From the Department of Biochemistry, Faculty of Medicine, National University of Singapore, 8 Medical Dr., Singapore 117597, Republic of Singapore
Received for publication, October 30, 2002, and in revised form, December 3, 2002
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ABSTRACT |
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Elevated levels of reactive nitrogen species
(RNS) such as peroxynitrite have been implicated in over 50 diverse
human diseases as measured by the formation of the RNS biomarker
3-nitrotyrosine. Recently, an additional RNS was postulated to
contribute to 3-nitrotyrosine formation in vivo; nitryl
chloride formed from the reaction of nitrite and neutrophil
myeloperoxidase-derived hypochlorous acid (HOCl). Whether nitryl
chloride nitrates intracellular protein is unknown. Therefore, we
exposed intact human HepG2 and SW1353 cells or cell lysates to HOCl and
nitrite and examined each for 3-nitrotyrosine formation by: 1) Western
blotting, 2) using a commercial 3-nitrotyrosine enzyme-linked
immunosorbent assay kit, 3) flow cytometric analysis, and 4) confocal
microscopic analysis. With each approach, no significant
3-nitrotyrosine formation was observed in either whole cells or cell
lysates. However, substantial 3-nitrotyrosine was observed when
peroxynitrite (100 µM) was added to cells or cell
lysates. These data suggest that nitryl chloride formed from the
reaction of nitrite with HOCl does not contribute to the elevated
levels of 3-nitrotyrosine observed in human diseases.
There is considerable interest in the role of reactive nitrogen
species (RNS)1 such as nitric
oxide (·NO) and peroxynitrite (ONOO Over 50 human disease conditions have elevated levels of
3-nitrotyrosine, a biomarker for RNS traditionally attributed to ONOO Recently, a further mechanism for 3-nitrotyrosine formation was
proposed (22, 23); the formation of nitryl chloride (NO2Cl) by reaction of myeloperoxidase-derived hypochlorous acid (HOCl) with
NO
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) in human
disease (reviewed in Refs. 1 and 2). Numerous cell types are capable of
producing high micromolar concentrations of nitric oxide (·NO)
through the activation of inducible nitric-oxide synthase (reviewed in Refs. 3 and 4). In vivo, ·NO is readily
oxidized via heme proteins to nitrite (NO
formation in vivo. These include
neurodegenerative, chronic inflammatory, gastrointestinal tract, and
cardiovascular disorders as well as viral and bacterial infections
(reviewed in Refs. 1 and 2). Recent research has shown that
3-nitrotyrosine formation is not solely a ONOO
-mediated
phenomenon. It is also observed with peroxidases such as eosinophil
peroxidase (18), myeloperoxidase (released by activated neutrophils at
sites of inflammation) (19), and other peroxidases (20) in the presence
of NO
It has been estimated that up to 80% of the
H2O2 generated by activated neutrophils during
the respiratory burst is used to form HOCl (25). Consequently,
NO2Cl formation from activated human neutrophils and
nitration of extracellular phenolics in the presence of added
NO
Although NO
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EXPERIMENTAL PROCEDURES |
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Materials--
Bovine serum albumin (BSA), oxidized glutathione
(GSSG), sodium nitrite (NaNO2), sodium nitrate
(NaNO3), sodium hypochlorite, and all other reagents were
purchased from Sigma-Aldrich (St. Louis, MO). HOCl concentration was
quantified immediately before use spectrophotometrically at 290 nm (pH
12, = 350 M
1 cm
1)
(29). Hydrogen peroxide-free peroxynitrite was synthesized as described
previously (30) and quantified in 1 N NaOH at 302 nm
(
= 1670 M
1 cm
1).
Rabbit polyclonal anti-nitrotyrosine antibodies were from either Upstate Biotechnology Inc. (#12-348) or BIOMOL (Plymouth Meeting, PA,
#SA-297). Mouse monoclonal anti-nitrotyrosine antibodies were obtained
from Calbiochem (La Jolla, CA, #487923) or Alexis (#804-204). Peroxidase-conjugated secondary antibodies for Western blotting were
purchased from Promega. Fluorescently labeled rhodamine anti-mouse IgG
(#12-329) was obtained from Calbiochem, and AlexaFluor 488 anti-rabbit
IgG (#A11008) was obtained from Molecular Probes (Eugene, OR). The
nitrotyrosine ELISA was purchased from Cambridge Biosciences
(Cambridge, England, #HK501).
