Abstract
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 microm) 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.
Highlights
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
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 hypochlorous acid (HOCl) does not contribute to the elevated levels of 3-nitrotyrosine observed in human diseases
There is a wealth of information on RNS-mediated processes, limited information is available on the consequences of HOCl and NO2Ϫ accumulation and resulting NO2Cl formation generated from this reaction [28, 32]
Summary
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 Confocal Microscopy—1 ϫ 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) NO2Ϫ-free PBS before addition of HOCl, NO2Ϫ, or ONOOϪ as described above. Cells were 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 washed three times in NO2Ϫ-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 added and incubated in PBS containing 1% (v/v) fetal bovine serum for 1 h at room temperature. 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)
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