Radiation protection by the ocimum flavonoids orientin and vicenin: mechanisms of action

In previous studies, flavonoids, orientin and vicenin, that were isolated from the leaf extract of Ocimum sanctum, were found to protect mice against radiation injury. Several flavonoids are known to be good antioxidants. Therefore, the effect of orientin and vicenin on radiation-induced lipid peroxidation in vivo and their antioxidant activity in vitro were studied. Adult mice were injected intraperitoneally with 50 microgram/kg of orientin or vicenin and exposed whole-body to 3 Gy of gamma radiation. Lipid peroxidation was measured in the liver 15 min to 8 h postirradiation. The antioxidant activity of orientin/vicenin (10-500 microM) was studied by measuring inhibition of hydroxyl radicals generated by the Fenton reaction (Fe(3+)-EDTA-ascorbic acid-H(2)O(2)) in vitro. The compounds were also tested for possible pro-oxidant and iron chelation activities at the above concentrations in the in vitro system. Orientin and vicenin provided almost equal protection against radiation-induced lipid peroxidation in mouse liver. Both compounds showed a significantly greater free radical-inhibiting activity in vitro than DMSO. Neither orientin nor vicenin showed any pro-oxidant activity at the concentrations tested. Both compounds inhibited free radical formation in the absence of EDTA. Free radical scavenging appears to be a likely mechanism of radiation protection by these flavonoids.     

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

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.     

Autoxidation and MAO-mediated metabolism of dopamine as a potential cause of oxidative stress: role of ferrous and ferric ions

The autoxidation and monoamine oxidase (MAO)-mediated metabolism of dopamine (3-hydroxytyramine; DA) cause a continuous production of hydroxyl radical (*OH), which is further enhanced by the presence of iron (ferrous iron, Fe(2+) and ferric ion, Fe(3+)). The accumulation of hydrogen peroxide (H2O2) in the presence of Fe(2+) appears to discard the involvement of the Fenton reaction in this process. It has been found that the presence of DA significantly reduces the formation of thiobarbituric acid reagent substances (TBARS), which under physiological conditions takes place in mitochondrial preparations. The presence of DA is also able to reduce TBARS formation in mitochondrial preparations even in the presence of iron (Fe(2+) and Fe(3+)). However, DA boosted the carbonyl content of mitochondrial proteins, which was further increased in the presence of iron (Fe(2+) and Fe(3+)). This latter effect is also accompanied by a significant reduction in thiol content of mitochondrial proteins. It has also been observed how the pre-incubation of mitochondria with pargyline, an acetylenic MAO inhibitor, reduces the production of *OH and increases the formation of TBARS. Although, the MAO-mediated metabolism of DA increases MAO-B activity, the presence of iron inhibits both MAO-A and MAO-B activities. Consequently, DA has been shown to be a double-edged sword, because it displays antioxidant properties in relation to both the Fenton reaction and lipid peroxidation and exhibits pro-oxidant properties by causing both generation *OH and oxidation of mitochondrial proteins. Evidently, these pro-oxidant properties of DA help explain the long-term side effects derived from l-DOPA treatment of Parkinson's disease and its exacerbation by the concomitant use of DA metabolism inhibitors.     

Antioxidant activities of ethanol extracts from seeds in fresh Bokbunja (Rubus coreanus Miq.) and wine processing waste

