EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on *OH formation by the Fenton reaction. (1/202)

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as beta-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against *OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for *OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of *OH from H2O2. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H2O2. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on *OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.  (+info)

The iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and its analogues prevent damage to 2-deoxyribose mediated by ferric iron plus ascorbate. (2/202)

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.  (+info)

Studies on mammalian ribonucleotide reductase inhibition by pyridoxal phosphate and the dialdehyde derivatives of adenosine, adenosine 5'-monophosphate, and adenosine 5'-triphosphate. (3/202)

Ribonucleotide reductase activity in a partially purified enzyme preparation from Ehrilich tumor cells was inhibited by the dialdehyde derivatives of adenosine, 5-adenylic acid, and adenosine 5-triphosphate (prepared by the periodate oxidation of adenosine 5-adenylic acid, and adenosine 5-triphosphate). The borohydride-reduced derivative of periodate-oxidized adenosine was not inhibitory to the ribonucleotide reductase activity, showing that the aldehyde moiety was important in the inhibitory interactions of these compounds. This suggested the formation of a Schiff base between the dialdehyde derivative and an amino group (presumably, the epsilon-amino group of lysine). Pyridoxal phosphate, which is known to inhibit enzymes that have lysyl residues in the catalytic or allosteric sites, was an inhibitor of ribonucleotide reductase. Pyridoxal, pyridoxamine phosphate, pyridoxamine, and pyridoxine were not inhibitors. Borohydride reduction of the enzyme in the presence of pyridoxal phosphate produced a protein fraction that had little reductase activity remaining. The inhibition by pyridoxal phosphate was not influenced by increasing the substrate concentration (cytidine 5-diphosphate or adenosine 5-diphosphate), but was diminished by increasing the ratio of allosteric effector to pyridoxal phosphate concentrations, suggesting an interaction of pyridoxal phosphate at the regulatory site of ribonucleotide reductase. The addition of adenosine 5-triphosphate to the pyridoxal phosphate-enzyme mixture, which was subsequently treated with borohydride, partially prevented the inhibition by pyridoxal phosphate. Heat treatment of the ribonucleotide reductase enzyme preparation in the presence of pyridoxal phosphate protected the enzyme against loss of cytidine 5-diphosphate and adenosine 5-diphosphate reductase activities.  (+info)

Development of potential iron chelators for the treatment of Friedreich's ataxia: ligands that mobilize mitochondrial iron. (4/202)

Friedreich's ataxia (FA) is a crippling neurodegenerative disease that is due to iron (Fe) overload within the mitochondrion. One therapeutic intervention may be the development of a chelator that could remove mitochondrial Fe. We have implemented the only well characterized model of mammalian mitochondrial Fe overload to examine the Fe chelation efficacy of novel chelators of the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) class. In this model we utilize reticulocytes treated with the haem synthesis inhibitor succinylacetone which results in mitochondrial Fe-loading. Our experiments demonstrate that in contrast to desferrioxamine, several of the PCIH analogues show very high activity at mobilizing (59)Fe from (59)Fe-loaded reticulocytes. Further studies on these ligands in animals are clearly warranted considering their potential to treat FA.  (+info)

The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents, IV: The mechanisms involved in inhibiting cell-cycle progression. (5/202)

Some chelators of the pyridoxal isonicotinoyl hydrazone class have antiproliferative activity that is far greater than desferrioxamine (DFO). In this study, DFO was compared with one of the most active chelators (311) on the expression of molecules that play key roles in cell-cycle control. This was vital for understanding the role of iron (Fe) in cell-cycle progression and for designing chelators to treat cancer. Incubating cells with DFO, and especially 311, resulted in a decrease in the hyperphosphorylated form of the retinoblastoma susceptibility gene product (pRb). Chelators also decreased cyclins D1, D2, and D3, which bind with cyclin-dependent kinase 4 (cdk4) to phosphorylate pRb. The levels of cdk2 also decreased after incubation with DFO, and especially 311, which may be important for explaining the decrease in hyperphosphorylated pRb. Cyclins A and B1 were also decreased after incubation with 311 and, to a lesser extent, DFO. In contrast, cyclin E levels increased. These effects were prevented by presaturating the chelators with Fe. In contrast to DFO and 311, the ribonucleotide reductase inhibitor hydroxyurea increased the expression of all cyclins. Hence, the effect of chelators on cyclin expression was not due to their ability to inhibit ribonucleotide reductase. Although chelators induced a marked increase in WAF1 and GADD45 mRNA transcripts, there was no appreciable increase in their protein levels. Failure to translate these cell-cycle inhibitors may contribute to dysregulation of the cell cycle after exposure to chelators. (Blood. 2001;98:842-850)  (+info)

Properties of 4-ethenyl and 4-ethynyl analogs of pyridoxal phosphate and their reactions with the apo form of asparatate aminotransferase. (6/202)

