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 *

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-nitroty-rosine formation in vivo ; nitryl chloride formed from the reaction of nitrite and neutrophil myeloperoxidase-de-rived 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 immu-nosorbent 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 (cid:1) M ) 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.

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 M) 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 ( ⅐ NO) and peroxynitrite (ONOO Ϫ ) in human disease (reviewed in Refs. 1 and 2). Numerous cell types are capable of producing high micromolar concentrations of nitric oxide ( ⅐ NO) through the activation of inducible nitric-oxide synthase (reviewed in Refs. 3 and 4). In vivo, ⅐ NO is readily oxidized via heme proteins to nitrite (NO 2 Ϫ ) and nitrate (NO 3 Ϫ ). Thus, evidence for an elevated production of ⅐ NO comes from the measurement of NO 2 Ϫ and NO 3 Ϫ in human body fluids such as plasma, cerebrospinal fluid, synovial fluid, respiratory tract lining fluid, saliva, and sputum. This has implicated ⅐ NO in a large number and diverse range of human diseases (reviewed in Ref. 4). Typically, levels of NO 2 Ϫ found in plasma taken from healthy human volunteers range between 0.5 and 21.0 M (5, 6), and levels are significantly elevated during inflammation, e.g. up to 36 M in patients with human immunodeficiency virus infection (7). Serum NO 2 Ϫ 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 2 Ϫ levels are reported to range from 0.3 to 15 M (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 (NHO 2 , pK a ϳ 3.4) (reviewed in Refs. 14 and 15). Furthermore, dietary NO 2 Ϫ has been proposed as an oral and gut anti-microbial agent (14,15) where salivary levels of NO 2 Ϫ of up to 98 M 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 Ϫ 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 Ϫ -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 2 Ϫ . In addition, hemoglobin and other heme proteins such as catalase may also serve as a mechanism for nitrating tyrosine residues in proteins using NO 2 Ϫ as a substrate (21).
It has been estimated that up to 80% of the H 2 O 2 generated by activated neutrophils during the respiratory burst is used to form HOCl (25). Consequently, NO 2 Cl formation from activated human neutrophils and nitration of extracellular phenolics in the presence of added NO 2 Ϫ have been demonstrated (23). Although NO 2 Ϫ , NO 3 Ϫ , 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 NO 2 Cl 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 2 Ϫ 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.
Exposure of Cells to NO 2 Ϫ 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% CO 2 :95% O 2 with ϳ95% humidity to 90% confluency before seeding into six-well plates (Falcon) overnight at a density of 1 ϫ 10 6 cells/well. Cells were washed three times in warm PBS and further incubated for 10 min with PBS containing increasing concentrations of NaNO 2 (10 M to 1 mM). After this time, HOCl was added to give final concentrations between 7 and 125 M. Cells were then incubated at 37°C for 5 min. In parallel experiments, cell lysates were obtained by freezethawing in 0.5 ml of PBS and sonication at 4°C for 10 min before addition of HOCl/NO 2 Ϫ or ONOO Ϫ for 5 min and the addition of protease inhibitors (1 g/ml aprotinin, 1 g/ml pepstatin A, 1 g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) as described (31).
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 g/ml aprotinin, 1 g/ml pepstatin A, 1 g/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 Ϫ was added to BSA (4 mg/ml) to generate nitrated BSA.
Confocal Microscopy-1 ϫ 10 5 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 2 Ϫ -free PBS before addition of HOCl, NO 2 Ϫ , or ONOO Ϫ 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 M NO 2 Ϫ 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 fluores-cence 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 ϫ 10 6 cells per well and treated with HOCl, NO 2 Ϫ , or ONOO Ϫ as described above. Cells were then washed three times in NO 2 Ϫ -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 Ϯ 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 Ͻ 0.1; **, p Ͻ 0.05; and ***, p Ͻ 0.01).

