ascorbic-acid and ferrous-chloride

Topics: Acetylcholinesterase; Animals; Antioxidants; Ascorbic Acid; Benzimidazoles; Brain; Butyrylcholinesterase; Cholinesterase Inhibitors; Dose-Response Relationship, Drug; Electrophorus; Ferrous Compounds; Horses; Mannich Bases; Molecular Docking Simulation; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Structure-Activity Relationship

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is a commonly formed DNA lesion that is useful as a biomarker for oxidative stress. Although methods for selective quantification of 8-oxodGuo exist, there is room for additional methods that are sensitive and utilize instrumentation that is widely available. We previously took advantage of the reported reactivity of 8-oxodGuo to develop a method for detecting the lesion by selectively covalently tagging it with a molecule equipped with a biotin label that can be used subsequently with a reporting method ( Xue , L. and Greenberg , M. M. ( 2007 ) J. Am. Chem. Soc. 129 , 7010 ). We now report a method that can detect as little as 14 amol of 8-oxodGuo by tagging DNA with a reagent containing a disulfide that reduces background due to nonspecific binding. The reagent also contains biotin that enables capturing target DNA on streptavidin-coated magnetic beads. The captured DNA is quantified using quantitative PCR. The method is validated by comparing the amount of 8-oxodGuo detected as a function of Fe(2+)/H2O2/ascorbate-dose to that reported previously using mass spectrometry.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Ascorbic Acid; Biotin; Deoxyguanosine; Disulfides; Ferrous Compounds; Hydrogen Peroxide; Plasmids; Polymerase Chain Reaction; Streptavidin; Tumor Suppressor Protein p53

This study was designed to investigate the potentially protective effects of glycyrrhetinic acid (GA) and the role of transcription factor nuclear factor-erythroid 2(NF-E2)-related factor 2 (Nrf2) signaling in the regulation of Carbon Tetrachloride (CCl(4))-induced chronic liver fibrosis in mice. The potentially protective effects of GA on CCl(4)-induced chronic liver fibrosis in mice were depicted histologically and biochemically. Firstly, histopathological changes including regenerative nodules, inflammatory cell infiltration and fibrosis were induced by CCl(4).Then, CCl(4) administration caused a marked increase in the levels of serum aminotransferases (GOT, GPT), serum monoamine oxidase (MAO) and lipid peroxidation (MDA) as well as MAO in the mice liver homogenates. Also, decreased nuclear Nrf2 expression, mRNA levels of its target genes such as superoxide dismutase 3 (SOD3), catalase (CAT), glutathione peroxidase 2 (GPX2), and activity of cellular antioxidant enzymes were found after CCl(4) exposure. All of these phenotypes were markedly reversed by the treatment of the mice with GA. In addition, GA exhibited the antioxidant effects in vitro by on FeCl(2)-ascorbate induced lipid peroxidation in mouse liver homogenates, and on DPPH scavenging activity. Taken together, these results suggested that GA can protect the liver from oxidative stress in mice, presumably through activating the nuclear translocation of Nrf2, enhancing the expression of its target genes and increasing the activity of the antioxidant enzymes. Therefore, GA may be an effective hepatoprotective agent and viable candidate for treating liver fibrosis and other oxidative stress-related diseases.

Topics: Animals; Antioxidants; Ascorbic Acid; Biphenyl Compounds; Chronic Disease; Cytoprotection; Ferrous Compounds; Fluorocarbons; Glycyrrhetinic Acid; Lipid Peroxidation; Liver; Liver Cirrhosis; Male; Mice; NF-E2-Related Factor 2; Oxidative Stress; Picrates; RNA, Messenger; Up-Regulation

Trimethylamine oxide (TMAO) in squid is demethylated to dimethylamine (DMA) and formaldehyde (FA) during storage and processing. This study examined the effects of thermal processing and various chemical substances on FA and DMA formation in squid.. The thermal conversion of TMAO was assessed by analysing four squid and four gadoid fish species, which revealed that FA, DMA and trimethylamine (TMA) were gradually produced in squid, whereas TMA increased and FA decreased in gadoid fish. A significant increase in both FA and DMA levels was observed in the supernatant of jumbo squid with increased heating temperature and extended heating time at pH 6-7. Ferrous chloride combined with cysteine and/or ascorbate had a significantly positive effect on FA formation in the heated supernatant of jumbo squid. No significant difference was observed in the levels of Cu and Fe in squid and gadoid fish. The capability of Fe(2+) to promote the formation of FA and DMA was not completely attributable to its reducing power in squid.. Non-enzymatic decomposition of TMAO was a key pathway during the thermal processing of jumbo squid, and Fe(2+) was a crucial activator in the formation of FA and DMA.

