
% CO
1
-1
1
(
5
2
at 37 °C and harvested with trypsin/EDTA (1:250) and
centrifugation at 800g for 5 min.
Ethidium Bromide DNA Release Assay. A7r5 cells or BCASMC
6
(
0.5 × 10 ) were allowed to adhere to 12-well tissue culture plates
for 2 h in the complete DMEM or BCASMC growth media at 37
C, respectively. The cells were washed twice with Hank’s buffered
°
salt solution (HBSS), before being treated with HOCl or N-
6
chloramines (0.25-4 µmol/10 cells) in HBSS for 2 or 4 h at 37
°
C. The extent of cell lysis was determined by the addition of
ethidium bromide (250 µM) and measuring the change in fluores-
cence at λEX 360 nm and λEM 580 nm (42, 43).
Chlorinated Base Production in Cells. A7r5 cells or BCASMC
6
(
4 × 10 ) were plated down overnight in complete DMEM or
BCASMC growth media. The cells were washed twice with PBS,
6
prior to treatment with HOCl (0.125-0.5 µmol/10 cells) or His-C
6
(
0.25-1 µmol/10 cells) in PBS for 4 h at 37 °C to prevent
confounding reactions of the oxidants with cell media components.
Cells were then harvested with trypsin/EDTA after a further wash
with PBS and centrifuged at 16000g for 5 min before discarding
the supernatant. Samples were resuspended in 200 µL of PBS and
the DNA and RNA extracted concurrently using the QIAamp DNA
mini kit (Qiagen). The DNA and RNA were then hydrolyzed and
dephosphorylated and nucleoside chlorination quantified by LC-
MS, as described above.
3
Quantification of Cellular ATP. A7r5 cells (50 × 10 ) were
plated down overnight in white 96-well plates to adhere. The cells
were washed twice with HBSS, before being treated with HOCl or
6
N-chloramine (0.05-2 µmol/10 cells) in HBSS for 2 h at 37 °C.
The ATP concentration was determined by ATPlite Luminescence
ATP Detection System (Perkin-Elmer), with the luminescence
measured by 10 s scans on a Fluoroskan Ascent plate reader
(
Thermo Electron Corp., Australia). ATP-free tips were used for
the dispensing of cells and reagents.
Statistics. All statistical analyses were performed using GraphPad
Prism 4.0 (GraphPad Software, San Diego, USA, www.graphpad.
com), with p < 0.05 taken as significant. Details of specific tests
are given in the text and figure legends.
Figure 1. Formation of chlorinated bases on treatment of nucleosides
with HOCl and N-chloramines. Nucleosides (dA, dC, dG; 500 µM,
1
2.5 nmol) were treated with (A) HOCl (B) His-C, or (C) Mono-C,
Gly-C, Lys-C, or Tau-C (250 µM, 6.25 nmol) for the time indicated
before quenching the reaction with methionine (10 mM) and quantifica-
tion of chlorinated bases by UV analysis at 260 nm, after separation
by HPLC. With HOCl and His-C, significant amounts of 8CldA (white);
5CldC (hatched); and 8CldG (black) were observed. With Mono-C
Results
HOCl and N-Chloramine Treatment of Isolated Nucleo-
sides and Nucleotides. Initially, the ability of HOCl to chlo-
rinate free nucleosides and nucleotides, both individually or
when present as an equimolar mixture, was studied. Reaction
of HOCl (250 µM, 6.25 nmol) with isolated nucleosides (dA,
dC, dG; 500 µM, 12.5 nmol) resulted in the loss of parent
nucleoside (with dG) and formation of chlorinated base products
(
white), Gly-C (hatched), Lys-C (checked), or Tau-C (Black), only
CldG was detected. * represents a significant increase in chlorinated
8
base formation, compared to that in the non-treated control, determined
by one-way ANOVA, with the Bonferroni post-test. Data represent the
mean ( SEM of n ) 3 experiments.
dA. On reaction of HOCl with the equimolar mixture of bases
(dA, dC, dG, and dT; each 500 µM, 12.5 nmol), 8CldG was
the only chlorinated base detected (Figure 2).
