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Matthew Whiteman

Publications and source records attributed to Matthew Whiteman.

44 records · Page 3Linked to original sources
PubMed2003

Nitrite-mediated protection against hypochlorous acid-induced chondrocyte toxicity: a novel cytoprotective role of nitric oxide in the inflamed joint?

OBJECTIVE: To examine the potential consequences of overproduction of nitric oxide (NO) and nitrite (NO(2) (-)) in the inflamed rheumatoid joint. METHODS: Human articular chondrocytes in culture were exposed to HOCl (hypochlorous acid, a physiologic oxidant formed in increased amounts at sites of chronic inflammation), and assays of cell viability, intracellular ATP and glutathione (GSH), and lactate dehydrogenase (LDH) were performed. HOCl-induced lipid peroxidation and activation of the MAP kinases ERK-1/2, JNK-1/2, and p38 were also measured. The modulatory effects of NO-derived nitrite (NO(2) (-)) and nitrate (NO(3) (-)) on HOCl-mediated chondrocyte toxicity were investigated. RESULTS: Exposure of human articular chondrocytes to HOCl resulted in a concentration- and time-dependent loss of viability, decrease in ATP and GSH levels, LDH leakage, and cell death. HOCl induced significant lipid peroxidation as well as activation of the MAP kinases ERK-1/2 and p38 but not JNK-1/2. However, the presence of NO(2) (-) but not NO(3) (-) substantially decreased HOCl-dependent cellular toxicity even when NO(2) (-) was added at low (microM) concentrations. In sharp contrast, NO(2) (-) (1 mM) did not inhibit superoxide-, hydroxyl radical-, H(2)O(2)-, or peroxynitrite-mediated cytotoxicity. Furthermore, culture media from cells treated with interleukin-1beta (to generate NO and NO(2) (-)) offered significantly more protection against HOCl-mediated cytotoxicity than culture media from untreated cells. CONCLUSION: These data suggest that NO(2) (-) accumulation at chronically inflamed sites where both HOCl and NO are overproduced may be cytoprotective against damage induced by HOCl. Accumulation of NO(2) (-) could represent a novel cytoprotective role of NO in inflamed joints. A mechanism for this is suggested.

Adenosine Triphosphate↗
PubMed2003

Factors affecting the ascorbate- and phenolic-dependent generation of hydrogen peroxide in Dulbecco's Modified Eagles Medium.

Ascorbate and several polyphenolic compounds have been reported to undergo oxidation in cell culture media to generate hydrogen peroxide (H2O2), but the mechanism underlying this has not been established. We therefore investigated the parameters affecting H2O2 production. H2O2 generation from ascorbate, gallic acid and other phenolic compounds in Dulbecco's Modified Eagles' Medium (DMEM) at 37 degrees C under 95% air - 5% CO2 was not significantly inhibited by high (5-10 mM) concentration of EGTA, o-phenanthroline or desferriox-amine, but partial inhibition by EDTA and diethylene-triaminepentaacetic acid (DTPA) was observed. Incubation of DMEM alone at 37 degrees C led to an upward drift of pH, even under an atmosphere of 95% air - 5% CO2. Prevention of this pH rise by increasing the concentration of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes) buffer lowered the levels of H2O2 generated by ascorbate and phenolic compounds, but there was still substantial H2O2 generated at pH 7.4. Mixtures of ascorbate and phenolic compounds led to less H2O2 generation than would be expected from the rates observed with ascorbate or phenolic compounds alone. Ascorbate prevented the loss of gallic acid incubated in DMEM. The role of metal ions and other constituents of the culture medium in promoting H2O2 generation is discussed.

Ascorbic Acid↗
PubMed2002

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-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.

Enzyme-Linked Immunosorbent Assay↗
PubMed2002

Inhibition of hypochlorous acid-induced cellular toxicity by nitrite.

