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Barry Halliwell

Publications and source records attributed to Barry Halliwell.

At least 55 records · Page 3Linked to original sources
PubMed2004

Sulfite-mediated oxidative stress in kidney cells.

BACKGROUND: Chronic renal failure has been associated with oxidative stress. Serum sulfite, sulfate, cysteine, homocysteine, cysteine sulfinic acid, and gamma-glutamylcysteine are elevated in patients on hemodialysis, suggesting an accelerated catabolism of sulfur-containing amino acids or a reduced elimination of sulfite/sulfate, or both. Administration of metabisulfite has also been shown to damage kidney cells. METHODS: Measurement of reactive oxygen species (ROS) was performed with the fluorescence of dichlorofluorescein (DCF), and that of intracellular ATP was by the luciferin-luciferase reaction. Oxidation of sulfite and succinate by isolated mitochondria from rat kidney was monitored polarographically. The fluorescent probe, 5, 5', 6, 6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) was employed to assess any loss in membrane potential in energized respiring mitochondria. Activities of glutamate and malate dehydrogenases (GDH, MDH, respectively) were assayed by the spectrophotometric measurement of NADH. Sulfite was determined by HPLC-fluorimetric measurement of monochlorobimane-sulfite and cell viability was by the MTT procedure. RESULTS: An immediate increase in ROS followed exposure of Madin-Darby canine kidney (MDCK), type II, and opossum kidney (OK) cells to 5-500 micromol/L sulfite. Depletion of intracellular ATP was also observed. A low rate of oxidation of 100 micromol/L sulfite was observed polarographically in isolated kidney mitochondria, but ADP-stimulated State 3 respiration was not apparent. ATP biosynthesis from the oxidation of glutamate in rat kidney mitochondria was significantly inhibited by coincubation with 100 micromol/L sulfite; this was not the case with malate, succinate, and TMPD/ascorbate. However, activities of both GDH and MDH in kidney mitochondrial extract were inhibited. The mitochondrial membrane potential and cell viability were not compromised. CONCLUSION: Micromolar sulfite elicited an immediate increase in ROS in MDCK, type II, and OK cells. This was accompanied by a depletion of intracellular ATP, which could be explained by its inhibitory effect on mitochondrial GDH. Although MDH was similarly inhibited, the impact was buffered by the high level of this enzyme in kidney mitochondria.

Adenosine Diphosphate↗
PubMed2004

Iron, atherosclerosis, and neurodegeneration: a key role for cholesterol in promoting iron-dependent oxidative damage?

This article reviews the roles and interactions of iron, atherosclerosis, and neurodegeneration. It highlights the importance of cholesterol in promoting iron-dependent oxidative damage. An intriguing possibility is that hypercholesterolemia can increase brain iron load and both the aggregation of beta-amyloid and the ability of iron on plaques to catalyze oxidative damage. This could explain why hypercholesterolemia is a risk factor for Alzheimer's disease. Further work is necessary to study the mechanism of increased iron transport across the blood brain barrier in atherosclerosis.

Animals↗
PubMed2004

Establishing biomarkers of oxidative stress: the measurement of hydrogen peroxide in human urine.

Hydrogen peroxide (H(2)O(2)) can be detected in freshly-voided human urine from healthy subjects and has been proposed as a "biomarker" of oxidative stress. This paper summarizes our studies to examine the extent to which urinary H(2)O(2) measurement fulfils the criteria for the "ideal biomarker". Levels of H(2)O(2), standardised for creatinine, varied widely between subjects. In most subjects, levels also varied considerably when measurements were made at different times and on different days. A reproducible increase in urinary H(2)O(2) was detected in all subjects examined after drinking coffee, a beverage rich in H(2)O(2). By contrast, green tea decreased urinary H(2)O(2) levels. We conclude that the H(2)O(2) in coffee is not excreted into urine. Instead, hydroxyhydroquinone from coffee is absorbed, excreted and oxidises in urine to produce H(2)O(2). No other confounders of urinary H(2)O(2) have been identified to date. Work is underway to compare H(2)O(2) levels with variations in other biomarkers of oxidative damage, to test the possibility that there are daily or other periodic variations in oxidative damage rates.

Adult↗
PubMed2003

Sulphite oxidase gene expression in human brain and in other human and rat tissues.

Sulphite oxidase (EC 1.8.3.1) is a molybdopterin-containing enzyme that catalyses the oxidation of sulphite to sulphate. Lack of active enzyme produces severe neurodegeneration and early death in humans, showing its essential role. Despite this, the expression of the sulphite oxidase gene in human and rat tissues (especially the brain) has not been elucidated. We therefore examined these tissues and found that the human liver, kidney, skeletal muscle, heart, placenta, and brain showed substantial expression while thymus, spleen, peripheral blood leucocytes, colon, small intestine, and lung showed little expression in humans. In rat, the liver, kidney, heart, brain, and lung (but not skeletal muscle) revealed a hybridization signal with the strongest signal in the liver. The spleen and testis also showed little expression. The differential expression of sulphite oxidase gene in various human brain regions was studied. Expression was seen in all brain regions examined (cerebellum, cerebral cortex, medulla, spinal cord, occipital pole, frontal lobe, amygdala, caudate nucleus, corpus callosum, hippocampus, thalamus, temporal lobe, and putamen). The cerebral cortex showed the highest level of expression.