(
Figure 1A). 8CldA, 5CldC, and 8CldG were all generated
within 5 min of incubation of HOCl with the respective
individual parent nucleosides. With the exception of 5CldC, no
further increase in base chlorination was observed on further
incubation of HOCl with the nucleoside. In the case of 5CldC,
an increase in product formation was observed on incubation
for 4 h compared to 5 min, suggesting that secondary products
such as dC-derived chloramines may be important in the
chlorination observed (Figure 1A). In experiments with HOCl,
dG was the most readily chlorinated base, followed by dC and
8CldG formation was also observed in analogous experiments
with His-C, Lys-C, Gly-C, Tau-C, and Mono-C (Figure 1B and
C). The concentration of 8CldG increased with longer incubation
time (Figure 1B and C), consistent with slow base chlorination
by the added N-chloramine. In each case, the extent of 8CldG
observed was significantly lower than that observed with HOCl.
With His-C, significant amounts of 8CldA or 5CldC were also
observed, whereas no evidence was obtained for the formation

1
296 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Stanley et al.
Figure 2. Formation of 8CldG on treatment of an equimolar mixture
of nucleosides with HOCl or His-C. Samples containing dA, dC, dG,
and dT (each 500 µM, 12.5 nmol) were treated with 250 µM (6.25
nmol) HOCl (white) or His-C (black) for the incubation times indicated
before quenching the reaction with methionine (10 mM). 8CldG was
quantified by UV analysis at 260 nm, after separation by HPLC. *
represents significant 8CldG formation, compared to that in non-treated
controls, determined by one-way ANOVA with the Bonferroni post-
test. Data represent the mean ( SEM of n ) 3 experiments.
Figure 4. Consumption of oxidant on treating DNA with HOCl or His-
C. Five hundred micromolar HOCl (A) and His-C (B) were incubated
in the presence (white bars) and absence (black bars) of DNA (50 µg;
from calf thymus) for the indicated incubation times before the removal
of DNA by filtration through 3 kDa cutoff filters and quantification of
the remaining oxidant by TNB assay. * represents a significant decrease
in oxidant concentration, compared to that in the control incubated in
the absence of DNA, determined by one-way ANOVA, with the
Bonferroni post-test. Data represent the mean ( SEM of n ) 3
experiments.
resulted in the formation of 8ClA, 5ClC, and 8ClG respectively
(
8
Figure 3A). However, with the nucleotides, 5ClC rather than
ClG, was the most prominent chlorinated base formed, with
only low levels of 8ClA detected. Reaction of HOCl and His-C
with an equimolar mixture of ATP, CTP, GTP, and TTP gave
only 8ClG, in accord with the nucleoside experiments. However,
the yield of 8ClG in the experiments with an equimolar mixture
of nucleotides was very low, compared to the experiments with
the individual nucleotides (Figure 3B). Experiments were not
performed with Mono-C, Gly-C, Lys-C, and Tau-C due to the
slow reaction of these oxidants with nucleotides and the
corresponding low yield of chlorinated bases observed (Figure
1
C).
HOCl and N-Chloramine Treatment of Isolated DNA.
Initial studies examined the extent of oxidant consumption on
reaction of HOCl and the N-chloramines with isolated DNA.
The DNA was removed by filtration prior to oxidant quantifica-
tion to prevent confounding results due to the presence of DNA-
derived chloramines. Both HOCl and His-C were rapidly
consumed by the DNA (Figure 4A and B). This loss in oxidant
occurred much more rapidly than the thermal decay of the
oxidant observed in the absence of DNA (Figure 4A and B). In
contrast, only a minor difference in the rate of Mono-C, Gly-
C, Lys-C, and Tau-C decay was observed in the presence or
absence of DNA, suggesting that they react very slowly with
DNA, in accord with the nucleoside experiments described
above (data not shown).
Reaction of isolated DNA (50 µg) with increasing concentra-
tions of HOCl (25 µM-1 mM, 0.625-25 nmol) for 5 min
resulted in a concentration-dependent loss of parent bases
(Figure 5A), with a corresponding increase in 8CldA and 5CldC
formation (Figure 5B and C). Evidence was also obtained for
the formation of low amounts of 8CldG (Figure 5D). The
percentage conversion of parent base lost to chlorinated product
formed was ca. 0.3, 5.6, and 0.01% for 8CldA, 5CldC, and
Figure 3. Formation of chlorinated bases on treatment of nucleotides
with HOCl or His-C. Individual nucleotides ATP, CTP, and GTP (500
µM, 12.5 nmol; A) or an equimolar mixture of these nucleotides and
TTP (each 125 µM, 3.125 nmol; B) were treated with 250 µM (6.25
nmol) HOCl (white) or His-C (black) for the incubation times indicated,
before quenching the reaction with methionine (2.5 mM) and dephos-
phorylating the samples by incubation with alkaline phosphatase (25
U) for 1 h at 37 °C. Chlorinated base formation was quantified by UV
analysis at 260 nm, after separation by HPLC. * represents a significant
increase in chlorinated base formation, compared to that in the non-
treated control, determined by one-way ANOVA, with the Bonferroni
post-test. Data represent the mean ( SEM of n ) 3 experiments.