The binding to apoaspartate aminotransferase of analogs of pyridoxal phosphate bearing vinyl, cis- and trans-methylvinyl, and ethynyl groups in place of the fomyl group of the coenzyme has been studied. Details of synthesis of the ethynyl compound are given. The absorption spectra of all of the compounds have been analyzed and pKa values have been determined. The positions of the absorption bands can be related to those of pyridoxine but with bathochromic shifts induced by the ethenyl and ethynyl groups. However, this shift is almost completely lacking for the cis-methylvinyl compound suggesting nonplanarity of the molecule. Binding of the analogs to the apoenzyme is accompanied by a strong bathochromic shift which, from a study of solvent effects on the free analogs, appears to indicate a hydrophobic environment on the enzyme. Nevertheless, the analogs are bound as dipolar ions exclusively. Binding is accompanied by a distinct perturbation of the protein spectrum in the aromatic region. An effect on the spectrum of 1 or more tryptophan residues is indicated. Bands of the bound analogs exhibit positive circular dichroism except for that of the 4-vinyl analog. The 4-ethynyl analog reacts in a more complex way, giving at least two successive products in addition to the initial complex. The final product is reducible by sodium borohydride, is released from the enzyme by boiling, and appears to have the properties of a Schiff base. We postulate that the addition of an amino group of the enzyme to the ethynyl group is followed by tautomeric rearrangement to a Schiff base in which the ring is in a p-quinonoid structure.  (+info)

Activation and inactivation of horse liver alcohol dehydrogenase with pyridoxal compounds. (7/202)

Pyridoxal compounds can either activate or inactivate horse liver alcohol dehydrogenase in differential labeling experiments. Amino groups outside of the active sites were modified with ethyl acetimidate, while the amino groups in the active sites were protected by the formation of the complex with NAD-plus and pyrazole. After removal of the NAD-plus and pyranzole, the partially acetimidylated enzyme was reductively alkylated with pyridoxal and NaBH4, with the incorporation of one pyridoxal group per subunit of the enzyme. The turnover numbers for the reaction of NAD-plus and ethanol increased by 15-fold, and for NADH and acetaldehyde by 32-fold. The Michaelis and inhibition constants increased 80-fold or more. Pyridoxal phosphate and NaBH4 also modified one group per subunit, but the turnover numbers decreased by 10-fold and the kinetic constants were intermediate between those obtained for pyridoxyl alcohol dehydrogenase and the partially acetimidylated enzyme. With native enzyme, the rates of dissociation of the enzyme-coenzyme complexes are rate-limiting in the catalytic reactions. The pyridoxyl enzyme is activated because the rates of dissociation of the enzyme-coenzyme complexes are increased. The rates of binding of coenzyme to phosphopyridoxyl enzyme have decreased due to the introduction of the negatively charged phosphate. The size of the group is not responsible for this decrease since these rates are not greatly decreased by the incorporation of pyridoxal. For both pyrodoxal and phosphopyridoxyl alcohol dehydrogenases, the interconversion of the ternary complex is at least partially rate-limiting. Chymotryptic-tryptic digestion of pryidoxyl enzyme produced a major peptide corresponding to residues 219 to 229, in which Lys 228 had reacted with pyridoxal. The same lysine residue reacted with pyridoxal phosphate.  (+info)

Technetium-99m-pyridoxylideneglutamate: a new hepatobiliary radiopharmaceutical. II. Clinical aspects. (8/202)

Technetium-99m-pyridoxylideneglutamate (99mTc-PG) is a nontoxic radiopharmaceutical that was found to undergo rapid biliary excretion in normal humans. The biliary tree and gallbladder were seen within 10-15 min of injection and by 20 min marked accumulation of radioactivity was noted in the gallbladder and gastrointestinal tract. Of ten "control" volunteers, seven had normal 99mTc-PG-cholescintigrams. In the remaining three, the gallbladder was not visualized. Gallbladder disease was not excluded in these three subjects. Of 24 patients referred for investigation of right upper quadrant abdominal pain, 13 proved to have gallbladder disease. All seven patients with acute cholecystitis and one of four patients with chronic cholecystitis had nonvisualization of the gallbladder on the cholescintigram whereas five patients with chronic cholecystitis or cholesterolosis had normal cholescintigrams. Six of the eight patients with nonvisualization of the gallbladder on cholescintigram had contrast radiologic studies (oral cholecystogram or intravenous cholangiogram or both), and in all six, nonvisualization of the gallbladder was also reported on the contrast study. cholescintigraphy was found to be greatly inferior to contrast radiologic studies in the detection of gallbladder stones. Eleven patients had complete extrahepatic biliary obstruction and this diagnosis was correctly made in all 11 by the cholescintigram. Fourteen patients had incomplete extrahepatic biliary obstruction. The correct diagnosis was made on the cholescintigram in seven but in the remaining seven it was not possible to distinguish between incomplete extrahepatic biliary obstruction and hepatocellular disease. Malignant lesions (carcinomas of head of pancreas, gallbladder, common bile duct or ampulla of Vater) were the cause of obstruction in 10 of the 25 patients with complete or incomplete obstruction and the diagnosis of obstruction due to malignancy was correctly made in 8 of these 10 by means of a scintigraphic equivalent to Courvoisier's sing. Finally, 11 patients had hepatocellular disease and a nonspecific pattern consistent with either imcomplete biliary obstruction or hepatocellular disease was observed on the cholescintigram in all 11. The 99mTc-PG cholescintigram is suggested for a role complementary to that of contrast radiologic studies in the preoperative investigation of patients with possible surgical disease of the biliary tract. Contrast radiologic techniques are advocated as being more appropriate in the nonjaundiced patient with suspected gallbladder disease whereas the 99mTc-PG cholescintigram is advocated as being more appropriate in the patient with jaundice. The value of the 99mTc-PG cholescintigram lies in the confidence with which complete extrahepatic biliary obstruction can be diagnosed. The "scintigraphic Courvoisier's sign" seems a useful indicator of malignant obstruction.  (+info)