The antioxidant potential of ethanol extracts from defatted Bokbunja seed wastes generated during wine processing were estimated by radical scavenging abilities (DPPH(*), H(2)O(2), and O(2)(*-)), retardation of lipid oxidation, and iron ion-chelating characteristics. For comparison, ethanol extracts from seeds of fresh ripe Bokbunja fruits were also used. The ethanol extracts from the wine seed waste always showed higher scavenging activities against DPPH(*), H(2)O(2), and O(2)(*-) than those from the fresh seeds. The oxidation of linoleic acid in dimethylsulfoxide at 105 degrees C revealed that the kinetic behavior clearly obeyed pseudo-zero-order regardless of the linoleic acid concentration. The Fe(II)-chelating capacity was determined by the Freundlich isotherm. The results showed high potential and favorability of the two extracts for Fe(II) chelation. The Freundlich chelation capacities (mg(1-1/n)L(1/n)/g) of both ethanol extracts from seeds of fresh Bokbunja and wine processing wastes, tannic acid, and proanthocyanidin from Pinus radiata bark were 100, 224, 260, and 307, respectively. The Fe(III)-chelating properties of the ethanol extracts were considered to be deeply associated with its 3',4',5'-trihydroxyl (galloyl) group as with tannic acid.     

The role of iron in oxygen radical mediated lipid peroxidation

The role of iron in the peroxidation of polyunsaturated fatty acids is reviewed, especially with respect to the involvement of oxygen radicals. The hydroxyl radical can be generated by a superoxide-driven Haber-Weiss reaction or by Fenton's reaction; and the hydroxyl radical can initiate lipid peroxidation. However, lipid peroxidation is frequently insensitive to hydroxyl radical scavengers or superoxide dismutase. We propose that the hydroxyl radical may not be involved in the peroxidation of membrane lipids, but instead lipid peroxidation requires both Fe2+ and Fe3+. The inability of superoxide dismutase to affect lipid peroxidation can be explained by the fact that the direct reduction of iron can occur, exemplified by rat liver microsomal NADPH-dependent lipid peroxidation. Catalase can be stimulatory, inhibitory or without affect because H2O2 may oxidize some Fe2+ to form the required Fe3+, or, alternatively, excess H2O2 may inhibit by excessive oxidation of the Fe2+. In an analogous manner reductants can form the initiating complex by reduction of Fe3+, but complete reduction would inhibit lipid peroxidation. All of these redox reactions would be influenced by iron chelation.     

In vitro antioxidant properties of the iron chelator pyridoxal isonicotinoyl hydrazone and some of its analogs

Since there are several problems with desferrioxamine (DFO) therapy, pyridoxal isonicotinoyl hydrazone (PIH) has been studied for more than 10 years as a promising new candidate for iron chelation therapy in iron-overload diseases. Iron chelation could also be helpful for experimental treatment of several other pathologies including rheumatoid arthritis and heart ischemia/reperfusion, due to the generation of oxyradicals and lipid peroxidation mediated by delocalized iron. We demonstrate here that sub-millimolar levels of PIH can inhibit the Fe(III)-EDTA/ascorbate-mediated formation of hydroxyl-like radicals as tested by the release of ethylene from 2-keto-4-methylthiobutyric acid (KMB assay) and the formation of malonaldehyde from 2-deoxyribose damage. PIH could also decrease the rates of Fe(III)-EDTA-mediated oxidation of ascorbate and block the peroxidation of liposomes of rat brain phospholipids induced by ferrous iron-EDTA. In all cases the in vitro antioxidant effectiveness of PIH was comparable to its analogs—including salicylaldehyde isonicotinoyl hydrazone—and to DFO. We conclude that PIH and its analogs are effective new candidates against iron-mediated oxidative stress for use in experimental medicine.     

Iron chelation and related properties of Podophyllum hexandrum, a possible role in radioprotection

Aqueous extract of Podophyllum species has been reported to render significant protection against radiation induced mortality, cytogenetic damage and cell death. In view of this, present study was undertaken to investigate its antioxidant properties. Chelation, oxidation and reduction of Fe2+ and Fe3+ were measured using chelating agents 2-2' bipiridyl and potassium thiocyanate respectively. Podophyllum extract, in a dose dependent manner, chelated Fe2+ more efficiently than Fe3+ and also modulated Fe2+/Fe3+ ratio. Homogenate of mouse liver was used to measure TBARS for estimating lipid peroxidation. Podophyllum extract also inhibited lipid peroxidation in a dose dependent manner and maximum inhibition (92%) was achieved at 1000 micrograms/ml concentration. These results demonstrates that Podophyllum exhibits antioxidant properties as seen through chelation and modulation of redox state of iron ions and these may primarily contribute towards its radioprotective manifestation.     