of these products with Mono-C, Gly-C, Lys-C, or Tau-C, even
after prolonged incubation (1 week). As with HOCl, 8CldG was
the only product detected on treating an equimolar mixture of
nucleosides with the N-chloramines (His-C shown in Figure 2).
Experiments with the isolated nucleotides ATP, CTP, or GTP
(
500 µM, 12.5 nmol) and HOCl or His-C (250 µM, 6.25 nmol)

DNA Chlorination by HOCl and N-Chloramines
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1297
Figure 5. Formation of chlorinated products on treatment of DNA with HOCl. Calf-thymus DNA (50 µg) was treated with increasing concentrations
of HOCl (25 µM-1 mM, 0.625-25 nmol) for varying times before precipitation, hydrolysis (with Nuclease P1; 10 U, 2 h at 37 °C), and
dephosphorylation (with alkaline phosphatase; 12 U, 1 h at 37 °C). Graph A represents the concentration of parent dA (white bars), dC (hatched
bars), and dG (black bars) remaining after the reaction with HOCl for 5 min. The concentration of chlorinated base products 8CldA (B), 5CldC (C),
and 8CldG (D) present after treatment for 5 min (white), 24 h (hatched), or 1 week (black) was quantified by selective reaction monitoring by
LC-MS-MS. * represents significant loss of parent base or chlorinated base formation, compared to that in the non-treated control, determined by
one-way ANOVA, with Dunnett’s multiple comparison test. Data represent the mean ( SEM of n ) 3 experiments.
8
CldG, respectively with 500 µM (12.5 nmol) HOCl. The
tifying the release of DNA from the cells using ethidium
bromide. With the exception of Mono-C, treatment of the
cells with up to 2 µmol HOCl or various N-chloramines (per
concentration of 8CldG decreased with increasing HOCl,
suggesting that this product is susceptible to further oxidation
6
(
Figure 5D). This was confirmed by the incubation of 8CldG
10 cells) for 2 or 4 h resulted in the lysis of e25% of the
with an equimolar concentration or 5-fold molar excess of HOCl
cells (Figure 7). The A7r5 cells lysed more readily in the
presence Mono-C, with ca. 13% and 28% lysis observed with
e0.2 µmol/10 cells on incubation for 2 or 4 h, respectively.
for 24 h at 20 °C, which resulted in a 40% or complete loss of
6
8
CldG, respectively (data not shown). A similar loss of 8CldA
and 5CldC was also observed on treating DNA with high (1
mM, 25 nmol) concentrations of HOCl, suggesting that these
products may also be susceptible to further oxidation in the
presence of a large excess of oxidant. In each case, there was
no significant increase in chlorinated base generation on reaction
of HOCl with DNA for increasing time periods, suggesting that
in general, the chlorination reactions occur within 5 min (Fig-
ure 5).
Experiments were not performed with higher concentrations
of Mono-C owing to the formation of autofluorescent
product(s). Lys-C and Tau-C induced very little cell lysis
(<5%), even at very high oxidant concentrations (1-4 µmol/
6
10 cells).
Experiments with HOCl and His-C were also performed with
6
primary BCASMC (0.25-2 µmol oxidant/10 cells) for 4 h. At
6
the lowest concentration of HOCl (0.25 µmol/10 cells), there
Reaction of DNA with His-C also resulted in the loss of
parent bases and the generation of 8CldA, 5CldC, and 8CldG
was no difference in the extent of cell lysis observed with the
two cell types (Figure 7C). However, at higher concentrations
of HOCl, significantly greater cell lysis was observed with
BCASMC, compared to that with A7r5 cells (67% and 78%,
respectively). Similarly, BCASMC were also more susceptible
to lysis induced by His-C compared to A7r5 cells, under similar
conditions (Figure 7C).
