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J. Biol. Chem., Vol. 278, Issue 10, 8380-8384, March 7, 2003

This Article Abstract Full Text (PDF) All Versions of this Article: 278/10/8380    most recent M211086200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Whiteman, M. Articles by Halliwell, B. Search for Related Content PubMed PubMed Citation Articles by Whiteman, M. Articles by Halliwell, B. Social Bookmarking                      What's this?

Lack of Tyrosine Nitration by Hypochlorous Acid in the Presence of Physiological Concentrations of Nitrite

IMPLICATIONS FOR THE ROLE OF NITRYL CHLORIDE IN TYROSINE NITRATION IN VIVO^*

Matthew Whiteman, Jia Ling Siau, and Barry Halliwell

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

There is considerable interest in the role of reactive nitrogen species (RNS)¹ such as nitric oxide (^·NO) and peroxynitrite (ONOO) 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) and nitrate (NO). Thus, evidence for an elevated production of ^·NO comes from the measurement of NO and NO in human body fluids such as plasma, cerebrospinal fluid, synovial fluid, respiratory tract lining fluid, saliva, and sputum. This has implicated ^·NO in a large number and diverse range of human diseases (reviewed in Ref. 4). Typically, levels of NO found in plasma taken from healthy human volunteers range between 0.5 and 21.0 µM (5, 6), and levels are significantly elevated during inflammation, e.g. up to 36 µM in patients with human immunodeficiency virus infection (7). Serum NO levels in patients with rheumatoid arthritis (8), systemic sclerosis (9), and systemic lupus erythematosus (10) are reported to be in the millimolar range, whereas in the synovial fluid of patients with rheumatoid arthritis NO levels are reported to range from 0.3 to 15 µM (11-13). Nitrite has been extensively used for decades in the food industry as a preservative and for curing meat. Approximately 5% of ingested nitrate is reduced to nitrite by oral microflora where it enters the gastrointestinal tract and protonates to form nitrous acid (NHO₂, pK_a ~ 3.4) (reviewed in Refs. 14 and 15). Furthermore, dietary NO has been proposed as an oral and gut anti-microbial agent (14, 15) where salivary levels of NO of up to 98 µM have been reported (16), and near millimolar concentrations are reported to be reached in the saliva of patients with systemic sclerosis (17).

Over 50 human disease conditions have elevated levels of 3-nitrotyrosine, a biomarker for RNS traditionally attributed to ONOO 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. In addition, hemoglobin and other heme proteins such as catalase may also serve as a mechanism for nitrating tyrosine residues in proteins using NO as a substrate (21).

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 (Reaction 1) (24).

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 have been demonstrated (23).

Although NO, NO, and HOCl are formed in substantial amounts during inflammation and the former two are normally present in saliva and are present in the gut at high concentrations, whether NO₂Cl plays any part in the tyrosine nitration observed in vivo is unclear. Therefore, in this report we investigated whether HOCl in the presence of NO could nitrate intracellular protein using HepG2 hepatoma and SW1353 chondrosarcoma cells as models of human liver (26) and cartilage cells (27, 28) exposed to RNS. We used four analytical approaches: immunochemistry with monoclonal and polyclonal antibodies from two commercial sources using confocal microscopy, flow cytometry, and Western blotting as well as a commercial ELISA kit.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 M¹ cm¹) (29). Hydrogen peroxide-free peroxynitrite was synthesized as described previously (30) and quantified in 1 N NaOH at 302 nm ( = 1670 M¹ cm¹). 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 and HOCl-- Human HepG2 hepatoma and SW1353 chondrosarcoma cells were obtained from the American Tissue Culture Collection (Gaithersburg, MD). HepG2 cells were grown in Minimum essential media, and SW1353 cells were grown in Dulbecco's modified Eagle's medium. Media were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and cells were grown at 37 °C in 5% CO₂:95% O₂ with ~95% humidity to 90% confluency before seeding into six-well plates (Falcon) overnight at a density of 1 × 10⁶ cells/well.

