ascorbic-acid and cupric-chloride

The oxidative modification of human low density lipoprotein (LDL) has been widely investigated. However, there are no data concerning the oxidation susceptibility of combined very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein fraction, although all of them are atherogenic and contain antioxidants such as alpha-tocopherol. We investigated the oxidation susceptibility and oxidation resistance of VLDL + LDL (including IDL) fraction by induction with CuCl2 and its relation to plasma alpha-tocopherol concentration and lipid standardised alpha-tocopherol concentration in 406 non-vitamin E-supplemented men from eastern Finland. Even thought we did not give oral vitamin E or any other antioxidant supplementation to our study participants, we observed a significant, consistent relationship between measurements of oxidation resistance and plasma content of vitamin E. In the multivariate regression model, a high plasma content of vitamin E or lipid standardised vitamin E concentration were the most important determinants of lag time to maximal oxidation rate (standardised regression coefficient = 0.244, P < 0.0001 for vitamin E and 0.211, P < 0.0001 for lipid standardised vitamin E). After statistical adjustment for age, use of cigarettes, hypolipidemic medication (yes vs. no), month of the measurements, plasma concentrations of total ascorbic acid (ascorbic acid + dehydroascorbic acid), beta-carotene and phospholipids, serum concentrations of LDL cholesterol and triglycerides and dietary intake of linoleic acid, the lag time to maximal oxidation rate was 10% (95% C.I. 6.0-13.5%) longer in men in the highest fifth than in the lowest fifth of plasma vitamin E content (P < 0.0001 for trend). When the fifths of lipid standardised vitamin E were compared, the lag time to maximal oxidation rate was 6% (95% C.I. 1.8-10.1%) longer in men in the highest than in the lowest fifth (P < 0.0001 for trend). Our data suggest that alpha-tocopherol is an important antioxidant preventing the in vitro oxidation of VLDL + LDL fraction even in non-supplemented subjects.

Topics: Adult; Anticholesteremic Agents; Antioxidants; Arteriosclerosis; Ascorbic Acid; beta Carotene; Cholesterol, LDL; Copper; Dietary Fats; Double-Blind Method; Humans; Hypercholesterolemia; Linoleic Acid; Linoleic Acids; Lipids; Lipoproteins, LDL; Lipoproteins, VLDL; Male; Middle Aged; Oxidation-Reduction; Pravastatin; Risk Factors; Smoking; Vitamin E

The disruption of copper homeostasis and the oxidative stress induced by Cu-amyloids are crucial features of Alzheimer's disease pathology. The copper specific N

Topics: Aminoquinolines; Amyloid beta-Peptides; Ascorbic Acid; Carrier Proteins; Chelating Agents; Chlorides; Copper; Homeostasis; Humans; Molecular Structure; Oxidation-Reduction; Oxidative Stress; Peptide Fragments; Reactive Oxygen Species; Zinc Compounds

Metal-catalyzed oxidation (MCO) of proteins is of primary concern in the development of biotherapeutics as it represents a prominent degradation pathway with potential undesired biological and biotherapeutic consequences.. We developed a fluorogenic derivatization methodology to study the MCO of IgG1 using a model oxidation system, CuCl2/L-ascorbic acid.. Besides the oxidation of Met, Trp and His residues, we detected significant oxidation of Phe and Tyr in IgG1.. The fluorogenic derivatization method provides an alternative approach for the rapid detection of oxidized Tyr and Phe as their benzoxazole derivatives by fluorescence spectrometry and size exclusion chromatography coupled to fluorescence detection.

Topics: Ascorbic Acid; Benzoxazoles; Copper; Immunoglobulin G; Oxidation-Reduction; Phenylalanine; Spectrometry, Fluorescence; Tyrosine

Human amyloid-β peptide 1-42 (Aβ) was subjected to a radical reaction by using ascorbic acid and CuCl(2). The percentage of D-aspartic acid (D-Asp) after 24 h had increased to 6.69 ± 0.09%, this being comparable with the reported D-Asp concentration of purified core amyloids in Alzheimer's disease patients. This racemization was significantly inhibited by radical scavengers. L-Alanine was also racemized during the same reaction.