Topics: Animals; Ascorbic Acid; Cysteine; Decapodiformes; Dimethylamines; Ferrous Compounds; Fishes; Food Handling; Formaldehyde; Hot Temperature; Humans; Hydrogen-Ion Concentration; Iron; Methylamines; Seafood

Few attempts have been made to improve the activity of plant compounds with low antimicrobial efficacy. (+)-Catechin, a weak antimicrobial tea flavanol, was combined with putative adjuncts and tested against different species of bacteria. Copper(II) sulphate enhanced (+)-catechin activity against Pseudomonas aeruginosa but not Staphylococcus aureus, Proteus mirabilis or Escherichia coli. Attempts to raise the activity of (+)-catechin against two unresponsive species, S. aureus and E. coli, with iron(II) sulphate, iron(III) chloride, and vitamin C, showed that iron(II) enhanced (+)-catechin against S. aureus, but not E. coli; neither iron(III) nor combined iron(II) and copper(II), enhanced (+)-catechin activity against either species. Vitamin C enhanced copper(II) containing combinations against both species in the absence of iron(II). Catalase or EDTA added to active samples removed viability effects suggesting that active mixtures had produced H(2)O(2)via the action of added metal(II) ions. H(2)O(2) generation by (+)-catechin plus copper(II) mixtures and copper(II) alone could account for the principal effect of bacterial growth inhibition following 30 minute exposures as well as the antimicrobial effect of (+)-catechin-iron(II) against S. aureus. These novel findings about a weak antimicrobial flavanol contrast with previous knowledge of more active flavanols with transition metal combinations. Weak antimicrobial compounds like (+)-catechin within enhancement mixtures may therefore be used as efficacious agents. (+)-Catechin may provide a means of lowering copper(II) or iron(II) contents in certain crop protection and other products.

Topics: Anti-Bacterial Agents; Antioxidants; Ascorbic Acid; Camellia sinensis; Catechin; Copper Sulfate; Drug Synergism; Escherichia coli; Ferrous Compounds; Proteus mirabilis; Pseudomonas aeruginosa; Staphylococcus aureus

Lycium barbarum (Fructus Lycii, Wolfberry, or Gouqi) belongs to the Solanaceae. The red-colored fruits of L. barbarum have been used for a long time as an ingredient in Chinese cuisine and brewing, and also in traditional Chinese herbal medicine for improving health. However, its effects on cognitive function have not been well studied. In the present study, prevention of a milk-based wolfberry preparation (WP) on cognitive dysfunction was tested in a prenatal stress model with rats and the antioxidant mechanism was tested by in vitro experiments. We found that prenatal stress caused a significant decrease in cognitive function (Morris water maze test) in female offspring. Pretreatment of the mother rats with WP significantly prevented the prenatal stress-induced cognitive dysfunction. In vitro studies showed that WP dose-dependently scavenged hydroxyl and superoxide radicals (determined by an electron spin resonance spectrometric assay), and inhibited FeCl(2)/ascorbic acid-induced dysfunction in brain tissue and tissue mitochondria, including increases in reactive oxygen species and lipid peroxidation and decreases in the activities of complex I, complex II, and glutamate cysteine ligase. These results suggest that dietary supplementation with WP may be an effective strategy for preventing the brain oxidative mitochondrial damage and cognitive dysfunction associated with prenatal stress.

