ascorbic-acid and 1-10-phenanthroline

In this work, we propose a method that employed a smartphone to capture images obtained from a colorimetric spot test to determine ascorbic acid (AA) in Brazilian Amazon native and exotic fruits. The spot test reaction was based on reduction of Fe(III) by AA and further complexation with 1,10-phenanthroline. After optimization, the limit of detection was 8.5 × 10

Topics: Ascorbic Acid; Brazil; Food Analysis; Fruit; Iron; Limit of Detection; Phenanthrolines; Smartphone

The discrepancy between the in vitro cytotoxic results and the in vivo performance of Pt56MeSS prompted us to look into its interactions and those of its Pt

Topics: Ascorbic Acid; Coordination Complexes; Cyclohexylamines; Drug Stability; Electrochemical Techniques; Humans; Magnetic Resonance Spectroscopy; Oxidation-Reduction; Phenanthrolines; Platinum; Prohibitins

Phytic acid is the major storage form of organic phosphorus in nature- and plant-based animal feed. It forms insoluble complexes with nutritionally important metals and proteins that are unavailable for monogastric or agastric animals. Phytases initiate the stepwise hydrolysis of phytic acid and release inorganic orthophosphate. In the present investigation, the phytase gene from a phytase producing Bacillus licheniformis strain PB-13 was successfully expressed in Escherichia coli BL21. Recombinant phytase 'rPhyPB13' was found to be catalytically active, with an activity of 0.97 U/mL and specific activity of 0.77 U/mg. The rPhyPB13 was purified to 14.10-fold using affinity chromatography. Similar to other β-propeller phytases, purified rPhyPB13 exhibited maximal activity at pH 6.0-6.5 and 60 °C in the presence of 1 mM Ca(2+) and was highly active over a wider pH range (pH 4.0-8.0) and high temperature (80 °C). It has shown maximum activity towards Na-phytate as substrate. The observed K m , V max and k cat of purified rPhyPB13 were 1.064 mM, 1.32 μmol/min/mg and 27.46 s(-1), respectively. PhyPB13 was resistant to trypsin inactivation, activated in presence of Ca(2+) and inhibited in presence of EDTA. Crude rPhyPB13 has good digestion efficiency for commercial feed and soybean meal. These results indicate that PhyPB13 is a β-propeller phytase that has application potential in aquaculture feed.

Topics: 6-Phytase; Animal Feed; Aquaculture; Ascorbic Acid; Bacillus; Digestion; Edetic Acid; Enzyme Stability; Escherichia coli; Hydrogen-Ion Concentration; Kinetics; Metals; Phenanthrolines; Recombinant Proteins; Substrate Specificity; Temperature; Trypsin

Topics: Antineoplastic Agents; Ascorbic Acid; DNA Cleavage; DNA, Superhelical; Hydrogen Peroxide; Phenanthrolines; Phthalic Acids; Plasmids; Time Factors

Ergothioneine (2-mercaptohistidine trimethylbetaine) is a naturally occurring amino acid analogue found in up to millimolar concentrations in several tissues and biological fluids. However, the biological functions of ergothioneine remain incompletely understood. In this study, we investigated the role of ergothioneine in copper-induced oxidative damage to DNA and protein, using two copper-containing systems: Cu(II) with ascorbate and Cu(II) with H(2)O(2) [0.1 mM Cu(II), 1 mM ascorbate, and 1 mM H(2)O(2)]. Oxidative damage to DNA and bovine serum albumin was measured as strand breakage and protein carbonyl formation, respectively. Ergothioneine (0.1-1.0 mM) provided strong, dose-dependent protection against oxidation of DNA and protein in both copper-containing systems. In contrast, only limited protection was observed with the purported hydroxyl radical scavengers, dimethyl sulfoxide and mannitol, even at concentrations as high as 100 mM. Ergothioneine also significantly inhibited copper-catalyzed oxidation of ascorbate and competed effectively with histidine and 1,10-phenanthroline for binding of cuprous copper, but not cupric copper, as demonstrated by UV-visible and low-temperature electron spin resonance techniques. We conclude that ergothioneine is a potent, natural sulfur-containing antioxidant that prevents copper-dependent oxidative damage to biological macromolecules by forming a redox-inactive ergothioneine-copper complex.

Topics: Animals; Ascorbic Acid; Cattle; Coordination Complexes; Copper; Dimethyl Sulfoxide; DNA; DNA Damage; Electron Spin Resonance Spectroscopy; Ergothioneine; Free Radical Scavengers; Histidine; Hydrogen Peroxide; Mannitol; Oxidation-Reduction; Phenanthrolines; Protein Carbonylation; Serum Albumin, Bovine

A chemometric-assisted kinetic spectrophotometric method has been developed for simultaneous determination of ascorbic acid (AA), uric acid (UA), and dopamine (DA). This method relies on the difference in the kinetic rates of the reactions of analytes with a common oxidizing agent, tris(1,10-phenanthroline) and iron(III) complex (ferritin, [Fe(phen)(3)](3+)) at pH 4.4. The changes in absorbance were monitored spectrophotometrically. The data obtained from the experiments were processed by chemometric methods of artificial neural network (ANN) and partial least squares (PLS). Acceptable techniques of prediction set, randomization t test, cross-validation, and Y randomization were applied for the selection of the best chemometric method. The results showed that feedforward artificial neural network (FFANN) is more efficient than the other chemometric methods. The parameters affecting the experimental conditions were optimized, and it was found that under optimal conditions Beer's law is followed in the concentration ranges of 4.3-74.1, 4.3-78.3, and 2.0-33.0 μM for AA, UA, and DA, respectively. The proposed method was successfully applied to the determination of analytes in serum and urine samples.