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 or ONOO 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, or NO/HOCl, cell viability was assessed using 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described (32).

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-free PBS before addition of HOCl, NO, or ONOO 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 alone were also performed (33). Cells were then incubated with either monoclonal or polyclonal anti-nitrotyrosine antibodies for 1 h at room temperature followed by rhodamine- or AlexaFluor 488-labeled secondary anti-IgG antibodies as described (33). Cells were processed and analyzed within 5 h of initial treatment. Laser confocal microscopy was performed with a Zeiss LSM410 confocal microscope. The red fluorescence of rhodamine and the green fluorescence of AlexaFlour 488 were excited with the 568- and 488-nm lines of an argon-krypton laser. Fluorescence was split by a 560-nm emission dichroic filter and collected by separate photomultipliers through 515- to 565-nm band pass and 590-nm long pass barrier filters with the following settings held constant: photo-multiplier tube (610), gain (4.2%), and offset (2%).

Flow Cytometry-- Cells were seeded overnight in six-well plates at a density of 1 × 10⁶ cells per well and treated with HOCl, NO, or ONOO as described above. Cells were then washed three times in NO-free PBS, scraped into 1.5 ml of PBS in Eppendorf tubes, centrifuged at 3000 rpm for 5 min, and fixed and permeabilized with 1 ml of ice-cold ethanol (70%, v/v) at 4 °C for 2 h as described (33, 34). Polyclonal or monoclonal anti-nitrotyrosine antibodies were then added and incubated in PBS containing 1% (v/v) fetal bovine serum for 1 h at room temperature. After washing, fluorescently labeled secondary antibodies (rhodamine or AlexaFluor 488) were then added for 1 h. Cells were then analyzed by flow cytometry using a Epics Elite flow cytometer (ESP, Coulter, Miami, FL) within 5 h of initial treatment. Data were analyzed from 20,000 cells using WinMDI 2.7 software (Scripps Institute, La Jolla, CA), and the percentage of nitrotyrosine-stained cells was determined from histogram analysis.

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).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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, or ONOO 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 did not show any significant increase in tyrosine nitration at any of the HOCl (7-125 µM) and NO (10 µM to 1 mM) concentrations used. Fig. 1A shows the results obtained with 125 µM HOCl and is representative of all the HOCl concentrations used. Similarly, the addition of HOCl and NO to freshly prepared cell lysates did not show any observable tyrosine nitration (Fig. 1B).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Analysis of tyrosine nitration by commercial ELISA. Cells (A) or fresh cell lysates (B) were incubated with NO at the concentrations stated and HOCl (125 µM) added for 5 min or treated with 100 µM ONOO for 5 min. Residual HOCl was quenched by the addition of GSSG, and the formation of 3-nitrotyrosine was analyzed by ELISA as described under "Experimental Procedures." Data are expressed as mean ± S.D. of six or more separate experiments.

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 did not result in any detectable tyrosine nitration with any of the commercial antibodies used. Fig. 2 (A and B) shows representative Western blots obtained using an Upstate Biotechnology rabbit polyclonal antibody (Fig. 2A) and the Calbiochem mouse monoclonal antibody (Fig. 2B) on whole cell extracts and cell lysates from SW1353 cells. It can be seen that, even after lengthy exposure (20 min) in ECL reagent, tyrosine nitration was only detected in the positive control (100 µM ONOO) using either monoclonal or polyclonal antibodies. The same results were obtained with HepG2 cells and cell lysates (data not shown).

