ascorbic-acid and pyridoxal-isonicotinoyl-hydrazone

The interaction of pyridoxal isonicotinoyl hydrazone (PIH) and salicylaldehyde isonicotinoyl hydrazone (SIH), two important biologically active chelators, with iron has been investigated by spectrophotometric methods. High iron(III) affinity constants were determined for PIH, logβ

Topics: Aldehydes; Ascorbic Acid; Edetic Acid; Electrochemical Techniques; Electrodes; Ferric Compounds; Ferrous Compounds; Hydrazones; Hydrogen-Ion Concentration; Iron Chelating Agents; Isoniazid; Kinetics; Oxidation-Reduction; Pyridoxal

Previous work from our laboratory demonstrated that pyridoxal isonicotinoyl hydrazone (PIH) has in vitro antioxidant activity against iron plus ascorbate-induced 2-deoxyribose degradation due to its ability to chelate iron; the resulting Fe(III)-PIH(2) complex is supposedly unable to catalyze oxyradical formation. A putative step in the antioxidant action of PIH is the inhibition of Fe(III)-mediated ascorbate oxidation, which yields the Fenton reagent Fe(II) [Biochim. Biophys. Acta 1523 (2000) 154]. In this work, we demonstrate that PIH inhibits Fe(III)-EDTA-mediated ascorbate oxidation (measured at 265 nm) and the formation of ascorbyl radical (in electron paramagnetic resonance (EPR) studies). The efficiency of PIH against ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation was dose dependent and directly proportional to the period of preincubation of PIH with Fe(III)-EDTA. The efficiency of PIH in inhibiting ascorbate oxidation and ascorbyl radical formation was also inversely proportional to the Fe(III)-EDTA concentration in the media. When EDTA was replaced by the weaker iron ligand nitrilotriacetic acid (NTA), PIH was much more effective in preventing ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation. Moreover, the replacement of EDTA with citrate, a physiological chelator with a low affinity for iron, also resulted in PIH having a higher efficiency in inhibiting iron-mediated ascorbate oxidation and 2-deoxyribose degradation. These results demonstrate that PIH removes iron from EDTA (or from either NTA or citrate), forming an iron-PIH complex that cannot induce ascorbate oxidation effectively, thus inhibiting iron-mediated oxyradical formation. These results are of pharmacological relevance because PIH has been considered for experimental chelating therapy in iron-overload diseases.

Topics: Ascorbic Acid; Chelating Agents; Deoxyribose; Edetic Acid; Electron Spin Resonance Spectroscopy; Ferric Compounds; Free Radicals; Hydroxyl Radical; Isoniazid; Nitrilotriacetic Acid; Oxidation-Reduction; Oxidative Stress; Pyridoxal

Pyridoxal isonicotinoyl hydrazone (PIH) analogues are effective iron chelators in vivo and in vitro, and may be of value for the treatment of secondary iron overload. The sensitivity of Jurkat cells to Fe-chelator complexes was enhanced several-fold by the depletion of the antioxidant glutathione, indicating the role of oxidative stress in their toxicity. K562 cells loaded with eicosapentaenoic acid, a fatty acid particularly susceptible to oxidation, were also more sensitive to the toxic effects of the Fe complexes, and toxicity was proportional to lipid peroxidation. Thus Fe-chelator complexes cause oxidative stress, which may be a major component of their toxicity. As was the case for their Fe complexes, the toxicity of PIH analogues was enhanced by glutathione depletion of Jurkat cells and eicosapentaenoic acid-loading of K562 cells. Thus the toxicity of the chelators themselves is also enhanced by compromised cellular redox status. In addition, the toxicity of the chelators was diminished by culturing Jurkat cells under hypoxic conditions, which may limit the production of the reactive oxygen species that initiate oxidative stress. A significant part of the toxicity of the chelators may be due to intracellular formation of Fe-chelator complexes, which oxidatively destroy the cell.

Topics: Ascorbic Acid; Cell Survival; Chelating Agents; Drug Design; Humans; Iron Chelating Agents; Isoniazid; Jurkat Cells; K562 Cells; Kinetics; Molecular Structure; Oxidation-Reduction; Oxidative Stress; Pyridoxal; Structure-Activity Relationship