(Figure 6). In this case, higher yields of 8CldA and 8CldG were
observed compared to HOCl, particularly on longer incubation
of His-C with DNA (Figure 6). In this case, the percentage
conversion of parent base lost to chlorinated product formed
with 500 µM (12.5 nmol) His-C was ca. 1.3, 5.8, and 0.06%
for 8CldA, 5CldC, and 8CldG, respectively. In contrast, only
low levels of 8CldG were observed in experiments with Mono-
C, Gly-C, Lys-C, and Tau-C, even on treatment for 1 week,
consistent with the very slow rate of oxidant consumption (data
not shown).
The ability of HOCl and His-C to chlorinate cellular RNA
and DNA was investigated under conditions where <20% cell
lysis was observed to minimize the loss of this material from
the cell. Reaction of A7r5 cells with HOCl (0.125-0.5 µmol/
6
10 cells) resulted in a loss of parent RNA and DNA bases,
Reactivity of HOCl and N-chloramines with Cells. The
extent of cell lysis observed on treating A7r5 cells with HOCl
and N-chloramines for 2 and 4 h was determined by quan-
and the formation of 5CldC (Figure 8A). No evidence for the
formation of 8CldA, 8CldG, or RNA base chlorination was
obtained in this case. Low levels of 5CldC were observed on

1
298 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Stanley et al.
Figure 6. Formation of chlorinated products on treatment of DNA with His-C. Calf-thymus DNA (50 µg) was treated with increasing concentrations
of His-C (25 µM-1 mM, 0.625-25 nmol) for varying times before precipitation, hydrolysis (with Nuclease P1; 10 U, 2 h at 37 °C), and
dephosphorylation (with alkaline phosphatase; 12 U, 1 h at 37 °C). Graph A represents the concentration of parent dA (white bars), dC (hatched
bars), and dG (black bars) remaining after the reaction with His-C for 5 min. The concentration of chlorinated base products 8CldA (B), 5CldC (C),
and 8CldG (D) after treatment for 5 min (white), 24 h (hatched), or 1 week (black) was quantified by selective reaction monitoring by LC-MS-MS.
*
represents significant loss of parent base or chlorinated base formation, compared to that in the non-treated control, determined by one-way
ANOVA, with Dunnett’s multiple comparison test. Data represent the mean ( SEM of n ) 3 experiments.
6
treatment of A7r5 cells with His-C (0.25-1 µmol/10 cells) for
Lys-C and Tau-C both induced a significant loss of cellular ATP,
whereas no evidence for cell lysis was obtained.
4
h (Figure 8A).
The formation of 5CldC was also observed on treating
6
BCASMC with HOCl (0.125-0.5 µmol/10 cells) or His-C
Discussion
6
(
0.25-1 µmol/10 cells) (Figure 8B).With HOCl, the level of
CldC observed with BCASMC was somewhat lower than that
5
The aim of this study was to investigate the ability of HOCl
and various N-chloramines to chlorinate nucleosides, nucle-
otides, and DNA, as N-chloramines are likely to be important
mediators of cellular damage under conditions of inflammation,
and little is known about their reactivity with DNA and related
materials. With the individual nucleosides, 8CldG was the
favored product, with reactivity in the order HOCl > His-C .
Lys-C ≈ Gly-C > Mono-C ≈ Tau-C. Formation of 5CldC and
8CldA was also observed in experiments with HOCl and His-
C. A similar pattern of reactivity of HOCl with nucleosides has
been reported previously (20, 44). Evidence for the formation
of 5CldC, 8CldG, and 8CldA was also obtained on treatment
of isolated DNA with HOCl and His-C but not Mono-C, Lys-
C, Gly-C, and Tau-C, which reacted very slowly with DNA
and gave low yields of 8CldG only.
The extent of conversion of each parent base to the respective
chlorinated product in the experiments with HOCl and calf
thymus DNA is similar to or greater than that observed
previously (20, 23, 26, 45). In general, the formation of
chlorinated products accounts for <10% of the HOCl consumed
by the DNA. Interestingly, the level of chlorinated base
generation observed on reaction of His-C with DNA is generally
greater than that observed in experiments with HOCl, particu-
larly with higher concentrations of oxidant. This is attributed
to the induction of further oxidation of the chlorinated products
with the A7r5 cells, whereas the extent of 5CldC formation by
His-C was similar for both cell types (Figure 8B). With
BCASMC, experiments with HOCl and His-C also resulted in
the formation of the chlorinated RNA bases, 5ClC (Figure 8C)
6
and 8ClA (4-50 lesions/10 Ado). The formation of chlorinated
RNA and DNA bases on treating cells with Mono-C, Gly-C,
Lys-C, and Tau-C was not investigated, owing to the lack of
reactivity observed in the experiments with isolated DNA.