Topics: Absorption; Ascorbic Acid; Calibration; Dopamine; Electrochemistry; Ferric Compounds; Humans; Hydrogen-Ion Concentration; Kinetics; Multivariate Analysis; Neural Networks, Computer; Phenanthrolines; Serum; Spectrophotometry; Uric Acid; Urine

Crystal structure analysis of the zinc complex establishes it as a distorted octahedral complex, bis(3-methylpicolinato-kappa(2) N,O)(2)(1,10-phenanthroline-kappa(2) N,N)-zinc(II) pentahydrate, [Zn(3-Me-pic)(2)(phen)]x5H(2)O. The trans-configuration of carbonyl oxygen atoms of the carboxylate moieties and orientation of the two planar picolinate ligands above and before the phen ligand plane seems to confer DNA sequence recognition to the complex. It cannot cleave DNA under hydrolytic condition but can slightly be activated by hydrogen peroxide or sodium ascorbate. Circular Dichroism and Fluorescence spectroscopic analysis of its interaction with various duplex polynucleotides reveals its binding mode as mainly intercalation. It shows distinct DNA sequence binding selectivity and the order of decreasing selectivity is ATAT > AATT > CGCG. Docking studies lead to the same conclusion on this sequence selectivity. It binds strongly with G-quadruplex with human tolemeric sequence 5'-AG(3)(T(2)AG(3))(3)-3', can inhibit topoisomerase I efficiently and is cytotoxic against MCF-7 cell line.

Topics: Antineoplastic Agents; Ascorbic Acid; Binding Sites; Cell Line, Tumor; Cell Proliferation; Computer Simulation; Crystallography, X-Ray; DNA Cleavage; DNA Topoisomerases, Type I; DNA, Superhelical; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Humans; Hydrogen Peroxide; Models, Chemical; Models, Molecular; Organometallic Compounds; Phenanthrolines; Picolinic Acids; Plasmids; Topoisomerase I Inhibitors; Zinc

The ability of the mycotoxin citrinin to act as an inhibitor of iron-induced lipoperoxidation of biological membranes prompted us to determine whether it could act as an iron chelating agent, interfering with iron redox reactions or acting as a free radical scavenger. The addition of Fe3+ to citrinin rapidly produced a chromogen, indicating the formation of citrinin-Fe3+ complexes. An EPR study confirms that citrinin acts as a ligand of Fe3+, the complexation depending on the [Fe3+]:[citrinin] ratios. Effects of citrinin on the iron redox cycle were evaluated by oxygen consumption or the o-phenanthroline test. No effect on EDTA-Fe2+-->EDTA-Fe3+ oxidation was observed in the presence of citrinin, but the mycotoxin inhibited, in a dose-dependent manner, the oxidation of Fe2+ to Fe3+ by hydrogen peroxide. Reducing agents such as ascorbic acid and DTT reduced the Fe3+-citrinin complex, but DTT did not cause reduction of Fe3+-EDTA, indicating that the redox potentials of Fe3+-citrinin and Fe3+-EDTA are not the same. The Fe2+ formed from the reduction of Fe3+-citrinin by reducing agents was not rapidly reoxidized to Fe3+ by atmospheric oxygen. Citrinin has no radical scavenger ability as demonstrated by the absence of DPPH reduction. However, a reaction between citrinin and hydrogen peroxide was observed by UV spectrum changes of citrinin after incubation with hydrogen peroxide. It was also observed that citrinin did not induce direct or reductive mobilization of iron from ferritin. These results indicate that the protective effect on iron-induced lipid peroxidation by citrinin occurs due to the formation of a redox inactive Fe3+-citrinin complex, as well as from the reaction of citrinin and hydrogen peroxide.

Topics: Ascorbic Acid; Citrinin; Dithiothreitol; Dose-Response Relationship, Drug; Drug Interactions; Edetic Acid; Electron Spin Resonance Spectroscopy; Ferric Compounds; Ferrous Compounds; Ferrozine; Free Radicals; Hydrogen Peroxide; Iron Chelating Agents; Kinetics; Ligands; Lipid Peroxidation; Oxidation-Reduction; Oxygen; Phenanthrolines; Spectrophotometry, Ultraviolet

Kojic acid, a fungal metabolic product, has been used as a skin-depigmenting agent in skin care products marketed in Japan. Iron in the skin is known to be involved in wrinkling as a result of chronic photodamage. Kojic acid was expected to have anti-wrinkling activity, since it possesses iron-chelating activity. We now evaluated the anti-wrinkling activity of kojic acid by using hairless mice exposed to chronic solar-simulating ultraviolet (UV) irradiation as model animal. At the end of a 20-week irradiation period, topical application of kojic acid before UV irradiation was observed to dramatically prevent: (1) the wrinkling, (2) hyperplasia of the epidermis, (3) fibrosis of the lower dermis, and (4) the increase of extracellular matrix components in the upper dermis. These findings indicate that kojic acid is a typical agent preventing wrinkling of the skin due to chronic photodamage.