Topics: Amyloid beta-Peptides; Ascorbic Acid; Aspartic Acid; Chromans; Copper; Free Radical Scavengers; Free Radicals; Humans; Mercaptoethanol; Peptide Fragments; Solutions; Stereoisomerism

Based on our previous findings, the present study has focused on free-radical-mediated degradation of the synovial biopolymer hyaluronan. The degradation was induced in vitro by the Weissberger's system comprising ascorbate plus cupric ions in the presence of oxygen, representing a model of the early phase of acute synovial joint inflammation. The study presents a novel strategy for hyaluronan protection against oxidative degradation with the use of cysteine-derived compounds. In particular, the work objectives were to evaluate potential protective effects of reduced form of L-glutathione, L-cysteine, N-acetyl-L-cysteine, and cysteamine, against free-oxygen-radical-mediated degradation of high-molar-mass hyaluronan in vitro. The hyaluronan degradation was influenced by variable activity of the tested thiol compounds, also in dependence of their concentration applied. It was found that L-glutathione exhibited the most significant protective and chain-breaking antioxidative effect against the hyaluronan degradation. Thiol antioxidative activity, in general, can be influenced by many factors such as various molecule geometry, type of functional groups, radical attack accessibility, redox potential, thiol concentration and pK(a), pH, ionic strength of solution, as well as different ability to interact with transition metals. Antioxidative activity was found to decrease in the following order: L-glutathione, cysteamine, N-acetyl-L-cysteine, and L-cysteine. These findings might be beneficial in future development of potential drugs in the treatment of synovial hyaluronan depletion-derived diseases.

Topics: Adjuvants, Immunologic; Antioxidants; Ascorbic Acid; Copper; Cysteamine; Cysteine; Free Radicals; Hyaluronic Acid; Oxidation-Reduction; Oxygen; Sulfhydryl Compounds

Free Cu(2+) is toxic due to the capacity of free copper ions to catalyze the production of reactive oxygen species (ROS) that can modify the structure and/or function of biomolecules. In addition, non-specific binding to enzymes, which modifies their catalytic activities, can occur. In this work, the mechanisms underlying the ability of copper to inhibit 3'-nucleotidase from Leishmania amazonensis (La3'-nucleotidase) were investigated. To that end, La3'-nucleotidase activity was assayed with CuCl(2) in the presence of ascorbate or hydrogen peroxide to discriminate non-specific binding effects from pro-oxidant effects of copper. Copper inhibitory effects were greater at more acidic pH than at alkaline pH. The addition of enzyme substrate, adenosine 3'-monophosphate (3'AMP), prevented the inhibition of enzyme activity by copper. Thiol-containing compounds were able to protect the enzyme activity against inhibition due to copper. The specific copper chelating agent bathocuproine sulphonate (BCS) restored enzyme activity after pre-treatment of the enzyme with copper. La3'-nucleotidase activity was found to be resistant to ROS generated during oxidation reactions of ascorbate and hydrogen peroxide catalyzed by copper. Our results suggest that Cu(2+) ions exert their inhibitory effects by binding to specific motifs of the 3'-nucleotidase protein and that the enzyme appears to be extremely resistant to ROS.

Topics: Animals; Ascorbic Acid; Copper; Cricetinae; Cysteine; Dithiothreitol; Dose-Response Relationship, Drug; Glutathione; Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Leishmania mexicana; Mercaptoethanol; Nucleotidases; Oxidation-Reduction; Oxygen; Phenanthrolines; Reactive Oxygen Species

The detection of DNA radicals by immuno-spin trapping (IST) is based on the trapping of radicals with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), forming stable nitrone adducts that are then detected using an anti-DMPO serum. DNA radicals are very reactive species, and because they are paramagnetic they have previously been detected only by electron spin resonance (ESR) with or without spin trapping, which is not available in most bioresearch laboratories. IST combines the simplicity, reliability, specificity and sensitivity of spin trapping with heterogeneous immunoassays for the detection of DNA radicals, and complements existing methods for the measurement of oxidatively generated DNA damage. Here we have used IST to demonstrate that DMPO traps Cu(II)-H(2)O(2)-induced DNA radicals in situ and in real time, forming DMPO-DNA nitrone adducts, but preventing both 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) formation and DNA fragmentation. We also applied IST to detect DNA radicals in rat hepatocytes exposed to Cu(II) and H(2)O(2) under nonlethal conditions.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Ascorbic Acid; Cell Nucleus; Chlorides; Copper; Cyanides; Cyclic N-Oxides; Deoxyguanosine; Deuterium Oxide; DNA; DNA Fragmentation; Enzyme-Linked Immunosorbent Assay; Female; Free Radicals; Glutathione; Hepatocytes; Histidine; Hydrogen Peroxide; Immunoassay; Nitrogen Oxides; Oxidation-Reduction; Rats; Rats, Sprague-Dawley; Spin Trapping; Thiourea; Zinc Compounds