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 M HOCl for 5 min resulted in a 8.8 Ϯ 3.4% and 5.6 Ϯ 4.8% reduction in SW1353 and HepG2 cell viability, respectively. The addition of HOCl, NO 2 Ϫ , or ONOO Ϫ 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 M ONOO Ϫ for 5 min. In sharp contrast, cells treated with HOCl and NO 2 Ϫ did not show any significant increase in tyrosine nitration at any of the HOCl (7-125 M) and NO 2 Ϫ (10 M to 1 mM) concentrations used. Fig. 1A shows the results obtained with 125 M HOCl and is representative of all the HOCl concentrations used. Similarly, the addition of HOCl and NO 2 Ϫ to freshly prepared cell lysates did not show any observable tyrosine nitration (Fig. 1B).
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 Ϫ (100 M, positive control) resulted in extensive tyrosine nitration in cell lysates. However, exposure of the cells or cell lysates to HOCl or HOCl and NO 2 Ϫ 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 M ONOO Ϫ ) using either monoclonal or polyclonal antibodies. The same results were obtained with HepG2 cells and cell lysates (data not shown).
Assessment of Intracellular Tyrosine Nitration by Flow Cytometry-Flow cytometric analysis of whole cells exposed to ONOO Ϫ , HOCl, and HOCl with NO 2 Ϫ 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 nitroty-rosine in HepG2 (Fig. 3, A-D) and SW1353 cells (Fig. 3, E-H). Treatment of cells with 100 M ONOO Ϫ 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 M 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 2 Ϫ (0 -1 mM) and HOCl (0 -125 M) tested, and, in contrast to ONOO Ϫ treatment, cells treated with HOCl in the presence of up to 1 mM NO 2 Ϫ did not show any observable nitrotyrosine formation (Fig. 3, D and H).
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 Ϫ or HOCl in the presence of NO 2 Ϫ . 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 Ϫ (100 M) 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 2 Ϫ followed by subsequent addition of HOCl (Fig. 4, C and F) did not result in any detectable nitrotyrosine formation at any of the NO 2 Ϫ concentrations (10 M to 1 mM) or HOCl concentrations (7-125 M) used. DISCUSSION 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 ⅐ NO and subsequent formation of highly reactive nitrogen species (RNS) usually attributed as ONOO Ϫ . However, an overproduction of ⅐ NO also results in the accumulation of NO 2 Ϫ , 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 2 Ϫ , forms an additional tyrosine-nitrating species, nitryl chloride (NO 2 Cl) (22,23). Although there is a wealth of information on RNS-mediated processes, limited information is available on the consequences of HOCl and NO 2 Ϫ accumulation and resulting NO 2 Cl formation generated from this reaction (28,32). The relatively fast second order rate constant of reaction of NO 2 Ϫ with HOCl (pH 7.2, 25°C, 7.4 Ϯ 1. Ϫ 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 2 Ϫ , 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 NO 2 Cl 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 2 Ϫ in vivo also confers HOCl-scavenging abilities on NO 2 Ϫ , 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 2 Ϫ 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 Ϫ added at sublethal concentrations (100 M). 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 2 Ϫ . Similarly, tyrosine nitration was only detected with ONOO Ϫ -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 2 Ϫ , but substantial nitrotyrosine formation was observed when the homogenates were exposed to ONOO Ϫ .
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 2 Ϫ reacts with HOCl rapidly (pH 7.2, 25°C, 7.4 Ϯ 1.3 ϫ 10 3 M Ϫ1 s Ϫ1 ) (24); 2) analysis of buffers after experimentation showed NO 2 Ϫ to be oxidized to NO 3 Ϫ (this reaction is stoichiometric (1 mol of NO 2 Ϫ consumed by 1 mol of HOCl to give 1 mol of NO 3 Ϫ ) (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 Ϫtreated 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 2 Ϫ (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 2 Ϫ could penetrate the cell membrane but are unlikely to cause tyrosine nitration, because it was only observed with the positive control (100 M ONOO Ϫ ) with each analytical technique used and not with mixtures of NO 2 Ϫ and HOCl. Therefore, it is possible that NO 2 Cl formed from the reaction of physiologically attainable concentrations of NO 2 Ϫ 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 Ϫ , peroxidase (18,20,23), or heme-mediated RNS (21). As with NO 2 Cl, the extent to which these contribute to intracellular tyrosine nitration is unknown and is currently being investigated by our laboratory.