View larger version (52K): [in this window] [in a new window]   Fig. 2.   Analysis of tyrosine nitration by Western blotting. SW1353 cells or fresh cell lysates were incubated with NO at the concentrations stated, and HOCl (125 µM) was added for 5 min or treated with 100 µM ONOO for 5 min. Lanes: (1) PBS, (2) whole cells exposed to 100 µM ONOO, (3) cell lysates exposed to 100 µM ONOO, (4) HOCl (125 µM), (5) HOCl + 125 µM NO, (6) HOCl + 250 µM NO, and (7) HOCl + 500 µM NO. Lanes 8-10: SW1353 cell lysates exposed to (8) HOCl + 125 µM NO, (9) HOCl + 250 µM NO, and (10) HOCl + 500 µM NO. Lane 11: BSA treated with 1 mM ONOO. Residual HOCl was quenched by the addition of GSSG, and the formation of 3-nitrotyrosine was analyzed by Western blotting as described under "Experimental Procedures." Data are representative of four separate experiments.

Assessment of Intracellular Tyrosine Nitration by Flow Cytometry-- Flow cytometric analysis of whole cells exposed to ONOO, HOCl, and HOCl with NO was also performed with monoclonal and polyclonal anti-nitrotyrosine antibodies. Fig. 3 is representative of results obtained using polyclonal (Fig. 3, A-D) and monoclonal antibodies (Fig. 3, E-H) against nitrotyrosine in HepG2 (Fig. 3, A-D) and SW1353 cells (Fig. 3, E-H). Treatment of cells with 100 µM ONOO 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 (0-1 mM) and HOCl (0-125 µM) tested, and, in contrast to ONOO treatment, cells treated with HOCl in the presence of up to 1 mM NO did not show any observable nitrotyrosine formation (Fig. 3, D and H).

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Analysis of tyrosine nitration by immunocytochemistry and flow cytometry. HepG2 cells were incubated with NO at the concentrations stated, and HOCl (125 µM) was added for 5 min or treated with 100 µM ONOO for 5 min. Residual HOCl was quenched by the addition of GSSG, and the formation of 3-nitrotyrosine was analyzed by flow cytometry using polyclonal (A-D) or monoclonal (E-H) anti-nitrotyrosine antibodies. In further control experiments, antibody binding was blocked with 10 mM nitrotyrosine (B and F). Experiments were conducted as described under "Experimental Procedures." Data are representative of four separate experiments.

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. HepG2 cells were immunostained with polyclonal anti-nitrotyrosine antibodies coupled to AlexaFluor 488-conjugated secondary anti-rabbit IgG (Fig. 4, A-C), and SW1353 cells were immunostained with monoclonal anti-nitrotyrosine antibodies coupled to rhodamine-conjugated anti-mouse IgG (Fig. 4, D and E). Substantial positive 3-nitrotyrosine immunostaining was observed in HepG2cells and SW1353 cells exposed to non-lethal concentrations of ONOO (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 followed by subsequent addition of HOCl (Fig. 4, C and F) did not result in any detectable nitrotyrosine formation at any of the NO concentrations (10 µM to 1 mM) or HOCl concentrations (7-125 µM) used.