The potential reactivity of HOCl and N-chloramines with
cellular ATP was investigated in A7r5 cells by quantifying the
concentration of cellular ATP, under conditions where minimal
cell lysis was observed. The analogous experiments were not
performed with BCASMC owing to the increased susceptibility
of these cells to HOCl and His-C mediated lysis (Figure 7C).
3
Treatment of A7r5 cells (50 × 10 ) with HOCl and N-
6
chloramines (0.05-2.0 µmol/10 cells) over 2 h resulted in a
significant decrease in the concentration of cellular ATP (Figure
9
). In each case, the loss of cellular ATP was more significant
than that observed in the control experiments with PBS or the
respective parent amine (Figure 9). The most rapid loss of ATP
was observed on treating the cells with Mono-C, with almost
complete loss of the ATP observed on addition of g0.2 µmol
6
Mono-C/10 cells (Figure 9A). Rapid loss of ATP was also
observed with HOCl, Gly-C, and His-C (Figure 9). Interestingly,

DNA Chlorination by HOCl and N-Chloramines
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1299
Figure 8. Formation of chlorinated bases on cellular DNA and RNA.
Figure 7. Cell lysis observed in smooth muscle cells treated with HOCl
and N-chloramines. Graphs A and B show the results of A7r5 cells
treated with increasing concentrations of HOCl (9), His-C (4), Mono-C
Graph A represents A7r5 cells, and graphs B and C represent BCASMC
6
treated with HOCl (0.125-0.5 µmol/10 cells; hatched bars) and His-C
6
(0.25-1 µmol/10 cells; black bars) in HBSS for 4 h at 37 °C. The
(
2), Gly-C, (O), Lys-C (b), or Tau-C (0) for (A) 2 h or (B) 4 h at 37
C, with the extent of cell lysis determined by the ethidium bromide
cellular DNA and RNA were extracted, hydrolyzed, and dephospho-
rylated to the individual nucleosides. The concentration of the
chlorinated base products was quantified by selective reaction monitor-
ing by LC-MS-MS. A and B show the formation of 5CldC from A7r5
cells and BCASMC, respectively; C shows the formation of 5ClC in
BCASMC. * represents significant chlorinated base formation, com-
pared to that in the non-treated control, determined by one-way
ANOVA, with Dunnett’s multiple comparison test. Data represent the
mean ( SEM of n ) 3 experiments.
°
DNA release assay. Graph C shows results from BCASMC treated with
HOCl (0) and His-C (2) for 4 h (solid line) and the resulting cell lysis
compared to analogous treatment of A7r5 cells (dashed line). Data
represent the mean ( SEM of n g 3 experiments. * represents a
significant difference in cellular integrity between A7r5 and BCASMC,
using two-way ANOVA with Bonferroni post-test.
with HOCl, as a similar loss of the parent bases is observed in
each case. The sensitivity of chlorinated and other modified
nucleosides to further oxidation resulting in the loss of these
materials has been reported previously (23, 46, 47).
more susceptible to His-C induced lysis. The greater oxidation
of dC compared to that of dA and dG on exposure of the cells
to HOCl agrees with previous data, though the lack of detectable
levels of 8CldA and 8CldG was unexpected (22, 27). This may
be associated with the loss of these materials via further
oxidation reactions, owing to the longer incubation conditions
and higher oxidant concentrations employed in the current study.
Evidence was also obtained for the formation of 5ClC and
8ClA on exposure of BCASMC to HOCl and His-C. In this
case, the concentration of chlorinated RNA products was
somewhat higher than the analogous DNA products (ca. 20-
Chlorination of DNA (and RNA) was also observed on
treatment of A7r5 and BCASMC with HOCl and His-C, with
evidence for the formation of 5CldC but not 8CldA or 8CldG,
in each case. With the A7r5 cells, the level of 5CldC observed
was greater in experiments with HOCl, whereas similar levels
of this chlorinated product were observed on exposure of
BCASMC to either HOCl or His-C. The reason for this
difference is not certain but may be associated with a greater
ability of His-C to permeate BCASMC, as this cell type is also
6
fold, with 0.5 µmol HOCl/10 cells). This is consistent with
previous reports that RNA is more sensitive to modification by

1
300 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Stanley et al.