Topics: Animals; Ascorbic Acid; Dose-Response Relationship, Drug; Edetic Acid; Female; Iron Chelating Agents; Lipid Peroxidation; Malondialdehyde; Mice; Oxidation-Reduction; Oximes; Phenanthrolines; Pyrones; Skin; Skin Aging; Sunscreening Agents; Ultraviolet Rays

Reactive oxygen species (ROS) are implicated in both cartilage aging and the pathogenesis of osteoarthritis. We developed an in vitro model to study the role of chondrocyte-derived ROS in cartilage matrix protein degradation. Matrix proteins in cultured primary articular chondrocytes were labeled with [(3)H]proline, and the washed cell matrix was returned to a serum-free balanced salt solution. Exposure to hydrogen peroxide resulted in oxidative damage to the cell matrix as established by monitoring the release of labeled material into the medium. Calcium ionophore treatment of chondrocytes, in a dose-dependent manner, significantly enhanced the release of labeled matrix, suggesting a chondrocyte-dependent mechanism of matrix degradation. Antioxidant enzymes such as catalase or superoxide dismutase did not influence matrix release by the calcium ionophore-activated chondrocytes. However, vitamin E, at physiological concentrations, significantly diminished the release of labeled matrix by activated chondrocytes. The fact that vitamin E is a chain-breaking antioxidant indicates that the mechanism of matrix degradation and release is mediated by the lipid peroxidation process. Lipid peroxidation was measured in chondrocytes loaded with cis-parinaric acid. Both resting and activated cells showed constitutive and enhanced levels of lipid peroxidation activity, which were significantly reduced in the presence of vitamin E. In an immunoblot analysis, malondialdehyde and hydroxynonenal adducts were observed in chondrocyte-matrix extracts, and the amount of adducts increased with calcium ionophore treatment. Furthermore, vitamin E diminished aldehyde-protein adduct formation in activated extracts, which suggests that vitamin E has an antioxidant role in preventing protein oxidation. This study provides in vitro evidence linking chondrocyte lipid peroxidation to cartilage matrix protein (collagen) oxidation and degradation and suggests that vitamin E has a preventive role. These observations indicate that chondrocyte lipid peroxidation may have a role in the pathogenesis of cartilage aging and osteoarthritis.

Topics: Aldehydes; Animals; Ascorbic Acid; Cartilage; Cells, Cultured; Chondrocytes; Collagen; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix Proteins; Glycoproteins; Immunoblotting; Ionophores; Lipid Peroxidation; Lipopolysaccharides; Matrilin Proteins; Metalloendopeptidases; Nitric Oxide; Osteoarthritis; Phagocytosis; Phenanthrolines; Protease Inhibitors; Rabbits; Reactive Oxygen Species; Time Factors; Vitamin E

Copper in the presence of excess 1,10-phenanthroline, a reducing agent, and H2O2 causes DNA base damage as well as strand breakage. We have reported in previous work that a strong chemiluminescence was followed by DNA base damage in this system, which is characteristic of guanine. In the present work, the mechanism of the chemiluminescence was studied. Results show that the luminescence was inhibited by all three classes of reactive oxygen species (*OH, O2-, (1)O2) scavengers to different degrees. Singlet oxygen scavengers showed the most powerful inhibition while the other two classes of scavengers were relatively weaker. The emission intensity in D2O was 3-fold that in H2O. Comparing the effect of scavengers on the luminescence of DNA with that of dGMP, the ratio of inhibition was similar. On the other hand, DNA breakage analysis showed that inhibition by the singlet oxygen scavenger NaN3 of strand breakage was strong and comparable to that of the scavengers of the two oxygen radicals. The results suggest that singlet oxygen may be a major factor for the chemiluminescence of guanine, while DNA strand breakage may be caused by many active species.

Topics: Ascorbic Acid; DNA Damage; Free Radical Scavengers; Hydrogen Peroxide; Hydroxyl Radical; Luminescent Measurements; Oxygen; Phenanthrolines

C6 glioma cells treated with 10 mM glutamate reduced intracellular GSH to one-seventh of the initial level, and induced cytolysis accompanied by apoptosis. The treated cells produced extracellular H2O2. The cytolysis of the C6 cells induced by glutamate was prevented by antioxidants such as N-acetylcysteine (NAC), ascorbic acid (ASC), catalase, and NaN3, iron chelators such as deferoxamine and 1,10-phenanthroline, and oxygen radical scavengers such as 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) and alpha-phenyl-tert-butyl nitrone (PBN). The effect of these antioxidants, iron chelators, and oxygen radical scavengers on the cytolysis of C6 cells was dependent on the dose and the intracellular GSH level. Furthermore, 1-2 Mbp chromosomal DNA (giant DNA) fragments were observed during cytolysis. The giant DNA fragments were further cleaved into smaller DNA fragments of 200-800 kbp, and then to fragments of less than 300 kbp in size including chromosomal ladder DNA fragments. Such serial chromosomal DNA degradations induced by glutamate were also inhibited by addition of these antioxidants, iron chelators, and oxygen radical scavengers. These findings suggest that glutamate induces GSH depletion, and consequently, apoptosis through endogenously produced active oxygen species in C6 glioma cells and that the apoptosis is accompanied by 1-2 Mbp giant DNA fragmentation prior to the internucleosomal DNA fragmentation.

Topics: Acetylcysteine; Animals; Antioxidants; Apoptosis; Ascorbic Acid; Catalase; Chromosomes; Deferoxamine; DNA Fragmentation; Free Radical Scavengers; Glioma; Glutamic Acid; Glutathione; Hydrogen Peroxide; Iron Chelating Agents; Oxidation-Reduction; Oxygen; Phenanthrolines; Rats; Tumor Cells, Cultured

The model quinone compound menadione has been used to study the effects of oxidative stress in mammalian cells, and to investigate the mechanism of action of the quinone nucleus which is present in many anti-cancer drugs. We have used the alkaline single cell gel electrophoresis assay (comet assay) to investigate the effects of low doses of this compound on isolated human lymphocytes. We found that concentrations of menadione as low as 1 microM were sufficient to induce strand breaks in these cells. Pre-incubation with the NAD(P)H quinone oxidoreductase inhibitor dicoumarol, enhanced the production of menadione-induced strand breaks. In contrast, the metal ion chelator 1,10-phenanthroline inhibited formation of strand breaks, although prolonged incubation with 1,10-phenanthroline in combination with menadione resulted in an increase in a population of very severely damaged nuclei. A marked variation in the response of lymphocytes from different donors to menadione, and in different samples from the same donor was also observed.