The characterisation of structural and antioxidant properties of isoflavone metal chelates

Isoflavone metal chelates are of interest as isoflavones act as oestrogen mimics. Metal interactions may enhance isoflavones biological properties so understanding isoflavone metal chelation is important for the commercial application of isoflavones. This work aimed to determine if isoflavones, daidzein (4',7-dihydroxyisoflavone) and genistein (4',5,7-trihydroxyisoflavone) could chelate with metals as isoflavone chelates. Biochanin A (4'-methoxy-5,7-dihydroxyisoflavone) was also examined for it's ability to chelate with Cu(II) and Fe(III). This study found daidzein does not chelate with Cu(II) and Fe(III) but genistein and biochanin A chelate with a 1:2 M/L stoichiometry. The copper and iron chelates were synthesised and characterised by elemental analysis, FTIR, thermogravimetric analysis (TGA) and electrospray ionisation mass spectrometry (ESI-MS). These studies indicated a 1:2 M/L stoichiometry and suggested the isoflavones bind with the metals at the 4-keto and the 5-OH site. 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition assays showed that copper isoflavone chelates have higher antioxidant activity than free isoflavones while the iron isoflavone chelates showed pro-oxidant activity compared to the free isoflavone. Synergistic DPPH studies with 0.02 mM ascorbic acid revealed copper chelates exhibit reduced antioxidant activity versus free isoflavones whereas the iron chelates showed lower pro-oxidant activity except at 1.0 mM.     

Polyphenol tannic acid inhibits hydroxyl radical formation from Fenton reaction by complexing ferrous ions

Tannic acid (TA), a plant polyphenol, has been described as having antimutagenic, anticarcinogenic and antioxidant activities. Since it is a potent chelator of iron ions, we decided to examine if the antioxidant activity of TA is related to its ability to chelate iron ions. The degradation of 2-deoxyribose induced by 6 microM Fe(II) plus 100 microM H2O2 was inhibited by TA, with an I50 value of 13 microM. Tannic acid was over three orders of magnitude more efficient in protecting against 2-deoxyribose degradation than classical *OH scavengers. The antioxidant potency of TA was inversely proportional to Fe(II) concentration, demonstrating a competition between H2O2 and AT for reaction with Fe(II). On the other hand, the efficiency of TA was nearly unchanged with increasing concentrations of the *OH detector molecule, 2-deoxyribose. These results indicate that the antioxidant activity of TA is mainly due to iron chelation rather than *OH scavenging. TA also inhibited 2-deoxyribose degradation mediated by Fe(III)-EDTA (iron = 50 microM) plus ascorbate. The protective action of TA was significantly higher with 50 microM EDTA than with 500 microM EDTA, suggesting that TA removes Fe(III) from EDTA and forms a complex with iron that cannot induce *OH formation. We also provided evidence that TA forms a stable complex with Fe(II), since excess ferrozine (14 mM) recovered 95-96% of the Fe(II) from 10 microM TA even after a 30-min exposure to 100-500 microM H2O2. Addition of Fe(III) to samples containing TA caused the formation of Fe(II)n-TA, complexes, as determined by ferrozine assays, indicating that TA is also capable of reducing Fe(III) ions. We propose that when Fe(II) is complexed to TA, it is unable to participate in Fenton reactions and mediate *OH formation. The antimutagenic and anticarcinogenic activity of TA, described elsewhere, may be explained (at least in part) by its capacity to prevent Fenton reactions.     

Cellular signalling and free-radical modulating activities of the novel peptidomimetic L-glutamyl-histamine.