The degradation of high-molar-mass hyaluronan (HA) by copper(II) chloride and ascorbate was studied by means of rotational viscometry. It was found that even small amounts of CuCl(2) present in the oxidative system led to the pronounced degradation of HA, reflected in a rapid decrease of the dynamic viscosity of the biopolymer solution. Such degradation was induced by free radicals generated in elevated amounts in the presence of copper ions. Electron paramagnetic resonance investigations performed on a model oxidative system containing Cu(II) and ascorbic acid proved the formation of relatively stable ascorbate anion radicals resulting from the reaction of ascorbic acid with hydroxyl radicals. In this way, by scavenging the hydroxyl radicals, ascorbic acid protected HA from their degradative action. Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry was applied to analyze the degraded HA. The results showed that only regular fragmentation of hyaluronan occurred using the mentioned oxidative system that led to the formation of HA oligomers with unaffected primary chemical structure.

Topics: Ascorbic Acid; Copper; Electron Spin Resonance Spectroscopy; Free Radicals; Hyaluronic Acid; Molecular Weight; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spin Trapping; Viscosity

Bacillus subtilis spores were killed by CuCl(2)-ascorbic acid, chloride ions were essential for killing of spores, and spores with defective coats were killed more rapidly. CuCl(2)-ascorbic acid did not damage spore DNA, and spores killed by this reagent initiated germination. However, spores killed by CuCl(2)-ascorbic acid may have damage to their inner membrane.

Topics: Ascorbic Acid; Bacillus subtilis; Copper; DNA Damage; DNA, Bacterial; Hydrogen Peroxide; Iron; Spores, Bacterial

An approach to decontamination of biological endospores is discussed. Specifically, the performance of an aqueous modified Fenton reagent is examined. A modified Fenton reagent formulation of cupric chloride, ascorbic acid, and sodium chloride is shown to be an effective sporicide under aerobic conditions. The traditional Fenton reaction involves the conversion of hydrogen peroxide to hydroxyl radical by aqueous ionic catalysts such as the transition metal ions. Our modified Fenton reaction involves the conversion of aqueous dissolved oxygen to hydrogen peroxide by an ionic catalyst (Cu(2+)) and then subsequent conversion to hydroxyl radicals. Results are given for the modified Fenton reagent deactivating spores of Bacillus globigii. A biocidal mechanism is proposed that is consistent with our experimental results and independently derived information found in the literature. This mechanism requires diffusion of relatively benign species into the interior of the spore, where dissolved O(2) is then converted through a series of reactions which ultimately produce hydroxyl radicals that perform the killing action.

Topics: Ascorbic Acid; Bacillus; Copper; Disinfection; Hydrogen Peroxide; Iron; Microscopy, Electron, Scanning; Oxygen; Sodium Chloride; Spores, Bacterial

The octapeptide repeat region of human prion protein is known to bind four Cu(II) ions per molecule. A peptide, Octa(4), representing this region was tested for inhibitory effects on copper-catalyzed oxidation of l-ascorbate or glutathione and on generation of OH(*) during the former reaction. The result indicated that the catalytic activity of the first Cu(II) ion bound to an Octa(4) molecule was completely suppressed. The valence state of the copper under reducing conditions was Cu(II), as determined by a newly developed method using bathocuproinedisulfonate under acidic conditions. Furthermore, it was shown that Escherichia coli cells expressing the octapeptide repeat region were significantly resistant to copper treatment compared with control cells. The results taken together indicate that prion protein can function to sequester copper ions in the redox-inactive state, rendering copper-induced generation of reactive oxygen species impossible.