Topics: Animals; Antioxidants; Ascorbic Acid; Cognition Disorders; Female; Ferrous Compounds; Free Radical Scavengers; Glutamate-Cysteine Ligase; Lipid Peroxidation; Lycium; Male; Maze Learning; Milk; Mitochondria; Oxidative Stress; Plant Extracts; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Sprague-Dawley; Restraint, Physical; Stress, Psychological

Hypoxia-inducible factor (HIF) plays an important role in determining patterns of gene expression in cancer. HIF is down-regulated in oxygenated cells by a series of Fe (II) and 2-oxoglutarate dependent dioxygenases that hydroxylate specific residues in the regulatory HIF-alpha subunits. Because these enzymes require ascorbate for activity in vitro we analyzed the effects of ascorbate on HIF in human cancer cell lines. Ascorbate at physiological concentrations (25 micro M) strikingly suppressed HIF-1alpha protein levels and HIF transcriptional targets, particularly when the system was oncogenically activated in normoxic cells. Similar results were obtained with iron supplementation. These results indicate that both ascorbate and iron availability have major effects on HIF, and imply that the system is commonly regulated by limiting hydroxylase activity under normoxic tissue culture conditions.

Topics: Adenocarcinoma; Ascorbic Acid; Breast Neoplasms; Cell Hypoxia; Endothelial Growth Factors; Female; Ferrous Compounds; Glucose Transporter Type 1; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Intercellular Signaling Peptides and Proteins; Lymphokines; Male; Monosaccharide Transport Proteins; Neoplasms; Ovarian Neoplasms; Procollagen-Proline Dioxygenase; Prostatic Neoplasms; RNA, Messenger; Transcription Factors; Transcriptional Activation; Transferrin; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Free radical production seems to be involved in the pathogenesis of a number of ocular diseases. Certain beta-adrenoceptor antagonists display antioxidant properties, but these have not been ascribed to any of the presently used ophthalmic beta-adrenoceptor antagonists. Therefore, we examined the influence of ophthalmic beta-adrenoceptor antagonists and other antiglaucoma drugs on stimulated lipid peroxidation in rat brain homogenates.. Lipid peroxidation in rat brain homogenates was stimulated by iron/ascorbate or sodium nitroprusside. Lipid peroxidation was assessed by the formation of thiobarbituric acid reactive species (TBARS).. Of the antiglaucoma drugs tested (brimonidine, carteolol, dorzolamide, latanoprost, levobetaxolol, levobunolol, metipranolol, pilocarpine, timolol, travoprost and unoprostone), only metipranolol and its active metabolite, desacetylmetipranolol, were found to significantly reduce iron/ascorbate-induced lipid peroxidation in rat brain homogenates with IC50 values of 6.9 and 1.1 microM, respectively. Metipranolol and desacetylmetipranolol also concentration-dependently inhibited sodium nitroprusside-stimulated lipid peroxidation in rat brain homogenates, displaying IC50 values of 25.1 and 2.6 microM, respectively.. These data indicate that metipranolol and desacetylmetipranolol exhibit remarkable antioxidant properties, with an effect not dissimilar from the reference antioxidant trolox.

Topics: Adrenergic beta-Antagonists; Animals; Antihypertensive Agents; Antioxidants; Ascorbic Acid; Brain; Dose-Response Relationship, Drug; Ferrous Compounds; Free Radical Scavengers; Lipid Peroxidation; Metipranolol; Nitroprusside; Ophthalmic Solutions; Rats; Rats, Wistar; Thiobarbituric Acid Reactive Substances

The antioxidant activities of hot water extracts (HWECC) and ethanol extracts (EECC) from the dry bark of Cinnamomum cassia Presl were evaluated in this study. Results showed that at 1.0 mg/mL, the ethanol extracts of C. cassia (96.30%) exhibited a greater inhibition than the alpha-tocopherol (93.74%) on FeCl(2)-ascorbic acid induced lipid peroxidation of rat liver homogenate in vitro. From 0.05 to 1.0 mg/mL, the EECC demonstrated the highest superoxide anions scavenging activity and the strongest anti-superoxide formation activity (p < 0.05). The same extract also showed an excellent antioxidant activity in enzymatic and nonenzymatic liver tissue oxidative systems. EECC revealed the strongest antioxidant activity followed by alpha-tocopherol and HWECC. Compared to alpha-tocopherol, the IC(50) values of EECC were found to be lower in thiobarbituric acid test (IC(50) = 0.24 mg/mL vs 0.37 mg/mL), in cytochrome c test (IC(50) = 0.16 mg/mL vs 0.27 mg/mL) and in xanthine oxidase inhibition test (IC(50) = 0.09 mg/mL vs 0.19 mg/mL). The present study concludes that EECC could be used as a good source of antioxidant in the dietary supplement.