Exposure of Cells to NO
Cells were washed three times in warm PBS and further incubated for 10 min with PBS containing increasing concentrations of NaNO2
(10 µM to 1 mM). After this time, HOCl was
added to give final concentrations between 7 and 125 µM.
Cells were then incubated at 37 °C for 5 min. In parallel
experiments, cell lysates were obtained by freeze-thawing in 0.5 ml of
PBS and sonication at 4 °C for 10 min before addition of
HOCl/NO for 5 min and the
addition of protease inhibitors (1 µg/ml aprotinin, 1 µg/ml
pepstatin A, 1 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride) as described (31).
After exposure to HOCl, ONOO, NO
Analysis of Nitrotyrosine-- After treatment, 10 mM GSSG was added to quench any unreacted HOCl, and cells were washed twice with warm (37 °C) PBS. Cells were then lysed with PBS containing 0.1% SDS and protease inhibitors (1 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) as described (31).
Western blotting for nitrotyrosine-containing proteins was conducted as
described (31) using polyclonal or monoclonal anti-nitrotyrosine antibodies with an enhanced chemiluminescence detection kit (Amersham Biosciences, Buckinghamshire, United Kingdom) followed by analysis using a Kodak image analyzer (IS440CF, PerkinElmer Life Sciences, Boston, MA), and captured images were analyzed using Kodak Digital Science one-dimensional image analysis software. The same samples obtained for Western blotting were also analyzed for nitrotyrosine content by commercial ELISA, prepared using the manufacturer's instructions. Protein concentration was determined using a commercial kit (Bio-Rad Dc protein assay), and samples were normalized for protein
content. As an additional control, 1 mM ONOO
was added to BSA (4 mg/ml) to generate nitrated BSA.
Confocal Microscopy--
1 × 105 cells were
seeded overnight in glass bottom Petri dishes (WillCo-dish, Willco
Wells, Amsterdam, The Netherlands) and washed three times in warm
(37 °C) NO as described above. Cells
were then fixed and permeabilized in 1 ml of ice-cold ethanol (70%
v/v) and incubated at 4 °C for 2 h. Control experiments using
100 µM NO
Flow Cytometry--
Cells were seeded overnight in six-well
plates at a density of 1 × 106 cells per well and
treated with HOCl, NO as
described above. Cells were then washed three times in
NO
Data Analysis--
All graphs were plotted with mean ± S.D. In all cases the mean values were calculated from data
taken from at least six separate experiments performed on separate days
using freshly prepared reagents. Where significance testing was
performed, an independent test (Student's t test, two
populations) was used (*, p < 0.1; **,
p < 0.05; and ***, p < 0.01).
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RESULTS |
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Assessment of Tyrosine Nitration by Commercial ELISA--
The
addition of HOCl to SW1353 and HepG2 cells for 5 min resulted in
negligible loss of cell viability as measured using MTT. For example,
the addition of 125 µM HOCl for 5 min resulted in a
8.8 ± 3.4% and 5.6 ± 4.8% reduction in SW1353 and HepG2
cell viability, respectively. The addition of HOCl,
NO did not significantly
alter the pH of the reaction mixture.
Using a commercially available nitrotyrosine ELISA kit, extensive
tyrosine nitration was observed after human HepG2 cells or SW1353 cells
(Fig. 1A) or cell lysates
(Fig. 1B) were exposed to 100 µM
ONOO for 5 min. In sharp contrast, cells treated with
HOCl and NO
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Assessment of Tyrosine Nitration by Western Blotting--
Western
blotting using monoclonal and polyclonal anti-nitrotyrosine antibodies
from several commercial sources was also performed. Treating cells or
freshly prepared cell lysates with ONOO (100 µM, positive control) resulted in extensive tyrosine
nitration in cell lysates. However, exposure of the cells or cell
lysates to HOCl or HOCl and NO
) using either monoclonal or
polyclonal antibodies. The same results were obtained with HepG2 cells
and cell lysates (data not shown).