A novel histamine-containing peptidomimetic, L-glutamyl-histamine (L-Glu-Hist), has been synthesized and characterized as a possible cytokine mimic which might lead to cellular responses of improved specificity. The energy-minimized 3-D conformations of L-Glu-Hist derived from its chemical structure stabilize Fe2+-chelating complexes. L-Glu-Hist concentration-dependently accelerates a decrease in ferrous iron in ferrous sulfate solution and shows ferroxidase-like activity at concentrations less than 3 mM in the phenanthroline assay, whereas in the concentration range 3-20 mM it restricts the availability of Fe2+ to phenanthroline by chelation of iron ions. At low concentrations (less than or about 1 mM), L-Glu-Hist stimulates peroxidation of phosphatidylcholine in liposomes catalyzed by a superoxide anion radical (O2)-generating system (Fe2+ + ascorbate) and, at high concentrations (*10 mM), it suppresses lipid peroxidation (LPO) in liposomes. The stimulation of LPO by L-Glu-Hist is related to its ability at low concentrations (*0.05 mM) to release O2 free radicals as determined by the superoxide dismutase-inhibitable reduction of cytochrome c. The release of O2 by L-Glu-Hist might result from its ferroxidase-like activity, while its inhibition of LPO is due to chelation of Fe2+, prevention of the formation of free radicals, and degradation of lipid hydroperoxides at 5-20 mM L-Glu-Hist concentrations. L-Glu-Hist releases O2 at concentrations which stimulate [3H]thymidine incorporation into DNA and proliferation of mouse spleen lymphocytes and also of mononuclear cells from human blood. The induction of lymphocyte proliferation by L-Glu-Hist is dose-dependent in the 0.01-0.05 mM concentration range, although the maximal stimulation of LPO in the O2-dependent system is observed at higher L-Glu-Hist concentrations (*1 mM). Thus, low concentrations of oxygen free radicals released by L-Glu-Hist may provide a very fast, specific, and sensitive trigger for lymphocyte proliferation and immunoregulation.     

Iron autoxidation and free radical generation: effects of buffers, ligands, and chelators.

The pH of the solution along with chelation and consequently coordination of iron regulate its reactivity. In this study we confirmed that, in general, the rate of Fe(II) autoxidation increases as the pH of the solution is increased, but chelators that provide oxygen ligands for the iron can override the affect of pH. Additionally, the stoichiometry of the Fe(II) autoxidation reaction varied from 2:1 to 4:1, dependent upon the rate of Fe(II) autoxidation, which is dependent upon the chelator. No partially reduced oxygen species were detected during the autoxidation of Fe(II) by ESR using DMPO as the spin trap. However, upon the addition of ethanol to the assay, the DMPO:hydroxyethyl radical adduct was detected. Additionally, the hydroxylation of terephthalic acid by various iron-chelator complexes during the autoxidation of Fe(II) was assessed by fluorometric techniques. The oxidant formed during the autoxidation of EDTA:Fe(II) was shown to have different reactivity than the hydroxyl radical, suggesting that some type of hypervalent iron complex was formed. Ferrous iron was shown to be able to directly reduce some quinones without the reduction of oxygen. In conclusion, this study demonstrates the complexity of iron chemistry, especially the chelation of iron and its subsequent reactivity.     

NADPH- and adriamycin-dependent microsomal release of iron and lipid peroxidation