Chronic inflammation results in increased nitrogen monoxide (.NO) formation and the accumulation of nitrite (NO(2-)). Neutrophils stimulated by various inflammatory mediators release myeloperoxidase to produce the cytotoxic agent hypochlorous acid (HOCl). Exposure of chondrocytic SW1353 cells to HOCl resulted in a concentration- and time-dependent loss in viability, ATP, and glutathione levels. Treatment of cells with NO(2-) but not nitrate (NO(3-)) substantially decreased HOCl-dependent cellular toxicity even when NO(2-) was added at low (microM) concentrations. In contrast, NO(2-) alone (even at 1 mM concentrations) did not affect cell viability or ATP and glutathione levels. These data suggest that NO(2-) accumulation at chronic inflammatory sites, where both HOCl and.NO are overproduced, may be cytoprotective against damage caused by HOCl. We propose that this is because HOCl is removed by reacting with NO(2-) to give nitryl chloride (NO2Cl), which is less damaging in our cell system.

Adenosine Triphosphate↗
PubMed2002

Loss of oxidized and chlorinated bases in DNA treated with reactive oxygen species: implications for assessment of oxidative damage in vivo.

Oxidative damage to DNA has been reported to occur in a wide variety of disease states. The most widely used "marker" for oxidative DNA damage is 8-hydroxyguanine. However, the use of only one marker has limitations. Exposure of calf thymus DNA to an .OH-generating system (CuCl(2), ascorbate, H(2)O(2)) or to hypochlorous acid (HOCl), led to the extensive production of multiple oxidized or chlorinated DNA base products, as measured by gas chromatography-mass spectrometry. The addition of peroxynitrite (ONOO(-)) (<200 microM) or SIN-1 (1mM) to oxidized DNA led to the extensive loss of 8-hydroxyguanine, 5-hydroxycytosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2-hydroxyadenine, 8-hydroxyadenine, and 4,6-diamino-5-formamidopyrimidine were lost at higher ONOO(-) concentrations (>200 microM). Exposure of DNA to HOCl led to the generation of 5-Cl uracil and 8-Cl adenine and addition of ONOO(-) (<200 microM) or SIN-1 (1mM) led to an extensive loss of 8-Cl adenine and a small loss of 5-Cl uracil at higher concentrations (>500 microM). An .OH-generating system (CuCl(2)/ascorbate/H(2)O(2)) could also destroy these chlorinated species. Treatment of oxidized or chlorinated DNA with acidified nitrite (NO(2)(-), pH 3) led to substantial loss of various base lesions, in particular 8-OH guanine, 5-OH cytosine, thymine glycol, and 8-Cl adenine. Our data indicate the possibility that when ONOO(-), nitrite in regions of low pH or .OH are produced at sites of inflammation, levels of certain damaged DNA bases could represent an underestimate of ongoing DNA damage. This study emphasizes the need to examine more than one modified DNA base when assessing the role of reactive species in human disease.

Animals↗
PubMed2002

5-s-Cysteinyl-conjugates of catecholamines induce cell damage, extensive DNA base modification and increases in caspase-3 activity in neurons.

A decrease in reduced glutathione levels in dopamine containing nigral cells in Parkinson's disease may result from the formation of cysteinyl-adducts of catecholamines, which in turn exert toxicity on nigral cells. We show that exposure of neurons (CSM 14.1) to 5-S-cysteinyl conjugates of dopamine, L-DOPA, DOPAC or DHMA causes neuronal damage, increases in oxidative DNA base modification and an elevation of caspase-3 activity in cells. Damage to neurons was apparent 12-48 h of post-exposure and there were increases in caspase-3 activity in neurons after 6 h. These changes were paralleled by large increases in pyrimidine and purine base oxidation products, such as 8-OH-guanine suggesting that 5-S-cysteinyl conjugates of catecholamines are capable of diffusing into cells and stimulating the formation of reactive oxygen species (ROS), which may then lead to a mechanism of cell damage involving caspase-3. Indeed, intracellular ROS were observed to rise sharply on exposure to the conjugates. These results suggest one mechanism by which oxidative stress may occur in the substantia nigra in Parkinson's disease.

3,4-Dihydroxyphenylacetic Acid↗