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Assessment of Intracellular Tyrosine Nitration by Flow
Cytometry--
Flow cytometric analysis of whole cells exposed to
ONOO, HOCl, and HOCl with NO
resulted in a substantial increase in the number of
nitrotyrosine-positive cells compared with untreated cells (Fig. 3,
A and E). Complete inhibition of antibody binding
was achieved by incubating the primary antibodies with 10 mM nitrotyrosine (Fig. 3, B and F). Incubation of cells with up to 125 µM HOCl for 5 min did
not result in the formation of intracellular 3-nitrotyrosine (Fig. 3,
C and G). Fig. 3 (D and H)
is representative of all the concentrations of NO
treatment, cells treated with HOCl in
the presence of up to 1 mM NO
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Assessment of Intracellular Tyrosine Nitration by Confocal
Microscopy--
Laser scanning confocal microscopy was also used to
assess intracellular tyrosine nitration. Fig.
4 is representative of data obtained when
cells were exposed to either ONOO or HOCl in the presence
of NO
(100 µM)
using either polyclonal (Fig. 4B) or monoclonal (Fig. 4E) anti-nitrotyrosine antibodies. In contrast, treating the
cells with PBS alone (Fig. 4, A and D) or
incubating cells with NO
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DISCUSSION |
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The formation of 3-nitrotyrosine has been observed in over 50 human disease conditions (reviewed in Refs. 1 and 2). The formation of
this biomarker has been attributed to an overproduction of ·NO
and subsequent formation of highly reactive nitrogen species (RNS)
usually attributed as ONOO. However, an overproduction of
·NO also results in the accumulation of NO
1
s
1) (24) and the high concentrations of HOCl and
accumulation of NO
-carotene and
-tocopherol depletion by NO
Using several monoclonal and polyclonal commercial antibodies,
substantial nitrotyrosine formation was observed only when cells or
cells lysates were exposed to ONOO added at sublethal
concentrations (100 µM). No formation of 3-nitrotyrosine was observed by Western blot with enhanced chemiluminescence detection (Fig. 2) in either cells or cell lysates exposed to
HOCl/NO
-treated cells using these antibodies
with flow cytometric or confocal microscopic analysis. In support,
Sampson et al. (20) also failed to detect tyrosine nitration
by Western blot in homogenates of horse hearts exposed to
HOCl/NO
.
It is unlikely that residual HOCl degraded any protein bound
nitrotyrosine formed, as we recently reported in vitro (42) for the following reasons: 1) NO1 s
1) (24); 2) analysis of
buffers after experimentation showed NO
-treated cells) performed adequately in each
technique used. Similarly, over this short time period there was
negligible loss of cell viability. However, for confocal microscopy,
even dead cells would have been fixed and would have immunostained
positively for nitrotyrosine if it had been formed. The possibility
that the levels of tyrosine nitration are too low for each of the four
separate techniques employed cannot be completely ruled out. However,
the majority of the published reports dealing with tyrosine nitration
in human disease have used the same techniques as described here
(reviewed in Ref. 2). The most commonly used detection of tyrosine
nitration in human disease lesions is immunohistochemistry with
colorimetric detection. This is less sensitive than fluorometric
immunohistochemistry detection with confocal microscopy and flow
cytometry used here. Similarly, Western blotting and ELISA are
routinely used techniques (2). Recently, Spencer et al. (23)
showed a potentiation of HOCl-mediated DNA base deamination and
inhibition of HOCl-mediated DNA strand breakage in HBE-1 cells with
high concentrations of NO
) with each analytical technique used and not with
mixtures of NO
Therefore, it is possible that NO2Cl formed from the
reaction of physiologically attainable concentrations of
NO, peroxidase (18, 20, 23), or heme-mediated RNS (21). As with NO2Cl, the extent to which these contribute to
intracellular tyrosine nitration is unknown and is currently
being investigated by our laboratory.
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ACKNOWLEDGEMENT |
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We are grateful to the National Medical Research Council of Singapore for generous research support.
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FOOTNOTES |
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* This work was supported by the National Medical Research Council of Singapore (Grants NMRC/0474/2000, NMRC/0481/2000, and NMRC/0635/2002).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.
To whom correspondence should be addressed. Tel.: 65-6874-8891;
Fax: 65-6779-1453; E-mail: bchwml@nus.edu.sg.
Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M211086200
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ABBREVIATIONS |
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The abbreviations used are: RNS, reactive nitrogen species; BSA, bovine serum albumin; GSSG, oxidized glutathione; HOCl, hypochlorous acid; MTT, 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline.
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