In a previous study (Minotti, G., 1989, Arch. Biochem. Biophys. 268, 398-403) NADPH-supplemented microsomes were found to reduce adriamycin (ADR) to semiquinone free radical (ADR-.), which in turn autoxidized at the expense of oxygen to regenerate ADR and form O2-. Redox cycling of ADR was paralleled by reductive release of membrane-bound nonheme iron, as evidenced by mobilization of bathophenanthroline-chelatable Fe2+. In the present study, iron release was found to increase with concentration of ADR in a superoxide dismutase- and catalase-insensitive manner. This suggested that membrane-bound iron was reduced by ADR-. with negligible contribution by O2-. or interference by its dismutation product H2O2. Following release from microsomes, Fe2+ was reconverted to Fe3+ via two distinct mechanisms: (i) catalase-inhibitable oxidation by H2O2 and (ii) catalase-insensitive autoxidation at the expense of oxygen, which occurred upon chelation by ADR and increased with the ADR:Fe2+ molar ratio. Malondialdehyde formation, indicative of membrane lipid peroxidation, was observed when approximately 50% of Fe2+ was converted to Fe3+. This occurred in presence of catalase and low concentrations of ADR, which prevented Fe2+ oxidation and favored only partial Fe2+ autoxidation, respectively. Lipid peroxidation was inhibited by superoxide dismutase via increased formation of H2O2 from O2-. and excessive Fe2+ oxidation. Lipid peroxidation was also inhibited by high concentrations of ADR, which favored maximum Fe2+ release but also caused excessive Fe2+ autoxidation via formation of very high ADR:Fe2+ molar ratios. These results highlighted multiple and diverging effects of ADR, O2-., and H2O2 on iron release, iron (auto-)oxidation and lipid peroxidation. Stimulation of malondialdehyde formation by catalase suggested that lipid peroxidation was not promoted by reaction of Fe2+ with H2O2 and formation of hydroxyl radical. The requirement for both Fe2+ and Fe3+ was indicative of initiation by some type of Fe2+/Fe3+ complex.     

Chemical properties of water-soluble porphyrins. 4. The reaction of a 'picket-fence-like' iron (III) complex with the superoxide oxygen couple

Solution properties of the iron-(III) 'picket-fence-like' porphyrin, Fe(III)-alpha,alpha,alpha, beta-tetra-ortho (N-methyl-isonicotinamidophenyl) porphyrin, (Fe(III)PFP) were investigated. These were acid/base properties of the aquo complex with pKa of 3.9 and its aggregation (formation of dimer with K = 1 X 10(-10) dm3 mol-1), complex formation with cyanide ions and 1-methyl imidazole (1-MeIm), spectral properties of the three iron complexes in their ferric and ferrous form and the one-electron reduction potential of these complexes. Knowing these properties, the reaction of the ferric complexes, aquo, dicyano and bis (1-MeIm), with the superoxide radical and other reducing radicals were studied using the pulse radiolysis technique. The second-order reaction rate constant of O2- with the iron (III) aquo complex which governs the catalytic efficiency of the metalloporphyrin upon the disproportionation of the superoxide radical was 7.6 X 10(7) dm3 mol-1 s-1, two orders of magnitude faster when compared to the reaction of each of the other complexes. The reduction by other radicals with all iron (III) complexes had similar second-order rate constants (10(9) to 10(10) dm3 mol-1 s-1). The reduction reaction in all cases produced Fe(II)PEP and no intermediate was found. The oxidation reaction of Fe(II)PEP by O2- was one order of magnitude faster when compared to the reduction of Fe(III)PFP by the same radical. Since the reactivity of O2- toward the three iron (III) porphyrin complexes follows their reduction potentials, it is suggesting the formation of a peroxo Fe(II) porphyrin as an intermediate. The reactions of the Fe(II)PFP complexes with dioxygen were also studied. The aquo complex was found to be first order in O2 and second order in Fe(II)PFP, suggesting the formation of a peroxo Fe(II) porphyrin as an intermediate. The intermediate formation was corroborated by evidence of the rapid CO binding reaction to the aquo complex of Fe(II)PFP. The two other complexes reacted very slowly with O2 as well as with CO.     

Silybin, a new iron-chelating agent.