Topics: alpha-Tocopherol; Animals; Antioxidants; Ascorbic Acid; Cinnamomum aromaticum; Dose-Response Relationship, Drug; Ferrous Compounds; Free Radical Scavengers; Inhibitory Concentration 50; Lipid Peroxidation; Liver; Male; Phytotherapy; Plant Bark; Plant Extracts; Rats; Rats, Wistar

Topics: Adult; Aged; Antioxidants; Ascorbic Acid; Chromatography, High Pressure Liquid; Female; Ferrous Compounds; Gastric Juice; Humans; Hydroxyl Radical; In Vitro Techniques; Male; Middle Aged

The present study examined the mechanisms by which 3,4-methylenedioxymethamphetamine (MDMA) produces long-term neurotoxicity of striatal dopamine neurones in mice and the protective action of the dopamine uptake inhibitor GBR 12909. MDMA (30 mg/kg, i.p.), given three times at 3-h intervals, produced a rapid increase in striatal dopamine release measured by in vivo microdialysis (maximum increase to 380 +/- 64% of baseline). This increase was enhanced to 576 +/- 109% of baseline by GBR 12909 (10 mg/kg, i.p.) administered 30 min before each dose of MDMA, supporting the contention that MDMA enters the terminal by diffusion and not via the dopamine uptake site. This, in addition to the fact that perfusion of the probe with a low Ca(2+) medium inhibited the MDMA-induced increase in extracellular dopamine, indicates that the neurotransmitter may be released by a Ca(2+) -dependent mechanism not related to the dopamine transporter. MDMA (30 mg/kg x 3) increased the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA) from salicylic acid perfused through a probe implanted in the striatum, indicating that MDMA increased free radical formation. GBR 12909 pre-treatment attenuated the MDMA-induced increase in 2,3-DHBA formation by approximately 50%, but had no significant intrinsic radical trapping activity. MDMA administration increased lipid peroxidation in striatal synaptosomes, an effect reduced by approximately 60% by GBR 12909 pre-treatment. GBR 12909 did not modify the MDMA-induced changes in body temperature. These data suggest that MDMA-induced toxicity of dopamine neurones in mice results from free radical formation which in turn induces an oxidative stress process. The data also indicate that the free radical formation is probably not associated with the MDMA-induced dopamine release and that MDMA does not induce dopamine release via an action at the dopamine transporter.

Topics: Animals; Ascorbic Acid; Body Temperature; Calcium; Corpus Striatum; Dopamine; Dopamine Uptake Inhibitors; Ferrous Compounds; Free Radicals; Hydroxybenzoates; Lipid Peroxidation; Male; Mice; Microdialysis; N-Methyl-3,4-methylenedioxyamphetamine; Piperazines; Rats; Salicylic Acid; Synaptosomes; Time Factors

Hypoxia-inducible factor (HIF) is a transcriptional complex that plays a central role in the regulation of gene expression by oxygen. In oxygenated and iron replete cells, HIF-alpha subunits are rapidly destroyed by a mechanism that involves ubiquitylation by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. Here we show that the interaction between human pVHL and a specific domain of the HIF-1alpha subunit is regulated through hydroxylation of a proline residue (HIF-1alpha P564) by an enzyme we have termed HIF-alpha prolyl-hydroxylase (HIF-PH). An absolute requirement for dioxygen as a cosubstrate and iron as cofactor suggests that HIF-PH functions directly as a cellular oxygen sensor.