cells can incorporate 5CldC into genomic DNA (54). Similarly,
5
ClU can be readily converted to 5CldU by thymidine phos-
phorylase, before incorporation into DNA via the action of DNA
polymerase (55, 56). 5CldU is also formed from 5CldC via
deamination mediated by cellular enzymes (54). This is
significant because 5CldU is a well-established thymidine
analogue mutagen that mispairs with guanosine, causing G·C
f A·T and A·T f G·C transitions (57), and can induce sister
chromatid exchanges (55, 56). 5CldC also perturbs epigenetic
signals by mimicking 5-methyl-cytosine and enhancing the
binding of methyl-CpG binding proteins (58). This is significant
as methylation of cytosine occurs predominantly in CpG
dinucleotides (where a cytosine nucleotide occurs next to a
guanine nucleotide in a linear sequence) in islands of concen-
trated CpG sequences located in promoter regions. Methyl-CpG
binding proteins facilitate the recruitment of histone modifying
enzymes, which triggers a cascade of events resulting in gene
silencing (e.g., of tumor suppressor genes) (59, 60). HOCl can
preferentially chlorinate cytosine at CpG dinucleotides (61). In
addition, treatment of cells with 8ClA results in the accumulation
of 8-chloro-ATP and incorporation of this modified base into
the cellular mRNA (62). This leads to the inhibition of mRNA
synthesis and eventual cell death via apoptosis (62).
In summary, we show that HOCl and His-C are capable of
chlorinating DNA and related materials, both in isolation and
in a cellular environment. His-C chlorinates DNA to a similar
or greater extent than HOCl, supporting a role of imidazole-
derived, N-chloramines as secondary oxidants. This is significant
as His residues are one of the most reactive amino acid side
chains and hence are likely to be favored sites for reaction of
HOCl under physiological conditions, owing to the abundance
of proteins (14). The formation of imidazole-derived chloram-
ines on histamine, a low-molecular mass species released by
inflammatory mediators (51), and/or intracellular proteins, for
example, may play a role in the chlorination of genetic material
observed under chronic inflammatory conditions.
Figure 9. Depletion of ATP in A7r5 cells treated with HOCl and
N-chloramines. A7r5 cells were treated with (A) increasing concentra-
tions of HOCl (0), His-C (4), and Mono-C (O), and (B) Gly-C (0),
Lys-C (4), or Tau-C (O), or the respective parent amine (solid symbols)
for 2 h at 37 °C in black 96-well plates. ATP concentration was
determined by ATPlite Luminescence ATP Detection Assay System.
Data represent the mean ( SEM of n ) 3 experiments.
oxidants compared to DNA, on account of the RNA being less
compartmentalized and mainly cytoplasmic in nature (27, 48).
Evidence for RNA, but not DNA, chlorination was also observed
on exposure of E. coli to HOCl generated via the MPO/H
Cl system (28).
2
O
2
/
-
These data show that His-C can chlorinate both isolated and
cellular DNA to a similar or greater extent than HOCl,
suggesting that it may be an important secondary oxidant in
MPO oxidant-mediated damage. This supports previous studies
demonstrating that His-C and related imidzole-derived N-
chloramines react readily with other protein components (49, 50)
and low-molecular mass antioxidants including ascorbate and
GSH (51). In contrast, Mono-C, Lys-C, Gly-C, and Tau-C are
poor chlorinating agents on exposure to both nucleosides and
DNA, though there is evidence that Lys-C and Tau-C can
mediate the formation of 5ClU (32). However, these N-
chloramines are able to induce cellular damage, as evidenced
by their ability to rapidly deplete intracellular ATP. This loss
in ATP may be due to the direct reaction of the oxidants with
ATP or by inhibition of cellular ATP generation and/or
regeneration capability, via the reaction with other targets. The
fact that significant ATP depletion and chlorination was only
observed on treatment of the isolated material with HOCl and
His-C suggests the latter is more likely. Indeed, treatment of E.
coli with HOCl resulted in the loss of ATPase activity of the
Acknowledgment. We are grateful to the National Health
and Medical Research Council (Australia), the National Heart
Foundation of Australia, and the Australian Research Council
(through the ARC Centres of Excellence program) for financial
support. N.R.S. gratefully acknowledges the receipt of a
University of Sydney Faculty of Medicine Scholarship. We also
thank Professor Michael Davies and Dr. Alison Heather for
helpful discussions.
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