Animals↗
PubMed2003

Contribution of hydrogen peroxide to the cytotoxicity of green tea and red wines.

Green tea and red wine are claimed to have health benefits because of their high content of polyphenolic compounds, but they have also been reported as mutagenic in some test systems. In this paper, we show that a commonly used cell culture medium, Dulbecco's modified Eagle's medium (DMEM), catalyses oxidation of green tea and red wines to generate H(2)O(2). The level of H(2)O(2) produced from green tea accounted for all of the cytotoxic effects of this beverage on PCl2 cells. By contrast, H(2)O(2) was only responsible for part of the cytotoxicity of the red wines examined. Our data illustrate the danger of extrapolating from cell culture studies to predict the effects of complex beverages in vivo.

Animals↗
PubMed2003

Inhibition of hypochlorous acid-induced oxidative reactions by nitrite: is nitrite an antioxidant?

Acute and chronic inflammation result in increased nitrogen monoxide (z.rad;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). At physiologically attainable concentrations, we found that NO(2)(-) significantly inhibits HOCl-mediated DNA strand breakage and ascorbate depletion. HOCl-mediated inactivation of pure alpha(1)-antiproteinase or of the elastase inhibitory capacity of human plasma was inhibited by the addition of NO(2)(-). NO(2)(-) was more effective than ascorbate, GSH, and urate at inhibiting HOCl-mediated toxicity to human HepG2 cells in culture. These data suggest that NO(2)(-) may act in an antioxidant manner by removing HOCl at sites of inflammation where both HOCl and z.rad;NO are overproduced.

Antioxidants↗
PubMed2003

Oxidative stress in cell culture: an under-appreciated problem?

Cell culture studies have given much valuable information about mechanisms of metabolism and signal transduction and of regulation of gene expression, proliferation, senescence, and death. However, cells in culture may behave differently from cells in vivo in many ways. One of these is that cell culture imposes a state of oxidative stress on cells. I argue that cells that survive and grow in culture might use ROS-dependent signal transduction pathways that rarely or never operate in vivo. A further problem is that cell culture media can catalyse the oxidation of compounds added to them, resulting in apparent cellular effects that are in fact due to oxidation products such as ROS. Such artefacts may have affected many studies on the effects of ascorbate, thiols, flavonoids and other polyphenolic compounds on cells in culture.

Animals↗
PubMed2003

Correlation of iron and zinc levels with lesion depth in newly formed atherosclerotic lesions.

Several studies have indicated a relationship between body iron content and cardiovascular disease, although other studies have not. There are also suggestions that zinc has an antioxidant and antiatherosclerotic effect. We have used Nuclear Microscopy, using the combination of Scanning Transmission Ion Microscopy (STIM), Rutherford Backscattering Spectrometry (RBS), and Proton Induced X-ray Emission (PIXE) to map and quantify iron and zinc levels in newly formed atherosclerotic lesions. Sixteen New Zealand White rabbits fed on a high cholesterol diet were divided into four groups of 4 rabbits each. Six weeks into the high cholesterol diet, two groups were treated with the iron chelating agent desferrioxamine, for 2 weeks and 4 weeks, respectively, by surgically implanting with Alzet osmotic pumps (Alza Corporation, Palo Alto, CA, USA) containing desferal (0.5 g/ml). The other two groups served as controls, and were surgically implanted with osmotic pumps containing saline. Tissue sections were taken from the aortic arch, flash frozen, and air-dried. Analysis of atherosclerotic lesions indicated a trend (p =.07) to a reduction in the progression of the lesion after 4 weeks of desferrioxamine treatment. For each of the control and desferrioxamine-treated animals however, the more extensive lesions contained a higher concentration of iron and a lower concentration of zinc. Our results are consistent with the view that early lesion formation may be accelerated by free radical production caused by increased iron levels, that zinc might antagonize such effects, and that more prolonged desferal treatment might have an antiatherosclerotic effect.

Animals↗
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

Functional significance of inducible nitric oxide synthase induction and protein nitration in the thermally injured cutaneous microvasculature.

Increased nitric oxide (NO) production after burn injury is well established. However, there is little information relating to the reactions that occur as a consequence of NO generation under such circumstances. We have investigated the synthesis and function of NO in a rat model of local cutaneous thermal injury. We show that NO levels are elevated from 3 hours after injury with a concomitant increase in protein nitration. A selective inducible nitric oxide synthase (iNOS) inhibitor (1400W) significantly attenuated NO synthesis, protein nitration, and neutrophil accumulation in this model, but had no effect on edema formation. The results also indicate that NO synthesis and protein nitration occurred independently of neutrophil accumulation because these parameters were unaffected by depletion of circulating neutrophils. 3-Chlorotyrosine, a marker of neutrophil/myeloperoxidase-mediated protein damage was significantly increased from 1 hour after burn. Our observations provide evidence for the involvement of reactive species in the inflammatory response after burn. The use of selective iNOS inhibitors may represent a novel approach for the management of human burn injuries.

