ascorbic-acid and bathophenanthroline-disulfonic-acid

While the naturally occurring reducing agents glutathione (GSH) and ascorbate (H2A) alone are ineffective at reducing iron(III) sequestered by the siderophore ferrioxamine B, the addition of an iron(II) chelator, sulfonated bathophenanthroline (BPDS), facilitates reduction by either reducing agent. A mechanism is described in which a ternary complex is formed between ferrioxamine B and BPDS in a rapidly established pre-equilibrium step, which is followed by rate limiting reduction of the ternary complex by glutathione or ascorbate. Spectral, thermodynamic, and kinetic evidence are given for ternary complex formation. Ascorbate was found to be slightly more efficient at reducing the ternary complex than glutathione (k4=2.1 x 10(-3) M(-1) s(-1) and k4=6.3 x 10(-4) M(-1) s(-1), respectively) at pH 7. Reduction is followed by a rapid ligand exchange step where iron is released from ferrioxamine B to form tris-(BPDS)iron(II). The implications of these results for siderophore mediated iron transport and release are discussed.

Topics: Algorithms; Ascorbic Acid; Deferoxamine; Ferric Compounds; Glutathione; Iron; Iron Chelating Agents; Kinetics; Models, Chemical; Molecular Structure; Oxidation-Reduction; Phenanthrolines; Siderophores; Thermodynamics

Perturbations to Fe species contributing to generation of DNA single-strand breaks (SSBs) and inhibition of growth by H2O2 were studied in HL-60 cells made Fe-deficient by 24 h pretreatment with 144 microM bathophenanthroline disulfonic acid and 400 microM ascorbic acid (Free Radic. Biol. Med. 20: 399; 1996). The diffusion distance for SSB generation (d) in Fe-deficient cells, measured via inhibition with the *OH scavenger Me2SO using alkaline elution, was 6.5 nm. This is similar to the d for Fe-normal cells reported previously. After 1 and 3 h in fresh RPMI 1640 medium containing 10% serum, SSB generation increased from 29 to 56 and 93% of control Fe-normal cells, respectively. The d of the major contributor to SSB generation at these two treatment times was 1.9 nm. This d resembled the d for Fe-ATP as determined in isolated Ehrlich cell nuclei. The association of ATP with Fe2+ was further supported by decreased SSB generation in cells in which ATP synthesis was inhibited. In contrast to SSB generation, H2O2-induced inhibition of growth of Fe-deficient cells treated immediately after placing in fresh medium was not appreciably different from Fe-normal cells. However, after 3 h, an approximately 70% greater concentration of H2O2 than for control, Fe-normal cells was required to inhibit growth. This increase in H2O2 concentration was associated with decreased generation of SSBs by H2O2 in isolated HL-60 cell nuclei. Thus, Fe bound to nuclear structures is more closely associated with inhibition of cell growth than apparent Fe-ATP species. In parallel experiments, changes in total cellular Fe assayed by ashing and complexing with ferrozine were consistent with a non-transferrin mode of acquisition. These short-term changes appear due to processes accompanying reestablishment of the Fe content and distribution normally observed during long-term growth.

Topics: Adenosine Triphosphate; Ascorbic Acid; Cell Division; Cell Nucleus; Chelating Agents; Diffusion; DNA Damage; Free Radicals; HL-60 Cells; Humans; Hydrogen Peroxide; Iron; Iron Deficiencies; Metals; Molecular Weight; Oxidation-Reduction; Phenanthrolines

The role of ascorbic acid in transferrin-independent ferric iron reduction and uptake was evaluated in cultured U-937 monocytic cells. Uptake of 55Fe by U-937 cells was doubled by 100 microM extracellular ascorbate, and by pre-incubation of cells with 100 microM dehydroascorbic acid, the two-electron-oxidized form of ascorbate. Reduction of extracellular ferric citrate also was enhanced by loading the cells with dehydroascorbic acid. Dehydroascorbic acid was taken up rapidly by the cells and reduced to ascorbate, such that the latter reached intracellular concentrations as high as 6 mM. However, some ascorbate did escape the cells and could be detected at concentrations of up to 1 microM in the incubation medium. Further, addition of ascorbate oxidase almost reversed the effects of dehydroascorbic acid on both 55Fe uptake and ferric citrate reduction. Thus, it is likely that extracellular ascorbate reduced ferric to ferrous iron, which was then taken up by the cells. This hypothesis also was supported by the finding that during loading with ferric citrate, only extracellular ascorbate increased the pool of intracellular ferrous iron that could be chelated with cell-penetrant ferrous iron chelators. In contrast to its inhibition of ascorbate-dependent ferric iron reduction, ascorbate oxidase was without effect on ascorbate-dependent reduction of extracellular ferricyanide. This indicates that the cells use different mechanisms for reduction of ferric iron and ferricyanide. Therefore, extracellular ascorbate derived from cells can enhance transferrin-independent iron uptake by reducing ferric to ferrous iron, but intracellular ascorbate neither contributes to this reduction nor modifies the redox status of intracellular free iron.

Topics: Ascorbic Acid; Chelating Agents; Dehydroascorbic Acid; Dose-Response Relationship, Drug; Humans; Iron; Oxidation-Reduction; Phenanthrolines; Transferrin; U937 Cells

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

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

An effort has been made to standardize the indirect iron saturation excess method for the determination of the serum unsaturated iron binding capacity (UIBC) and thus to relinquish the direct adsorption methods for the assay of the serum total iron binding capacity (TIBC) which give falsely high results due to unspecific binding of the saturating iron to serum proteins. In order to eliminate the interfering effects of hydrolytic polymerization of iron(III) on the saturation of apotransferrin in serum and on the colorimetric determination of the unbound iron excess at pH 8.3, conditions have been studied for the preparation of the iron-nitrilotriacetate-complex (Fe(NTA)2) solution at pH 8.3 with respect to its reactivity with the reductant sodium ascorbate and with the chromogen bathophenanthroline-disulfonate in photometric standards and in samples containing iron-saturated serum. The validity of the results for the UIBC thus obtained has been investigated (1) by direct spectrophotometric titration with Fe(NTA)2 of the apotransferrin in serum by measuring the absorbance of transferrin at 470 nm in 50-mm cuvettes, and of the UIBC using the modified indirect iron saturation excess assay, both of which gave the same saturation points, and (2) by the correlation of the TIBC obtained from serum iron determinations and the UIBC, with the transferrin concentration measured by the radial immunodiffusion assay. Results of UIBC determinations are presented along with serum iron concentration, TIBC, and transferrin saturation in groups of subjects with normal iron stores and prelatent, latent, and manifest iron deficiency.

Topics: Anemia, Hypochromic; Apoproteins; Ascorbic Acid; Carrier Proteins; Chromogenic Compounds; False Positive Reactions; Humans; Iron; Iron-Binding Proteins; Methods; Nitrilotriacetic Acid; Phenanthrolines; Photometry; Transferrin; Transferrin-Binding Proteins

Two spectrophotometric methods for the assay of serum iron without deproteinisation are compared: 1. Liberation of iron by a detergent (Teepol SHELL), reduction by dithionite, chelation by bathopehanthroline disulfonate; 2. Liberation of iron by 6 mol/l guanidine, reduction by ascorbic acid, chelation by 3-(2-pyridyl)-5,6-bis-(4-phenyl sulfonic acid)-1,2,4-triaxine (Ferrozine).

Topics: Ascorbic Acid; Chelating Agents; Detergents; Dithionite; Fatty Alcohols; Ferrozine; Guanidines; Humans; Iron; Oxidation-Reduction; Phenanthrolines; Spectrophotometry