Pyridoxal isonicotinoyl hydrazone (PIH) is an iron chelator with antioxidant activity, low toxicity and is useful in the experimental treatment of iron-overload diseases. Previous studies on x-ray diffraction have revealed that PIH also forms a complex with Cu(II). Since the main drug of choice for the treatment of Wilson's disease, d-penicillamine, causes a series of side effects, there is an urgent need for the development of alternative copper chelating agents for clinical use. These chelators must also have antioxidant activity because oxidative stress is associated with brain and liver copper-overload. In this work we tested the ability of PIH to prevent in vitro free radical formation mediated by Cu(II), ascorbate and dissolved O2. Degradation of 2-deoxyribose mediated by 10 microM Cu(II) and 3 mM ascorbate was fully inhibited by 10 microM PIH (I50 = 6 microM) or 20 microM d-penicillamine (I50 = 10 microM). The antioxidant efficiency of PIH remained unchanged with increasing concentrations (from 1 to 15 mM) of the hydroxyl radical detector molecule, 2-deoxyribose, indicating that PIH does not act as a hydroxyl scavenger. On the other hand, the efficiency of PIH (against copper-mediated 2-deoxyribose degradation and ascorbate oxidation) was inversely proportional to the Cu(II) concentration, suggesting a competition between PIH and ascorbate for complexation with Cu(lI). An almost full inhibitory effect by PIH was observed when the ratio PIH:copper was 1:1. A similar result was obtained with the measurement of copper plus ascorbate-mediated O2 uptake. Moreover, spectral studies of the copper and PIH interaction showed a peak at 455 nm and also indicated the formation of a stable Cu(II) complex with PIH with a 1:1 ratio. These data demonstrated that PIH prevents hydroxyl radical formation and oxidative damage to 2-deoxyribose by forming a complex with Cu(II) that is not reactive with ascorbate (first step of the reactions leading to hydroxyl radical formation from Cu(II), ascorbate and O2) and does not participate in Haber-Weiss reactions.

Topics: Ascorbic Acid; Copper Sulfate; Deoxyribose; Free Radical Scavengers; Free Radicals; Hydroxyl Radical; In Vitro Techniques; Iron Chelating Agents; Isoniazid; Kinetics; Oxidation-Reduction; Oxygen; Pyridoxal

Iron chelating agents are essential for treating iron overload in diseases such as beta-thalassemia and are potentially useful for therapy in non-iron overload conditions, including free radical mediated tissue injury. Deferoxamine (DFO), the only drug available for iron chelation therapy, has a number of disadvantages (e.g., lack of intestinal absorption and high cost). The tridentate chelator pyridoxal isonicotinoyl hydrazone (PIH) has high iron chelation efficacy in vitro and in vivo with high selectivity and affinity for iron. It is relatively non-toxic, economical to synthesize and orally effective. We previously demonstrated that submillimolar levels of PIH and some of its analogues inhibit lipid peroxidation, ascorbate oxidation, 2-deoxyribose degradation, plasmid DNA strand breaks and 5,5-dimethylpyrroline-N-oxide (DMPO) hydroxylation mediated by either Fe(II) plus H(2)O(2) or Fe(III)-EDTA plus ascorbate. To further characterize the mechanism of PIH action, we studied the effects of PIH and some of its analogues on the degradation of 2-deoxyribose induced by Fe(III)-EDTA plus ascorbate. Compared with hydroxyl radical scavengers (DMSO, salicylate and mannitol), PIH was about two orders of magnitude more active in protecting 2-deoxyribose from degradation, which was comparable with some of its analogues and DFO. Competition experiments using two different concentrations of 2-deoxyribose (15 vs. 1.5 mM) revealed that hydroxyl radical scavengers (at 20 or 60 mM) were significantly less effective in preventing degradation of 2-deoxyribose at 15 mM than 2-deoxyribose at 1.5 mM. In contrast, 400 microM PIH was equally effective in preventing degradation of both 15 mM and 1.5 mM 2-deoxyribose. At a fixed Fe(III) concentration, increasing the concentration of ligands (either EDTA or NTA) caused a significant reduction in the protective effect of PIH towards 2-deoxyribose degradation. We also observed that PIH and DFO prevent 2-deoxyribose degradation induced by hypoxanthine, xanthine oxidase and Fe(III)-EDTA. The efficacy of PIH or DFO was inversely related to the EDTA concentration. Taken together, these results indicate that PIH (and its analogues) works by a mechanism different than the hydroxyl radical scavengers. It is likely that PIH removes Fe(III) from the chelates (either Fe(III)-EDTA or Fe(III)-NTA) and forms a Fe(III)-PIH(2) complex that does not catalyze oxyradical formation.

Topics: Ascorbic Acid; Chelating Agents; Deoxyribose; Dimethyl Sulfoxide; DNA Damage; Edetic Acid; Ferric Compounds; Free Radical Scavengers; Hydroxyl Radical; Isoniazid; Kinetics; Models, Chemical; Plasmids; Pyridoxal; Structure-Activity Relationship