Topics: Antioxidants; Ascorbic Acid; Cell Survival; Chelating Agents; Dicumarol; Dimethyl Sulfoxide; DNA Damage; DNA Fragmentation; Electrophoresis, Agar Gel; Enzyme Inhibitors; Humans; Lymphocytes; Male; NAD(P)H Dehydrogenase (Quinone); Oxidative Stress; Phenanthrolines; Salicylates; Salicylic Acid; Vitamin K

A new method was developed that reduces the intracellular iron content of cells grown in serum-containing culture without involving the significant uptake of iron-chelating agents into cells. Negatively charged bathophenanthrolinedisulfonate (BPS), together with ascorbate, caused cells to lose much of their cellular iron without causing much depression in HL-60 or H9c2 (2-1) cell proliferation over a 48-h period. When added to serum supplemented RPMI-1640 culture media, BPS and ascorbate efficiently reduced and competed for iron in Fe(III) transferrin to form Fe(II)(BPS)3. The reaction also occurred with purified human iron-transferrin. When cells were incubated with growth medium containing serum that had been treated with BPS and ascorbate for 24 h, little or no BPS2- or Fe(II)(BPS)(4-)3 entered the cells, according to direct measurements and in agreement with the highly unfavorable 1-octanol/water partition coefficients for these molecules. However, iron was mobilized out of both cell types. After 24 h incubation of cells in this medium, there was no change in the activities of catalase and superoxide dismutase, or in the concentration of glutathione. Glutathione peroxidase was elevated 9%. Using HL-60 and H9c2 (2-1) cells made iron deficient with BPS and ascorbate, HL-60 cells grown in defined-growth media in the absence of iron-pyridoxal isonicotinoyl hydrazone, or Euglena gracilis cells maintained in a defined medium that was rigorously depleted of iron, it was shown that the cytotoxicity of adriamycin is markedly dependent on the presence of iron in each type of cell. Similar results were obtained when HL-60 cells were grown in RPMI-1640 culture medium and serum that had been incubated for 24 h in BPS and ascorbate and then chromatographed over a Bio-Rad desalting column to remove small molecules including BPS, ascorbate, and Fe(II)(BPS)3.

Topics: Animals; Ascorbic Acid; Catalase; Cell Survival; Cells, Cultured; Chelating Agents; Doxorubicin; Euglena gracilis; Glutathione; Glutathione Disulfide; Glutathione Peroxidase; HL-60 Cells; Humans; Iron; Iron Chelating Agents; Kinetics; Myocardium; Phenanthrolines; Rats; Superoxide Dismutase

Some of the properties of cellular iron species which react with H2O2 to cause DNA single-strand breaks in HL-60 cells were characterized in control cells and in cells made deficient of iron using 4,7-phenylsulfonyl-1,10-phenanthroline (bathophenanthroline disulfonic acid or BPS) and ascorbate. Single-strand breaks were measured using alkaline elution of DNA of cells treated at 4 degrees to minimize repair during treatment. Strand breakage in the presence of 10% serum was only 40% of that in the absence of serum. This effect was traced to reaction of H2O2 with metals, most likely iron, in serum. Dimethyl sulfoxide (Me2SO) inhibited a maximum of 65% of breaks in control cells. The diffusion distance from the site of generation of hydroxyl radicals to the site of reaction with DNA for the Me2SO-inhibitable fraction was 6.9 nm. There was no significant alteration in the fraction of Me2SO-inhibitable strand breaks or in diffusion distance in iron-deficient cells, though total strand breaks decreased by 70%. When the effect of extracellular iron in serum was taken into account, 60 microM orthophenanthroline (OP) inhibited a maximum of 85% of strand breaks. In cells pretreated with 60 microM OP, the Me2SO-inhibitable fraction of the remaining strand breaks decreased to 32%, while the diffusion distance decreased to 4.1 nm. These data indicate the existence of a number of different iron species, as characterized by overlapping but not coincidental inhibition by OP and Me2SO, and by differing diffusion distances.

Topics: Ascorbic Acid; Chelating Agents; Dimethyl Sulfoxide; DNA Damage; DNA, Neoplasm; DNA, Single-Stranded; Dose-Response Relationship, Drug; HL-60 Cells; Humans; Hydrogen Peroxide; Hydroxyl Radical; Iron; Iron Chelating Agents; Kinetics; Phenanthrolines

Sorbitol formation in rat lenses incubated with high levels of glucose was related to activation of aldose reductase (AR). The hyperglycaemia-activated aldose reductase was inhibited by alpha-lipoic (thioctic) acid, O-phenanthroline and aldose reductase inhibitors (ARIs) including Zeopolastat (ZPLS), Sorbinil (SBN) and AL-1576. This study also examined ARIs for the ability to chelate metal ions. We found that ARIs suppress copper-dependent ascorbate oxidation, lipid peroxidation and hydrogen peroxide production in erythrocytes. ARIs also increased partition of copper ions into noctanol, which indicates formation of lipophilic complexes. Our data support the hypothesis that transition metals may be involved in activation of the polyol (aldose reductase) pathway. Also, ARIs function as metal-chelating antioxidants that may contribute to their therapeutic role for diabetic complications.