Topics: Ascorbic Acid; Binding Sites; Cloning, Molecular; Copper; Escherichia coli; Glutathione; Humans; Kinetics; Oxidation-Reduction; Peptide Fragments; Prions; Recombinant Proteins; Repetitive Sequences, Amino Acid

Previous cell biological studies demonstrated that S100B protein enhances neurite extension of cortical neurons and stimulates proliferation of glial cells. Although these activities of the protein are ascribed to its disulfide-linked dimeric form, there have been no indications as to how the dimer is formed in vivo. We have found by an in vitro study that it is produced by copper-dependent oxidation of noncovalent S100B dimer. The disulfide-linked dimer markedly stimulated nitric oxide production in a microglial cell line, BV2. Interestingly, the disulfide-linked dimer formation was found to be prevented by ascorbic acid. The copper-dependent formation of the dimer may not happen in vivo under normal conditions; however, under pathological conditions where copper is likely to be released from tissues and catalyze autoxidation of ascorbic acid, the dimer formation may proceed, resulting in the stimulated production of nitric oxide that would induce toxic signaling pathways.

Topics: Animals; Ascorbic Acid; Autoantigens; Calcium-Binding Proteins; Cattle; Cell Line; Copper; Dimerization; Disulfides; Kinetics; Mice; Microglia; Nerve Growth Factors; S100 Calcium Binding Protein beta Subunit; S100 Proteins

The metal-catalyzed oxidation (ascorbate/cupric chloride/oxygen) of recombinant human relaxin (rhRlx, type II) was shown by Li et al. [Li, S., Nguyen, T. H., Schöneich, C., and Borchardt, R. T. (1995) Biochemistry 34, 5762-5772] to result in the chemical modification of His A(12), Met B(4), and Met B(25). Considering the fact that His A(12) exists in an extended loop that joins two alpha-helices in this protein, we hypothesized that oxidation of this specific amino acid leads to alterations in the secondary and tertiary structures of the protein, resulting in the pH-dependent aggregation/precipitation phenomena observed in our earlier studies (i.e., at pH >6.0 most of the degradants of rhRlx are insoluble). Evidence obtained in the current study that supports this hypothesis includes the following: (i) oxidation of rhRlx with hydrogen peroxide (H(2)O(2)), which leads only to modification of Met B(4) and Met B(25), does not result in the pH-dependent aggregation/precipitation of the protein; and (ii) metal-catalyzed oxidation of porcine relaxin (pRlx), which does not contain His at position A(12), leads to chemical degradation of the protein [e.g., Met A(2) is oxidized] but produces only slight pH-dependent aggregation/precipitation of the protein. In addition, experimental evidence is provided to show that the physical instability of rhRlx observed at pH >6.0 does not appear to be related to the pH-dependent solubility of a common protein degradant. Instead, it appears that several oxidation products of His A(12) are produced in a pH-dependent manner and that these oxidation products produce different effects on the physical stability of the protein. Evidence in support of this conclusion includes the observation that the soluble degradants of rhRlx showed reduced levels of His, reduced levels of the T(2)-T(7) tryptic fragment that contained His A(12), and the presence of 2-oxo-His. Similarly, the precipitated degradants of rhRlx showed reduced levels of His but no 2-oxo-His. In addition, the soluble degradants, which contain 2-oxo-His, appear to exist as monomers having an average molecular weight similar to that of rhRlx. These results suggest that the metal-catalyzed oxidation of His A(12) leads to other, as yet unidentified oxidation products of His A(12) that affect the secondary/tertiary structure of the protein more significantly than does 2-oxo-His and ultimately lead to the physical instability of the protein observed at higher pH values.

Topics: Amino Acids; Animals; Ascorbic Acid; Catalysis; Chemical Precipitation; Chromatography, Liquid; Copper; Histidine; Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Mass Spectrometry; Oxidation-Reduction; Peptide Fragments; Peptide Mapping; Recombinant Proteins; Relaxin; Solubility; Swine