Topics: Amino Acid Sequence; Ascorbic Acid; Cell Hypoxia; Deferoxamine; DNA-Binding Proteins; Ferrous Compounds; Humans; Hydroxylation; Hydroxyproline; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Ligases; Molecular Sequence Data; Nuclear Proteins; Oxygen; Point Mutation; Procollagen-Proline Dioxygenase; Protein Structure, Tertiary; Proteins; Recombinant Fusion Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transcription Factors; Tumor Cells, Cultured; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Ubiquitins; Von Hippel-Lindau Tumor Suppressor Protein

Cystinuria, an inherited disease, is clinically diagnosed by detecting cystine in urine. A colorimetric method using sodium cyanide and sodium nitroprusside is a simple qualitative test used to detect cystinuria. Several colorimetric methods have been proposed for quantitative analysis of cystine; however, we found that none of them were satisfactory because the results were not reproducible. The causes of non-reproducible results were: (1) insufficient reduction time for conversion of cystine to cysteine, and (2) the interference of creatinine. In this report, we present a method to quantitate cystine in urine. We also found that ascorbic acid and ferric chloride, but not zinc chloride, interfered with the color reaction. Using this method, 15 normal urine samples (10 males and 5 females) and 12 cystine stone forming patients' (5 males and 7 females) urine were analyzed. The method was compared to commercially available urine controls. Only captopril showed a dose dependent response and color intensity at 521 nm. Thiola and D-penicillamine showed little effect on cystine determination.

Topics: Adult; Artifacts; Ascorbic Acid; Captopril; Chlorides; Colorimetry; Creatinine; Cyanides; Cysteine; Cystinuria; Dose-Response Relationship, Drug; Female; Ferrous Compounds; Humans; Male; Middle Aged; Nitroprusside; Penicillamine; Reference Values; Reproducibility of Results; Serum Albumin; Tiopronin; Zinc Compounds

In this study, the relationship between liver protective effects and antioxidant activity of Boehmeria nivea var. nivea (= B. nivea) and B. nivea var. tenacissima (= B. frutescens) was investigated. The water extracts of both plants exhibited a hepatoprotective activity against CCl4-induced liver injury. B. nivea var. nivea and B. nivea var. tenacissima, also showed anti-oxidant effects in FeCl2-ascorbate induced lipid peroxidation in rat liver homogenate. Moreover, the active oxygen species scavenging potencies were evaluated by an electron spin resonance (ESR) spin-trapping technique. B. nivea var. tenacissima displayed better superoxide radical scavenging activity than B. nivea. Based on these findings, we suggest that in the liver protective and antioxidative effects of B. nivea var. nivea and B. nivea var. tenacissima, possibly involve mechanisms related to free radical scavenging effects.

Topics: Animals; Antioxidants; Ascorbic Acid; Carbon Tetrachloride; Drug Evaluation; Electron Spin Resonance Spectroscopy; Ferrous Compounds; Free Radical Scavengers; Lipid Peroxidation; Liver; Male; Plant Extracts; Rats; Rats, Wistar; Superoxide Dismutase; Superoxides

Oral administration of the adrenal steroid dehydroepiandrosterone (DHEA), a peroxisome proliferator and hepatocarcinogen in the rat, caused an increase in NADPH-dependent lipid peroxidation in microsomes isolated from rat liver and kidney cortex, but not from brain. The increase of liver microsomal lipid peroxidation was greater in male than in female rats. the effect of DHEA on lipid peroxidation became discernible after feeding steroid-containing diet (0.6%) to male and female rats for 2 and 3 days and reached maximal levels at 1 and 2 weeks, respectively. The increase of microsomal lipid peroxidation reached a plateau stimulation at 0.05% in the diet. The addition of DHEA in the concentration range 0.1-100 microM to microsomes isolated from control rats had no effect on lipid peroxidation. Furthermore, a significant increase of the endogenous concentration of thiobarbituric acid reactive substances was found in microsomes after DHEA-administration at 0.05% in the diet. These results provide in vivo evidence that DHEA can cause lipid peroxidation in rat liver. Administration of DHEA at 0.6% in the diet for 7 consecutive days also significantly enhanced NADH- and ascorbate-dependent lipid peroxidation in liver microsomes. The DHEA-stimulated rat liver microsomal lipid peroxidation was completely inhibited by EDTA but not by superoxide dismutase, catalase or mannitol applied as OH-radical scavenger. The findings indicate that membrane lipid peroxidation is an early effect of DHEA, and that this process may be involved in the steroid-induced carcinogenesis in rats.