Topics: 1-Octanol; Aldehyde Reductase; Animals; Ascorbic Acid; Chelating Agents; Copper; Enzyme Activation; Enzyme Inhibitors; Erythrocytes; Fluorenes; Glucose; Humans; Hydantoins; Hydrogen Peroxide; Hyperglycemia; Imidazoles; Imidazolidines; In Vitro Techniques; Ions; Lens, Crystalline; Lipid Peroxidation; NADP; Octanols; Oxidation-Reduction; Phenanthrolines; Rats; Thioctic Acid

Topics: Animals; Ascorbic Acid; Cattle; DNA; DNA Damage; Enzyme-Linked Immunosorbent Assay; Hydrogen Peroxide; Immunochemistry; In Vitro Techniques; Phenanthrolines; Reactive Oxygen Species; Ultraviolet Rays

Topics: Ascorbic Acid; Bleomycin; Catalase; Chromatography, High Pressure Liquid; Copper; DNA; DNA Damage; Intercalating Agents; Iron; Magnetic Resonance Spectroscopy; Molecular Structure; Phenanthrolines; Sensitivity and Specificity; Spectrometry, Fluorescence

In many peptide hormones and neuropeptides, the carboxyl-terminal alpha-amide structure is essential in eliciting their biological activity. In the present study, an enzymatic activity capable of converting 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly(Dabsyl-Gly-Phe -Gly) to 4-dimethylaminoazo-benzene-4'-sulfonyl-Gly-L-Phe-NH2(Dabsyl- Gly-Phe-NH2) was investigated in bovine hypothalamus. The concentrations of copper ion and ascorbic acid required for maximal enzyme activity were 16 microM and 2 mM, respectively. Amidating activity showed a pH profile with two pH optima at acidic pH (around 6.0) and neutral pH (around 7.5). Kinetic studies with the enzyme obtained from bovine hypothalamus demonstrated two distinct Km and Vmax values. The first Km and Vmax values were 142.9 microM and 22.2 pmol/microgram/h and the second Km and Vmax values were 22.7 microM and 4.44 pmol/microgram/h, respectively. Two molecular forms of amidating activity were identified by size-exclusion chromatography and the molecular weight of the two enzymes were estimated to be 49 kDa and 69 kDa.

Topics: Amino Acid Sequence; Animals; Ascorbic Acid; Cattle; Chromatography, Gel; Copper; Copper Sulfate; Edetic Acid; Hydrogen-Ion Concentration; Hypothalamus; Kinetics; Mixed Function Oxygenases; Molecular Sequence Data; Molecular Weight; Multienzyme Complexes; Phenanthrolines

To elucidate the mechanism of cytotoxicity of H2O2, we selected H2O2-resistant Chinese hamster V79 cells by single-step selection from a pool of spontaneous variants. The resistant cells showed bimodal sensitivity to H2O2 without exhibiting a significantly higher level of the detoxicating enzymes, catalase, glutathione peroxidase and superoxide dismutase. Mode-one and mode-two killing were observed at lower (< 300 microM) and higher (> 2 mM) H2O2 concentrations, respectively. Mode-one but not mode-two killing was prevented by iron chelators. Pretreatment with low concentrations of ascorbic acid preferentially enhanced the killing at higher H2O2 concentrations. These resistant cells were cross-resistant to t-butyl hydroperoxide and cumene hydroperoxide.

Topics: Animals; Ascorbic Acid; Benzene Derivatives; Catalase; Cell Line; Cell Survival; Chelating Agents; Clone Cells; Cricetinae; Cricetulus; Deferoxamine; Dose-Response Relationship, Drug; Drug Resistance; Glucosephosphate Dehydrogenase; Glutathione Peroxidase; Hydrogen Peroxide; Kinetics; Peroxides; Phenanthrolines; Reactive Oxygen Species; Superoxide Dismutase; tert-Butylhydroperoxide

We introduced eight bases, including four base analogs, into 15-mer triplex-forming oligonucleotides (TFOs) [d-psTTTCTTTNTTTTCTT; ps = thiophosphate; N = A, G, C, T, 2'-deoxyinosine (I), 2'-deoxyxanthosine (X), 5-methyl-2'-deoxycytidine (m5C), or 5-bromo-2'-deoxyuridine(br5U)] to investigate the Hoogsteen-like hydrogen bonding to the base in the target 34-mer strand (d-TGAGTGAGTAAAGAAARAAAAGAATGAGTGCCAA.d-TTGGCACTCATTCTTTTYTTTCT TTACTCACTCA; RY = AT, GC, TA, or CG). We examined the thermal stability of 15-mer triplexes in buffer containing 100 mM sodium acetate and 1 M NaCl at pH 5.0. The triplexes with typical triplets of T.AT (51.3 degrees C), br5U.AT (52.4 degrees C), C+.GC (66.7 degrees C), and m5C+.GC (66.8 degrees C) at the central position showed relatively higher Tm values, as expected. The relatively high stability of the X.AT triplex (39.8 degrees C) was observed. Among the N.TA triplets, G.TA (44.8 degrees C) was thermally the most stable, and moreover, the data showed that the N.TA triplet was also stabilized by I in the N position (40.7 degrees C). Furthermore, the TFOs were converted to DNA-cleaving molecules by introducing a newly synthesized 1,10-phenanthroline (OP) derivative on the thiophosphate group at the 5' end. Cleavage reactions of the 32P-labeled DNA (34-mer) were carried out. The cleavage efficiencies were compared to the Tm values of triplexes with or without an OP derivative. Results showed that the increased cleavage yields reflect the higher thermal stability of the triplex formed in most cases, but a few exceptional cases existed. Especially, the G-containing TFO did not show the above correlation between thermal stability and cleavage yield.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3-Mercaptopropionic Acid; Ascorbic Acid; Base Sequence; Binding Sites; Chelating Agents; Copper; Cysteine; Dithiothreitol; DNA; Drug Stability; Glutathione; Hot Temperature; Hydrogen Bonding; Molecular Sequence Data; Molecular Structure; Nucleic Acid Conformation; Nucleic Acid Denaturation; Oligodeoxyribonucleotides; Phenanthrolines