Animals↗
PubMed2003

Proteasomal inhibition causes the formation of protein aggregates containing a wide range of proteins, including nitrated proteins.

Mutations in Cu,Zn-superoxide dismutase (SOD-1) are associated with some familial cases of amyotrophic lateral sclerosis (ALS), but it is not known how they result in cell death. We examined effects of overexpression of wild-type SOD-1 or the G37R or G85R mutations on the accumulation of ubiquitinated and nitrated proteins, and on loss of cell viability induced by the proteasome inhibitor, lactacystin. Wild-type SOD-1 had no effect on proteasomal activity, but the mutants decreased it somewhat. Treatment with lactacystin (1 micro m) caused only limited cell viability loss, even though it induced a marked inhibition of proteasomal activities. However, viability loss due to apoptosis was substantial in response to lactacystin when cells were overexpressing a mutant SOD-1. The frequency of cells showing immunoreactivity against ubiquitinated- or nitrated-proteins was enhanced when wild-type and mutant SOD-1 s were overexpressed. Ubiquitinated or nitrated alpha-tubulin, SOD-1, alpha-synuclein and 68K neurofilaments were observed in the aggregates. Similar aggregates were observed in cells overexpressing mutant parkin (Del3-5, T240R and Q311'X). The nitric oxide synthase inhibitor, l-NAME, decreased viability loss and aggregation, suggesting that nitration of proteins may play an important role in aggregation and in the cell death accompanying it.

Acetylcysteine↗
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↗
PubMed2003

Increase in cholesterol and cholesterol oxidation products, and role of cholesterol oxidation products in kainate-induced neuronal injury.

Little is known about changes in sterols, in particular cholesterol, and cholesterol oxidation products (COPs) in oxidative injury in neural tissues. We have therefore examined changes in cholesterol and COPs using a model of excitotoxic injury. Intracerebroventricular injections of kainate in rats resulted in an increase in immunoreactivity to cholesterol in the affected CA fields of the hippocampus. The increase was confirmed by increased filipin staining of cholesterol in adjacent sections from the same animals, and in hippocampal slice or neuronal cultures after kainate treatment. In neuronal cultures, addition of lovastatin, an inhibitor of cholesterol synthesis, attenuated the increased filipin staining after kainate treatment, indicating that the increase in cholesterol could involve increased cholesterol synthesis. Furthermore, gas chromatographic mass spectrometric (GC/MS) analysis of cholesterol and COPs in kainate-injected rat brain showed a marked increase in cholesterol and COPs including 7-ketocholesterol, 3 days after kainate treatment. The addition of some COPs, including 7-ketocholesterol and cholesterol epoxides to hippocampal slices resulted in neuronal injury as reflected by decreased staining of a neuronal marker in the affected CA fields. The ability of these COPs to produce neuronal injury was attenuated by glutathione, suggesting that oxidative mechanisms are involved in neuronal injury induced by these products. These results, together with GC/MS results that showed significant increase in 7-ketocholesterol at 3 days post-kainate injury suggest that 7-ketocholesterol may be a factor in aggravating oxidative damage to neurons, after the initial stages of kainate-induced neuronal injury.

Animals↗
PubMed2003

Oxidative DNA damage in peripheral leukocytes and its association with expression and polymorphisms of hOGG1: a study of adolescents in a high risk region for hepatocellular carcinoma in China.

AIM: To study the oxidative DNA damage to adolescents of hepatocellular carcinoma (HCC) families in Guangxi Zhuang Autonomous Region, China. METHODS: Peripheral leukocytes' DNA 7, 8-dihydro-8-oxoguanine (8-oxoG) and repair enzyme hOGG1 were quantified by flow-cytometry. hOGG1-Cys326Ser single nucleotide polymorphism (SNP) was distinguished by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) assay. RESULTS: There was a positive correlation between 8-oxoG and repair enzyme hOGG1 expression (P<0.001). HCC children (n=21) in Fusui county had a higher level of hOGG1 (P<0.01) and a lower level of 8-oxoG (P<0.05) than the controls (n=63) in Nanning city. Children in Nanning exposed to passive-smoking had a higher hOGG1 expression (P<0.05) than the non-exposers. 8-oxoG and hOGG1 were negatively correlated with body mass index, while hOGG1 was positively correlated with age. There was a peak of 8-oxoG level nearby the 12 year point. Individuals with the hOGG1 326Ser allele had a significantly marginal higher concentration of leukocyte 8-oxoG level than hOGG1 326Cys allele. CONCLUSION: This is the first report using flow-cytometry to simultaneously quantify both the DNA oxidative damage and its repairing enzyme hOGG1. The results provide new insights towards a better understanding of the mechanisms of oxidative stress in a population highly susceptible to hepatocarcinogenesis.

Adolescent↗
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↗