Topics: Adenosine Diphosphate; Administration, Oral; Animals; Ascorbic Acid; Brain; Catalase; Dehydroepiandrosterone; Dose-Response Relationship, Drug; Female; Ferrous Compounds; Kidney Cortex; Lipid Peroxidation; Male; Microsomes, Liver; NAD; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances

The in vitro effects of membrane lipid peroxidation on ATPase-ADPase activities in synaptic plasma membranes from rat forebrain were investigated. Treatment of synaptic plasma membranes with an oxidant generating system (H(2)0(2)/Fe(2+)/ascorbate) resulted in lipid peroxidation and inhibition of the enzyme activity. Besides, trolox as a water soluble vitamin E analogue totally prevented lipid peroxidation and the inhibition of enzyme activity. These results demonstrate the susceptibility of ATPase-ADPase activities of synaptic plasma membranes to free radicals and suggest that the protective effect against lipid peroxidation by trolox prevents the inhibition of enzyme activity. Thus, inhibition of ATPase-ADPase activities of synaptic plasma membranes in cerebral oxidative stress probably is related to lipid peroxidation in the brain.

Topics: Adenosine Triphosphatases; Animals; Antioxidants; Apyrase; Ascorbic Acid; Chromans; Ferrous Compounds; Hydrogen Peroxide; Kinetics; Lipid Peroxidation; Male; Prosencephalon; Rats; Rats, Wistar; Synaptic Membranes; Vitamin E

Oxidative stress and adenine nucleotide catabolism occur concomitantly in several disease states, such as cardiac ischaemia-reperfusion, and may act as synergistic determinants of tissue injury. However, the mechanisms underlying this potential interaction remain ill-defined. We examined the influence of oxidative stress on the molecular, kinetic and regulatory properties of a ubiquitous AMP-catabolizing enzyme, adenylate deaminase (AMPD) (EC 3.5.4.6). To this intent, rabbit heart AMPD and an H2O2/ascorbate/iron oxidation system were employed. Enzyme exposure to the complete oxidation system acutely impaired its catalytic activity, lowered the Vmax. by 7-fold within 5 min, and rendered the enzyme unresponsive to nucleotide effectors. Irreversible AMPD inactivation resulted within about 15 min of oxidative insult and was not prevented by free-radical scavengers. Oxidative stress did not affect the molecular mass, tetrameric nature, Km, immunoreactivity or trypsinolytic pattern of the enzyme; nor did it induce carbonyl formation, Zn2+ release from the holoenzyme or net AMPD S-thiolation. This injury pattern is inconsistent with a radical-fragmentation mechanism as the basis for the oxidative AMPD inactivation observed. Rather, the sensitivity of the enzyme to both S-thiolation and thiol alkylation and the significant (3 of 9/mol of denatured enzyme) net loss of DTNB-reactive thiols on exposure to oxidant strongly implicate the conversion of essential thiol moieties into stable higher-oxidation states in the oxidative inactivation of cardiac AMPD. The altered thiol status of the enzyme on oxidative insult may prohibit a catalytically permissible conformation and, in so doing, increase AMP availability to 5'-nucleotidase in vivo.

Topics: 5'-Nucleotidase; Alkylation; AMP Deaminase; Animals; Ascorbic Acid; Catalysis; Chlorides; Dithionitrobenzoic Acid; Ferric Compounds; Ferrous Compounds; Hydrogen Peroxide; Kinetics; Macromolecular Substances; Molecular Weight; Myocardium; Oxidative Stress; Rabbits; Sulfhydryl Compounds; Trypsin; Zinc