To elucidate the major degradation products of the metal-catalyzed oxidation of (cyclo S-S) AcCys-Ala-X-Val-Gly-CysNH2 (X = His, cyclic-His peptide), which is a fragment of the protein relaxin, and the effect of this oxidation on its solution structure.. The cyclic-His peptide and its potential oxidative degradation products, cyclic-Asp peptide (X = Asp) and cyclic-Asn peptide (X = Asn), were prepared by using solid phase peptide synthesis and purified by preparative HPLC. The degradation of the cyclic-His peptide was investigated at pH 5.3 and 7.4 in an ascorbate/cupric chloride/oxygen [ascorbate/Cu(II)/O2] system in the absence or presence of catalase (CAT), superoxide dismutase (SOD), isopropanol, and thiourea. The oxidation of the cyclic-His peptide was also studied in the presence of hydrogen peroxide (H2O2). All reactions were monitored by reversed-phase HPLC. The main degradation product of the cyclic-His peptide formed at pH 7.4 in the presence of ascorbate/Cu(II)/O2 was isolated by preparative HPLC and identified by 1H NMR and electrospray mass spectrometry. The complexation of Cu(II) with the cyclic-His peptide was determined with 1H NMR. The solution structure of the cyclic-His peptide in the presence and absence of Cu(II) at pH 5.3 and 7.4 and the solution structure of the main degradation product were determined using circular dichroism (CD).. CAT and thiourea were effective in stabilizing the cyclic-His peptide to oxidation by ascorbate/Cu(II)/O2, while SOD and isopropanol were ineffective. Cyclic-Asp and cyclic-Asn peptides were not observed as degradation products of the cyclic-His peptide oxidized at pH 5.3 and 7.4 in an ascorbate/Cu(II)/O2 system. The main degradation product formed at pH 7.4 was the cyclic 2-oxo-His peptide (X = 2-oxo-His). At pH 5.3, numerous degradation products were formed in low yields, including the cyclic 2-oxo-His peptide. The cyclic 2-oxo-His peptide appeared to have a different secondary structure than did the cyclic-His peptide as determined by CD. 1H NMR results indicate complexation between the cyclic-His peptide and Cu(II). CD results indicated that the solution structure of the cyclic-His peptide in the presence of Cu(II) at pH 5.3 was different than the solution structure observed at pH 7.4.. H2O2 and superoxide anion radical (O(*-)2) were deduced to be the intermediates involved in the ascorbate/Cu(II)/O2-induced oxidation of cyclic-His peptide. H2O2 degradation by a Fenton-type reaction appears to form secondary reactive-oxygen species (i.e., hydroxyl radical generated within complex forms or metal-bound forms of hydroxyl radical) that react with the peptide before they diffuse into the bulk solution. CD results indicate that different complexes are formed between the cyclic-His peptide and Cu(II) at pH 5.3 and pH 7.4. These different complexes may favor the formation of different degradation products. The apparent structural differences between the cyclic-His peptide and the cyclic 2-oxo-His peptide indicate that conformation of the cyclic-His peptide was impacted by metal-catalyzed oxidation.

Topics: Ascorbic Acid; Copper; Histidine; Humans; Hydrogen-Ion Concentration; Metals; Oxidants; Peptides, Cyclic; Reactive Oxygen Species; Relaxin

Ascorbic acid and its oxidation products have been implicated in non-enzymatic modification of proteins in aging and diseases of oxidative stress. We have studied the feasibility of using 6-deoxy-6-fluoroascorbic acid (6) for identification of ascorbic acid degradation products by 19F NMR spectroscopy. Crystals of compound 6 from nitromethane belonged to the space group P2(1) with a = 5.547(2), b = 6.769(3), c = 9.302(2) A, beta = 91.80(3) degrees and Z = 2. Atomic coordinates, bond lengths and angles, hydrogen coordinates, anisotropic and isotropic displacement parameters were similar if not identical with those of native ascorbic acid. Similarly, UV properties and oxidation kinetics by CuCl2 at different pH values were essentially identical with ascorbic acid. Using 750 MHz 19F NMR spectroscopy, five to six new fluorinated products were detected after overnight oxidation of 6 with Cu2+, suggesting that 6 may be a powerful and sensitive tool for assessment of its catabolism in vivo.

Topics: Ascorbic Acid; Biomarkers; Copper; Crystallization; Crystallography, X-Ray; Feasibility Studies; Hydrogen-Ion Concentration; Kinetics; Magnetic Resonance Spectroscopy; Molecular Structure; Oxidation-Reduction