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Analysis of tyrosine nitration by immunocytochemistry and confocal microscopy. HepG2 cells (A-C) or SW1353 cells (D and E) were incubated with PBS (A and D), 100 µM ONOO (B and E), or NO and 125 µM HOCl (C and F) for 5 min. Residual HOCl was quenched by the addition of GSSG, and the formation of 3-nitrotyrosine was analyzed by confocal microscopy using polyclonal (A-C) or monoclonal (D and E) anti-nitrotyrosine antibodies performed as described under "Experimental Procedures." Data are representative of four separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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, which has been reported to reach millimolar concentrations in certain disease conditions (8-10). Nitrite also serves as a substrate for peroxidases (20) such as myeloperoxidase (20, 23) and eosinophil peroxidase (18) as well as heme proteins (21) to generate tyrosine-nitrating species. At sites of chronic inflammation, neutrophils produce the oxidant HOCl, which in the presence of NO, forms an additional tyrosine-nitrating species, nitryl chloride (NO₂Cl) (22, 23). Although there is a wealth of information on RNS-mediated processes, limited information is available on the consequences of HOCl and NO accumulation and resulting NO₂Cl formation generated from this reaction (28, 32). The relatively fast second order rate constant of reaction of NO with HOCl (pH 7.2, 25 °C, 7.4 ± 1.3 × 10³ M¹ s¹) (24) and the high concentrations of HOCl and accumulation of NO at sites of chronic inflammation or in the gut after a meal (14, 15), suggest this reaction is plausible in vivo. Recently, Panasenko et al. (24) demonstrated protein modification, low density lipoprotein oxidation, and -carotene and -tocopherol depletion by NO and HOCl mixtures. NO also enhanced formation of some DNA base damage products in HOCl-treated isolated calf thymus DNA (35) as well as in DNA isolated from human bronchial epithelial cells exposed to HOCl (32). The majority of reports thus far have focused on the potentiation of oxidative and chlorinative reactions of HOCl by NO, and the data on nitration of phenolics have been conducted on cell media or buffer (23, 24) rather than the effects on cells themselves. The extent to which NO₂Cl penetrates the cell membrane and reacts with intracellular tyrosine residues to contribute to the tyrosine nitration observed in the diverse and large number of human diseases is unknown. The relatively fast rate of reaction and high concentrations of NO in vivo also confers HOCl-scavenging abilities on NO, such as inhibition of HOCl-mediated anti-microbial activity (36-38) and cell toxicity (28). Therefore, using two human cells lines as models of human cells exposed to RNS in vivo, the extent of tyrosine nitration induced by HOCl/NO was investigated using several established analytical techniques.

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. Similarly, tyrosine nitration was only detected with ONOO-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, but substantial nitrotyrosine formation was observed when the homogenates were exposed to ONOO.

It is unlikely that residual HOCl degraded any protein bound nitrotyrosine formed, as we recently reported in vitro (42) for the following reasons: 1) NO reacts with HOCl rapidly (pH 7.2, 25 °C, 7.4 ± 1.3 × 10³ M¹ s¹) (24); 2) analysis of buffers after experimentation showed NO to be oxidized to NO (this reaction is stoichiometric (1 mol of NO consumed by 1 mol of HOCl to give 1 mol of NO) (24, 28); and 3) the time course of exposure used was short (5 min). HOCl-mediated loss of 3-nitrotyrosine in proteins over the same time is negligible (42). The presence of a variety of protease inhibitors during Western blotting and ELISA and fixation in ethanol for flow cytometry and confocal microscopy techniques should have inhibited any endogenous nitrotyrosine-removing entities. In any case these reactions appear slow and incomplete such as those observed in dog prostate tissue (39), rat brain and heart homogenates (40), and skin (41). Furthermore, the positive control (ONOO-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 (1 mM) when added to high concentrations of HOCl (1 mM) followed by a prolonged period of exposure (2 h). Therefore, reactive entities from the reaction of HOCl and NO could penetrate the cell membrane but are unlikely to cause tyrosine nitration, because it was only observed with the positive control (100 µM ONOO) with each analytical technique used and not with mixtures of NO and HOCl.

Therefore, it is possible that NO₂Cl formed from the reaction of physiologically attainable concentrations of NO and HOCl in vivo contributes minimally to the formation of intracellular tyrosine nitration observed in human diseases and animal models. Consequently, the cellular nitration reported in human disease (1, 2) is more likely to originate from ONOO, 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.

	ACKNOWLEDGEMENT

We are grateful to the National Medical Research Council of Singapore for generous research support.

	FOOTNOTES

^* 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

	ABBREVIATIONS

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.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 278/10/8380    most recent M211086200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Whiteman, M. Articles by Halliwell, B. Search for Related Content PubMed PubMed Citation Articles by Whiteman, M. Articles by Halliwell, B. Social Bookmarking                      What's this?