The effects of antioxidants and various other modifying agents on oxygen-radical-generated DNA damage in human lymphocytes have been investigated using the COMET assay. Hydrogen peroxide (H2O2) and bleomycin (BLM) have produced clear dose-related responses. In 38 independent experiments, there was consistency between the two donors used in the study for the negative and positive control data. The endogenous antioxidant catalase abolished effects with H2O2, but only slightly affected the response with BLM. Superoxide dismutase did not alter the response with H2O2 and only slightly affected BLM. The exogenous antioxidant vitamin C produced a clear dose-related response on its own. In combination with H2O2, there were small protective effects at low doses and exacerbating effects at high doses, but these were within the inter-experimental variability range. Vitamin E (trolox) produced no effects with either H2O2 or BLM, or on its own. Silymarin protected against the effect due to H2O2. Other modifying agents such as apo-transferrin and deferoxamine mesylate produced a clear dose-related protection of effects due to BLM. This protection was less due to H2O2. In the presence of ferrous chloride, the effect due to BLM was exacerbated. In a small sample of 6 smokers and 6 non-smokers, responses from smokers approached borderline significance (P = 0.054) by comparison with non-smokers. These observations would suggest that the COMET assay is a useful tool for examining issues related to oxidative stress in human lymphocytes.

Topics: Adult; Antioxidants; Apoproteins; Ascorbic Acid; Bleomycin; Catalase; Cells, Cultured; Deferoxamine; DNA Damage; Drug Interactions; Female; Ferrous Compounds; Humans; Hydrogen Peroxide; Lymphocytes; Male; Mannitol; Mutagenicity Tests; Reactive Oxygen Species; Silymarin; Smoking; Superoxide Dismutase; Transferrin; Vitamin E

Rat heart myocardial membranes exposed to the free radical generating system, Fe2+/ascorbate, undergo lipid peroxidation as evidenced by the accumulation of thiobarbituric acid-reactive substances, loss of polyunsaturated fatty acids from phospholipids, and formation of conjugated dienes and fluorescent substances. In addition, the treated membranes exhibit a dramatic decrease in extractable phospholipids. This decrease is even more pronounced in individual phospholipid classes isolated by high-performance liquid chromatography. The decrease in lipid phosphorus under oxidant stress is accompanied by an increase in the phosphorus content of the aqueous phase after Folch extraction and by an even greater increase of phosphorus in the protein residue. In addition, increased amounts of saturated and monounsaturated fatty acyl groups are found in the protein residue of Fe2+/ascorbate-treated membranes. Extraction of the oxidant-treated membranes with acidic solvents does not enhance the recovery of phospholipids and neither does treatment with detergents, trypsin, and chymotrypsin prior to lipid extraction. However, treatment with the bacterial protease, Pronase, markedly enhances the recovery of phospholipids from the peroxidized membranes. These results indicate that membrane phospholipids undergoing free radical-induced peroxidation may form lipid-protein adducts, which renders them inextractable with lipid solvents.

Topics: Animals; Ascorbic Acid; Cell Membrane; Ferrous Compounds; Free Radicals; Kinetics; Lipid Peroxidation; Male; Membrane Lipids; Myocardium; Phospholipids; Rats; Rats, Inbred Strains

The effect of lipid peroxidation on the Mg2(+)-independent and Mg2(+)-dependent activity of brain cell membrane 5'-nucleotidase was determined and the affinity of the active sites of Mg2(+)-dependent enzyme for 5'-AMP (substrate) and Mg2+ (activator) was examined. Brain cell membranes were peroxidized at 37 degrees C in the presence of 100 microM ascorbate and 25 microM FeCl2 (resultant) for 10 min. The activity of 5'-nucleotidase and lipid peroxidation products (thiobarbituric acid reactive substances) were determined. At 10 min, the level of lipid peroxidation products increased from 0.20 +/- 0.10 to 17.5 +/- 1.5 nmoles malonaldehyde/mg membrane protein. The activity of Mg2(+)-independent 5'-nucleotidase increased from 0.201 +/- 0.020 in controls to 0.305 +/- 0.028 mumol Pi/mg protein/hr in peroxidized membranes. In the presence of 10 mM Mg2+, the activity increased by 5.8-fold in the peroxidized membrane preparation in comparison to 14-fold in control. In peroxidized preparation, the affinity of active site of Mg2(+)-dependent 5'-nucleotidase for 5'-AMP tripled, as indicated by a significant decrease in Km (Km = 95 +/- 2 microM AMP for control; Km = 32 +/- 2 microM AMP for peroxidized). Vmax was significantly reduced from 3.35 +/- 0.16 in control to 1.70 +/- 0.9 mumoles Pi/mg protein in peroxidized membranes. The affinity of the active site for Mg2+ significantly increased (Km = 6.17 +/- 0.37 mM Mg2+ for control; Km = 4.0 +/- 0.31 peroxidized).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 5'-Nucleotidase; Adenosine Monophosphate; Animals; Ascorbic Acid; Binding Sites; Brain; Cell Membrane; Ferrous Compounds; Lipid Peroxidation; Magnesium; Malondialdehyde; Swine; Thiobarbiturates