To elucidate the nature of the reactive oxygen species (i.e., superoxide anion radical, hydroxyl radical, and hydrogen peroxide) involved in the metal-catalyzed oxidation of histidine (His) in two model peptides.. The degradation of AcAla-His-ValNH2 (Ala-peptide) and AcCysNH2-S-S-AcCys-His-VaNH2 (Cys-peptide) was investigated at pH 5.3 and 7.4 in an ascorbate/cupric chloride/oxygen (ascorbate/ Cu(II)/O2) system, both in the absence and presence of selective scavengers (i.e., catalase, superoxide dismutase, mannitol, sodium formate, isopropanol, and thiourea) of the reactive oxygen species. All reactions were monitored by HPLC. The major degradation products were characterized by electrospray mass spectrometry.. The Cys-peptide was more stable than the Ala-peptide at pH 5.3 and 7.4. Both peptides displayed greater stability at pH 5.3 than at 7.4. At pH 5.3, 35 +/- 0.7% of the Cys-peptide and 18 +/- 1% of the Ala-peptide remained after 7 hours, whereas at pH 7.4, 16 +/- 3% of the Cys-peptide and 4 +/- 1% of the Ala-peptide remained. Catalase, thiourea, bicinchoninic acid, and ethylenediaminetetraacetate were effective at stabilizing both peptides toward oxidation, while superoxide dismutase, mannitol, isopropanol, and sodium formate were ineffective. The main degradation products of the Ala- and Cys-peptides at pH 7.4 appeared to be AcAla-2-oxo-His-ValNH2 and AcCysNH2-S-S-AcCys-2-oxo-His-ValNH2, respectively.. Hydrogen peroxide, Cu(I), and superoxide anion radical were deduced to be intermediates involved in the oxidation of the Ala- and Cys-peptides. Hydrogen peroxide degradation to secondary reactive oxygen species may have led to the oxidation of the peptides. The degradation of hydrogen peroxide by a Fenton-type reaction was speculated to form a complexed form of hydroxyl radical that reacts with the peptide before diffusion into the bulk solution.

Topics: Ascorbic Acid; Copper; Histidine; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydroxyl Radical; Oxidation-Reduction; Oxygen; Peptide Fragments; Reactive Oxygen Species; Relaxin; Superoxides

The ability of Cu(II) and Fe(III) to promote site-specific DNA damage in the presence of endogenous reductants was investigated by using 32P-5'-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. Ascorbate induced metal-dependent DNA damage most efficiently (ascorbate > GSH > NADH). Cu(II) induced endogenous reductants-dependent DNA damage more efficiently than Fe(III). Endogenous reductants plus Fe(III) caused DNA cleavage at every nucleotide, without marked site preference. DNA damage by Fe(III) was inhibited by hydroxyl free radical (.OH) scavengers and catalase. These results suggest that endogenous reductants plus Fe(III) generate free or extremely near free .OH via H2O2 formation, and that .OH causes DNA damage. In the presence of 50 microM Cu(II) in bicarbonate buffer, ascorbate caused DNA cleavage frequently at sites of two or more adjacent guanine residues. In contrast, in the presence of 20 microM Cu(II), ascorbate caused DNA cleavage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited DNA damage by Cu(II), whereas .OH scavengers did not. Fe(III)-dependent 8-oxo-7,8-dihydro-2'-deoxyguanosine formation was inhibited by .OH scavengers, whereas no inhibition by .OH scavengers was observed with Cu(II). These results suggest that .OH is the main active species formed with Fe(III), whereas copper-peroxide complexes with a reactivity similar to .OH participate in Cu(II)-dependent DNA damage. The polyguanosine sequence specificity of DNA damage in the presence of high concentrations of Cu(II) can be explained by the preferential binding of Cu(II) to guanine residues.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Ascorbic Acid; Cattle; Chlorides; Copper; Deoxyguanosine; DNA Damage; DNA Fragmentation; Ferric Compounds; Free Radical Scavengers; Hydrogen Peroxide; Hydroxyl Radical; NAD; Oxidation-Reduction

Potential antioxidant properties of therapeutically achievable concentrations of the protonated, active form of omeprazole (OM) were investigated in vitro at specific acidic pH values to mimic intragastric conditions in the clinical setting. We found that OM is a powerful scavenger of hypochlorous acid (HOCl) even at a drug concentration of 10 microM at pH 5.3 or 3.5. This effect is also evident in the presence of the physiological HOCl scavenger ascorbate. Moreover, 10 and 50 microM OM inhibit significantly both iron- and copper-driven oxidant damage at pH 5.3 and 3.5, respectively. Since oxidative stress is involved the gastric injury of peptic ulcer and gastritis, it may be hypothesized that some therapeutical effects of OM could also be related to its antioxidant properties.