The exposure of DNA to H2O2 in the presence of Cu(II) and a reducing agent is known to result in the induction of a variety of oxidative lesions, including DNA strand breaks and base modifications. Since the reducing agent glutathione occurs in cell nuclei at relatively high concentrations, and copper exists in nuclei associated with chromatin, the present study was undertaken to evaluate the ability of GSH to promote copper-mediated free radical damage to DNA. When compared with ascorbate, GSH was found to be inefficient in the promotion of damage to DNA. Parallel ESR spin trapping measurements indicated that GSH inhibits free radical formation by copper ions in the presence of H2O2, ascorbate, and DNA. The protective effect of GSH is attributed to its stabilization of copper in the +1 oxidation state, thereby compromising its ability to participate in free radical generating reactions. Consequently, it is suggested that the GSH in cell nuclei serves to prevent, rather than promote, copper-dependent damage to DNA. In contrast, in the presence of 1,10-phenanthroline, GSH stimulated free radical formation and DNA damage. This is attributed to the failure of GSH to remove copper(I) from 1,10-phenanthroline. Therefore, under these conditions, GSH serves primarily to redox cycle the reactive 1,10-phenanthroline-copper complex.

Topics: Ascorbic Acid; Catalase; Chelating Agents; Copper; DNA; DNA Damage; Glutathione; In Vitro Techniques; Oxidation-Reduction; Phenanthrolines

Structural luteolysis was found decades ago to be induced by PRL in the hypophysectomized rat, but the mechanisms of this process are unknown. To gain information on mechanisms of luteal involution, we developed an animal model that circumvented complex surgery and provided ample tissue for analyses. Gonadotropin-synchronized ovulation and luteinization were induced in immature rats, followed by treatment with ergot alkaloid and PRL. PRL-induced structural luteolysis, as shown by loss of luteal weight, protein, and DNA after pretreatment with ergot alkaloid, was evident after 36 h. Ascorbic acid depletion was rapid, severe, and lasting in luteal tissue during structural luteolysis, but lipid peroxidation or depletion of vitamin E was not evident. PRL treatment of animals with functional corpora lutea did not induce luteal involution. Significantly, after natural functional luteolysis occurred, PRL was highly effective in inducing structural luteolysis. Thus, either natural or ergot-induced functional luteolysis permitted the luteolytic expression of PRL. A greater depletion of protein than DNA was seen during PRL-induced structural luteolysis and was associated with a significant increase in neutral caseinase activity in luteal extracts. Caseinase activity was markedly reduced by calcium chelators and profoundly inhibited by the chelator orthophenanthroline; only slightly reduced activity was seen with serine, aspartate, or cysteine proteinase inhibitors. These findings implicate metalloproteinase (MMP) as the relevant caseinase that was increased during structural luteolysis. The major proteinase identified by zymography had apparent sizes of 72 and 66 kilodaltons (kDa), and slight but detectable activity was also seen at 92 and 84 kDa. Organomercurial treatment caused a major shift of the 72-kDa band to 66 kDa and the 92-kDa band to 84 kDa, confirming MMP-2 and MMP-9 by activation of latent activity of each MMP, respectively. Structural luteolysis caused a significant increase in the activated 66-kDa form and the latent 72-kDa form of MMP-2, which occurred before a loss of luteal weight or protein. As MMP-2 degrades collagen (type IV) in basement membranes, we conclude that an early event in PRL-induced structural luteolysis is the degradation of extracellular matrix. This conclusion is further emphasized by the marked and lasting depletion of ascorbic acid, a vitamin long known to serve an essential role in collagen synthesis.

Topics: Animals; Ascorbic Acid; Caseins; Corpus Luteum; DNA; Edetic Acid; Egtazic Acid; Enzyme Activation; Enzyme Induction; Ergot Alkaloids; Female; Immunosuppressive Agents; Kinetics; Metalloendopeptidases; Molecular Weight; Phenanthrolines; Prolactin; Proteins; Rats; Rats, Sprague-Dawley

Normal human plasma does not contain low molecular mass iron because the iron-binding protein transferrin retains a considerable iron-binding capacity. In conditions of iron-overload, however, low molecular mass iron can be detected in plasma. Plasma contains several molecules capable of reducing ferric complexes to the ferrous state and this could lead to oxidative damage through reactions dependent on Fenton chemistry and lipid peroxidation. It seems likely that ascorbate and urate would reduce ferric complexes present in plasma during iron-overload. However, the plasma, 'ferroxidase' protein caeruloplasmin protects the extracellular environment by catalytically oxidising ferrous complexes back to the less reactive ferric state.