The glutamine synthetase of Neurospora crassa, either purified or in cell extracts, was inactivated by ascorbate plus FeCl3 and by H2O2 plus FeSO4. The inactivation reaction was oxygen dependent, inhibited by MnCl2 and EDTA, and stimulated in cell extracts by sodium azide. This inactivation could also be brought about by adding NADPH to the cell extract. The alpha and beta polypeptides of the active glutamine synthetase were modified by these inactivating reactions, giving rise to two novel acidic polypeptides. These modifications were observed with the purified enzyme, with cell extracts, and under in vivo conditions in which glutamine synthetase is degraded. The modified glutamine synthetase was more susceptible to endogenous phenylmethylsulfonyl fluoride-insensitive proteolytic activity, which was inhibited by MnCl2 and stimulated by EDTA. The possible physiological relevance of enzyme oxidation is discussed.

Topics: Ascorbic Acid; Chlorides; Edetic Acid; Ferrous Compounds; Glutamate-Ammonia Ligase; Hydrogen Peroxide; Macromolecular Substances; Manganese; Manganese Compounds; NADP; Neurospora; Neurospora crassa; Oxidation-Reduction; Peptide Hydrolases

Radioiron uptake from 59FeCl3 by Streptococcus mutans OMZ176 was increased by anaerobiosis, sodium ascorbate, and phenazine methosulfate (PMS), although there was a 10-min lag before PMS stimulation was evident. The reductant ascorbate may have provided ferrous iron. The PMS was reduced by the cells, and the reduced PMS then may have generated ferrous iron for transport; reduced PMS also may have depleted dissolved oxygen. We conclude that S. mutans transports only ferrous iron, utilizing reductants furnished by glucose metabolism to reduce iron prior to its uptake.

Topics: Anaerobiosis; Ascorbic Acid; Biological Transport; Ferrous Compounds; Iron Chelating Agents; Methylphenazonium Methosulfate; Oxidation-Reduction; Siderophores; Streptococcus mutans

Alveolar surfactant is known to be impaired after inhalation of various oxidizing agents (NO2, ozone) as well as in inflammatory lung processes, in which leucocyte-derived active oxygen species or arachidonic acid oxygenation products may be involved. The effect of lipid peroxidation, oxygen-free radicals and oxygenated versus native arachidonic acid on the surface tension behaviour of natural surfactant was tested in vitro. The studies were performed on pooled surfactant material, obtained from bronchoalveolar lavage of rabbit lungs, in a Langmuir trough/Wilhelmy balance system. Initiation of lipid peroxidation with FeCl3/ascorbate or UV radiation and the generation of OH.(FeCl2/EDTA/H2O2), O2-. (xanthine/xanthine oxidase) and 1O2 (NaOCl/H2O2) provoked a common profile of changes: delayed reduction of surface tension during compression with an increase in minimal compressibility accelerated decrease of film pressure during expansion, reduction of hysteresis area and markedly augmented monolayer collapse rate. Addition of arachidonic acid resulted in decreased minimal compressibility, stability index and hysteresis area. Incubation with the arachidonic acid cyclooxygenase products, prostaglandin E2, I2, F2 alpha or thromboxane B2, with soybean lipoxygenase or with H2O2 and O2-exposure caused only moderate or no alteration of surfactant behaviour in vitro.. oxidative stress, but not arachidonic acid oxygenation products, provoked altered surface tension behaviour of natural surfactant in vitro.

Topics: Arachidonic Acid; Arachidonic Acids; Ascorbic Acid; Ferrous Compounds; Free Radicals; Humans; Hydrogen Peroxide; In Vitro Techniques; Lipid Peroxides; Lung; Oxygen; Prostaglandin-Endoperoxide Synthases; Pulmonary Surfactants; Ultraviolet Rays