Topics: Anti-Ulcer Agents; Antioxidants; Ascorbic Acid; beta Carotene; Carotenoids; Chlorides; Copper; Deoxyribose; Ferric Compounds; Hydrogen-Ion Concentration; Hypochlorous Acid; Omeprazole; Oxidation-Reduction

Iron and copper ions mediate generation of reactive oxygen radicals from O2 and H2O2 by the Fenton reaction: these radicals are capable of damaging DNA. We studied (a) the ability of these metals to induce double-strand breaks in DNA in vitro in the presence of H2O2 and ascorbic acid as donors of reactive oxygen, and (b) the ability of the metal-binding protein metallothionein (MT) to protect DNA from damage. Strand cleavage was measured by loss of fluorescence after binding to ethidium bromide and by increased mobility of DNA in agarose. The results show that Cu(II), Fe(II) and Fe(III) all can induce damage to calf thymus DNA under our experimental conditions. Cu(II)-induced DNA damage was dose-dependent and the degree of damage was proportional to the concentration of H2O2. On the other hand, DNA fragmentation was significant only in the presence of high concentrations of Fe(II) or Fe(III). Addition of Zn-MT to the reaction mixture prior to addition of Cu(II) inhibited fragmentation of DNA in a dose-dependent manner but had little effect on iron induced damage. Other proteins (histone or albumin) were not effective in protecting DNA from Cu-induced damage, as compared to Zn-MT. The formation of Cu(I) from Cu(II) in the presence of hydrogen peroxide and ascorbate was also inhibited by addition of Zn-MT. Thus, MT may protect DNA from damage by free radicals by sequestering copper and preventing its participation in redox reactions.

Topics: Animals; Ascorbic Acid; Copper; DNA Damage; Dose-Response Relationship, Drug; Drug Synergism; Electrophoresis, Agar Gel; Ethidium; Ferric Compounds; Ferrous Compounds; Free Radicals; Hydrogen Peroxide; In Vitro Techniques; Metallothionein; Oxidation-Reduction; Reactive Oxygen Species; Zinc

Tamrabhasma is a traditional copper oxide-containing Indian preparation which has anti-ulcer activity. We have studied the effect of tamrabhasma and CuCl2.2H2O on prostaglandin formation by human gastric and colonic mucosa and submucosa, as prostaglandins have mucosal protective activity, and their release may contribute to the anti-ulcer effect. With the gastric mucosa, tamrabhasma 10 micrograms mL-1, but not 0.1 or 1 microgram mL-1, increased prostaglandin E (PGE) concentration by 38% (P < 0.05), with little or no effect on 6-keto-PGF1 alpha, thromboxane B2 or the leukotriene (LT) LTC4/LTD4. CuCl2.2H2O (10 micrograms mL-1) increased 6-keto-PGF1 alpha by 46% (P < 0.05), but 0.1, 1, 50 and 250 micrograms mL-1 did not significantly affect any of the prostanoids, and only the highest concentration reduced the amount of LTC4/LTD4. In the colon mucosa, tamrabhasma (0.1-10 micrograms mL-1) or CuCl2.2H2O (10-50 micrograms mL-1) increased all the prostanoids and this effect was greater than in the gastric mucosa but there was no significant change in LTC4/LTD4. CuCl2.2H2O showed a bell-shaped concentration-effect curve, with no significant effect at lower and higher amounts. Indomethacin (0.1-10 micrograms mL-1) caused a concentration-dependent reduction in the prostanoid amounts. The effect of tamrabhasma was probably not only due to the presence of Cu2+, as tamrabhasma was more effective than CuCl2.2H2O alone; in addition the solubility of CuO is very low. Increased prostanoid levels might explain, at least partly, the gastric mucosal protection by tamrabhasma. The results in the colon, however, raise the possibility that tamrabhasma should be examined for the treatment of inflammatory bowel disease.

Topics: Anti-Ulcer Agents; Ascorbic Acid; Copper; Drug Combinations; Eicosanoids; Ferrous Compounds; Gastric Mucosa; Humans; In Vitro Techniques; Indomethacin; Intestinal Mucosa; Prostaglandins; Radioimmunoassay

Topics: Ascorbic Acid; Copper; Kinetics; Oxidation-Reduction; Thyroxine; Vitamins