Topics: 2,2'-Dipyridyl; Ascorbic Acid; Ceruloplasmin; Ferric Compounds; Ferrozine; Humans; Iron; Molecular Weight; Oxidation-Reduction; Phenanthrolines; Uric Acid

Copper in the presence of excess 1,10-phenanthroline, a reducing agent, and molecular oxygen causes cleavage of DNA with a preference for T-3',5'-A-steps, particularly in TAT triplets. The active molecular species is commonly thought to be the bis-(1,10-phenanthroline)Cu(I) complex, (Phen)2Cu(I), regardless of the reducing agent type. We have found that (Phen)2Cu(I) is not the predominant copper complex when 3-mercaptopropionic acid (MPA) or 2-mercaptoethanol are used as the reducing agents, but (Phen)2Cu(I) predominates when ascorbate is used as the reducing agent. Substitution of ascorbate for thiol significantly enhances the rate of DNA cleavage by 1,10-phenanthroline + copper, without altering the sequence selectivity. We show that (Phen)2Cu(I) is the complex responsible for DNA cleavage, regardless of reducing agent, and that 1,10-phenanthroline and MPA compete for copper coordination sites. DNA cleavage in the presence of ascorbate also occurs under conditions where the mono-(1,10-phenanthroline)Cu(I) complex predominates (1:1 phenanthroline:copper ratio), but preferential cleavage was observed at a CCGG sequence and not at TAT sequences. The second phenanthroline ring of the (Phen)2Cu(I) complex appears essential for determining the T-3',5'-A sequence preferences of phenanthroline + copper when phenanthroline is in excess.

Topics: 3-Mercaptopropionic Acid; Ascorbic Acid; Base Sequence; Copper; DNA; Kinetics; Mercaptoethanol; Molecular Sequence Data; Phenanthrolines; Spectrum Analysis

Incubation of rat-liver microsomes, previously azide-treated to inhibit catalase, with H2O2 caused a loss of cytochrome P-450 but not of cytochrome b5. This loss of P-450 was not prevented by scavengers of hydroxyl radical, chain-breaking antioxidants or metal ion-chelating agents. Application of the thiobarbituric acid (TBA) assay to the reaction mixture suggested that H2O2 induces lipid peroxidation, but this was found to be due largely or completely to an effect of H2O2 on the TBA assay. By contrast, addition of ascorbic acid and Fe(III) to the microsomes led to lipid peroxidation and P-450 degradation: both processes were inhibited by chelating agents and chain-breaking antioxidants, but not by hydroxyl radical scavengers. H2O2 inhibited ascorbate/Fe(III)-induced microsomal lipid peroxidation, but part of this effect was dues to an action of H2O2 in the TBA test itself. H2O2 also decreased the colour measured after carrying out the TBA test upon authentic malondialdehyde, tetraethoxypropane, a DNA-Cu2+/o-phenanthroline system in the presence of a reducing agent, ox-brain phospholipid liposomes in the presence of Fe(III) and ascorbate, or a bleomycin-ion iron/DNA/ascorbate system. Caution must be used in interpreting the results of TBA tests upon systems containing H2O2.

Topics: Animals; Ascorbic Acid; Bleomycin; Chlorides; Cytochrome P-450 Enzyme Inhibitors; Deferoxamine; DNA; DNA Damage; Edetic Acid; Ferric Compounds; Hydrogen Peroxide; Lipid Peroxidation; Liposomes; Male; Microsomes, Liver; Phenanthrolines; Rats; Thiobarbiturates

Na(+)-dependent Ca2+ uptake in rat brain microsomal membrane vesicles was inhibited by preincubating the vesicles with ascorbic acid at 0.1-10 mM. The inhibitory effect of ascorbate was blocked by simultaneous addition of ascorbate oxidase. The decrease in activity was not reversed upon removing the ascorbate. The kinetic study showed that the treatment with ascorbate decreased Bmax without a change in Km for Ca2+. The inhibitory effect by ascorbate was also observed in membrane vesicles derived from osmotically shocked synaptosomes and in reconstituted membrane vesicles. The effect by ascorbate was specific: it did not affect either ATP-dependent Ca2+ uptake in the presence of o-phenanthroline, an inhibitor of lipid peroxidation, or Na(+)-dependent glutamate uptake in the membrane vesicles. The activity of Na(+)-Ca2+ exchange was also decreased by isoascorbic acid, but not by ascorbate 2-sulfate at 1 mM. The treatment with glutathione or 2-mercaptoethanol did not affect the Na(+)-Ca2+ exchange activity, while 1 mM dithiothreitol caused the inhibition which was completely blocked by o-phenanthroline. The effect of ascorbate on Na(+)-dependent Ca2+ uptake was observed even under the conditions which suppress peroxidation of membrane phospholipids.

Topics: Animals; Ascorbate Oxidase; Ascorbic Acid; Brain; Calcium; Dithiothreitol; Glutathione; In Vitro Techniques; Intracellular Membranes; Mercaptoethanol; Microsomes; Phenanthrolines; Rats; Rats, Inbred Strains; Sodium; Synaptosomes

Pea seed ferritin is able to incorporate ferrous iron into the mineral core. Fe2+ may be formed by reduction of exogenous Fe3+ with ascorbate or by photoreduction by ferritin and by ferric citrate. In our experimental conditions the bulk of the photoreduction is carried out by ferritin, which is able to photoreduce its endogenous iron. Citrate does not enhance the photoreduction capacity of ferritin, and exogenous ferric citrate improves the yield of the reaction by about 30%. The mineral core of the ferritin is shown to photoreduce actively, and the protein shell does not participate directly in the photoreduction. Low light intensities and low concentration of reducing agents do not allow a release of iron from ferritins, but induce a 'redox mill' of photoreduction and simultaneous ferroxidase-mediated incorporation. High ascorbate concentrations induce the release of ferritin iron. These reactions are accompanied by the correlated occurrence of damage caused by radicals arising from Fenton reactions, leading to specific cleavages in the 28 kDa phytoferritin subunit. This damage caused by radicals occurs during the oxidative incorporation into the mineral core and is prevented by o-phenanthroline or by keeping the samples in the dark.

Topics: Ascorbic Acid; Chelating Agents; Citrates; Citric Acid; Fabaceae; Ferric Compounds; Ferritins; Ferrous Compounds; Free Radicals; Iron; Iron Radioisotopes; Oxidation-Reduction; Oxygen; Phenanthrolines; Photochemistry; Plants, Medicinal; Seeds

The characteristics of the photocurrent response activated by continuous illumination of planar bilayer membranes containing bacterial reaction centers have been resolved by voltage clamp methods. The photocurrent response to a long light pulse consists of an initial spike arising from the fast, quasi-synchronous electron transfer from the reaction center bacteriochlorophyll dimer, BChl2, to the primary quinone QA. This is followed by a slow relaxation of the current to that promoted by secondary, asynchronous multiple electron transfers from the reduced cytochrome c through the reaction centers to the ubiquinone-10 pool. Currents derived from cytochrome c oxidation that occurs when cytochrome c is associated with the reaction center or when limited by diffusional interaction from solution are recognized. Changes of the ionic strength and pH in the aqueous phase, and the clamped membrane potential (+/- 150 mV), affect the electron-transfer rate between cytochrome c and BChl2. In contrast, the primary light-induced charge separation between BChl2 and QA, or electron transfer between QA on the ubiquinone pool are unaffected. During illumination of reaction center membranes supplemented with cytochrome c and a ubiquinone pool, there is a small but significant steady-state current which is considered to be caused by the re-oxidation of photoreduced quinone by molecular oxygen. In the dark, after illumination of reaction centers supplemented with cytochrome c and a ubiquinone pool, there is a small amount of reverse current resulting from the movement of charges back across the membrane. This reverse current is observed maximally after 400 ms illumination while prolonged illumination diminishes the effect. The source of this current is uncertain, but it is considered to be due to the flux of anionic semiquinone within the membrane profile; this may also be the species that interacts with oxygen giving rise to the steady-state current. It is postulated that when the reaction centers are contained in an alkane-containing phospholipid membrane, in contrast to the in vivo situation, the semiquinone anion formed in the QB site is not tightly bound to the site and can, by exchange-diffusion with the membrane-quinone pool, move away from the site and accumulate in the membrane. However, in the absence, more quantitative work superoxide anion, resulting from O2 interaction with semiquinone of QA, QB or pool cannot be excluded.

Topics: Ascorbic Acid; Bacterial Proteins; Cytochrome c Group; Electric Stimulation; Electron Transport; Ferricyanides; Hydrogen-Ion Concentration; Light-Harvesting Protein Complexes; Lipid Bilayers; Membrane Potentials; Osmolar Concentration; Oxidation-Reduction; Phenanthrolines; Photic Stimulation; Photochemistry; Photosynthetic Reaction Center Complex Proteins; Ubiquinone

The asorbic acid (AH-) auto-oxidation rates catalyzed by copper chelates of 1,10-phenanthroline (OP) or by iron chelates of bleomycin (BLM) are only slightly higher than the oxidation rates catalyzed by the metal ions. AH- oxidation in the presence of DNA is accompanied by degradation of the DNA. The rates of DNA scission by the metal chelates are markedly higher than the rates induced by the free metal ions. AH- oxidation is slowed down in the presence of DNA which forms ternary complexes with the chelates. The ternary complexes react slowly with AH- but induce DNA double strand breaks more efficiently than the free metal chelates. With OP, DNA is degraded by the reaction of the ternary complex, DNA-(OP)2Cu(I), with H2O2. AH- oxidation in the presence of DNA was biphasic, showing a marked rate increase after DNA was cleaved. We suggest that this sigmoidal pattern of the oxidation curves reflects the low initial oxidative activity of the ternary complexes, accelerating as DNA is degraded. Using O2- produced by pulse radiolysis as a reductant, we found that AH- oxidation with (OP)2Cu(II) induced more DNA double strand breaks per single strand break than bipyridine-copper. The site specific DNA damaging reactions indicated by these results are relevant to the mechanism of cytotoxic activities of bleomycin and similar antibiotics or cytotoxic agents.

Topics: Ascorbic Acid; Bleomycin; Catalysis; Copper; DNA; DNA Damage; Hydrogen Peroxide; Iron Chelating Agents; Kinetics; Osmolar Concentration; Oxidation-Reduction; Phenanthrolines; Spectrophotometry, Ultraviolet; Superoxides

The DNA-cleavage specificity of ascorbate in the presence of copper ion is analyzed with end-labeled pBR322 DNA fragments. The nonenzymatic reaction of Cu(II)/ascorbate and DNA shows certain degrees of cleavage preference toward purine-containing short segments in the labeled DNA under mild conditions (at 0 degrees C and 10 min). The segments of pyrimidine clusters are least susceptible to cleavage. The DNA scission cannot be detected in the absence of metal ions, and is greatly diminished in the presence of EDTA and metal-chelating peptide. It is more specific than the nuclease-like scission activity induced by cuprous-phenanthroline complex. This scission activity in relation to the antiviral and antitumor activities of vitamin C reported in the literature deserves a crucial consideration.

Topics: Ascorbic Acid; Base Sequence; Chelating Agents; Copper; DNA, Bacterial; Edetic Acid; Oligopeptides; Oxidation-Reduction; Phenanthrolines; Plasmids