Silybin, a natural occurring flavolignan isolated from the fruits of Silibum marianum, has been reported to exert antioxidant and free radical scavenging abilities. It was suggested to act also as an iron chelator. The complexation and protonation equilibria of the ferric complex of this compound have been studied by potentiometric, spectrophotometric and electrochemical techniques. The formation of the complex silybin-Ga(III) in anhydrous DMSO-d6 has been studied by 1H NMR spectroscopy. Mass spectrometry and infrared spectroscopy on silybin-Fe(III) complex confirm all data obtained by 1H NMR spectroscopy. The experimental results show that silybin binds Fe(III) even at acidic pH. Different ternary complexes were observed at increasing methoxide ion concentration and their stability constants have been calculated. The results show the possible role of silybin in relation to the chelation therapy of chronic iron overload, as occurs in the treatment of Cooley's anemia.     

Oxalate modulates thiobarbituric acid reactive species (TBARS) production in supernatants of homogenates from rat brain, liver and kidney: effect of diphenyl diselenide and diphenyl ditelluride

The aim of this paper was to investigate the mechanism(s) involved in the sodium oxalate pro-oxidative activity in vitro and the potential protection by diphenyl diselenide ((PhSe)(2)) and diphenyl ditelluride ((PhTe)(2)) using supernatants of homogenates from brain, liver and kidney. Oxalate causes a significant increase in the TBARS (thiobarbituric acid reactive species) production up to 4mmol/l and it had antioxidant activity from 8 to 16mmol/l in the brain and liver. Oxalate had no effect in kidney homogenates. The difference among tissues may be related to the formation of insoluble crystal of oxalate in kidney, but not in liver and brain homogenates. (PhSe)(2) and (PhTe)(2) reduced both basal and oxalate-induced TBARS in rat brain homogenates, whereas in liver homogenates they were antioxidant only on oxalate-induced TBARS production. (PhSe)(2) showed a modest effect on renal TBARS production, whereas (PhTe)(2) did not modulate TBARS in kidney preparations. Oxalate at 2mmol/l did not change deoxyribose degradation induced by Fe(2+) plus H(2)O(2), whereas at 20mmol/l it significantly prevents its degradation. Oxalate (up to 4mmol/l) did not alter iron (10micromol/l)-induced TBARS production in the brain preparations, whereas at 8mmol/l onwards it prevents iron effect. In liver preparations, oxalate amplifies iron pro-oxidant activity up to 4mmol/l, preventing iron-induced TBARS production at 16mmol/l onwards. These results support the antioxidant effect of organochalcogens against oxalate-induced TBARS production. In addition, our results suggest that oxalate pro- and antioxidant activity in vitro could be related to its interactions with iron ions.     

Unprecedented oxidation of a biologically active aroylhydrazone chelator catalysed by iron(III): serendipitous identification of diacylhydrazine ligands with high iron chelation efficacy

Ligands of the 2-pyridylcarbaldehyde isonicotinoylhydrazone class show high iron (Fe) sequestering efficacy and have potential as agents for the treatment of Fe overload disease. We have investigated the mechanisms responsible for their high activity. X-ray crystallography studies show that the tridentate chelate 2-pyridylcarbaldehyde isonicotinoylhydrazone undergoes an unexpected oxidation to isonicotinoyl(picolinoyl)hydrazine when complexed with FeIII. In contrast, in the absence of FeIII, the parent hydrazone is not oxidized in aerobic aqueous solution. To examine whether the diacylhydrazine could be responsible for the biological effects of 2-pyridylcarbaldehyde isonicotinoylhydrazone, their Fe chelation efficacy was compared. In contrast to its parent hydrazone, the diacylhydrazine showed little Fe chelation activity. Potentiometric titrations suggested that this might be because the diacylhydrazine was charged at physiological pH, hindering its access across membranes to intracellular Fe pools. In contrast, the Fe complex of this diacylhydrazine was charge neutral, which may allow facile movement through membranes. These data allow a model of Fe chelation for this compound to be proposed: the parent aroylhydrazone diffuses through cell membranes to bind Fe and is subsequently oxidized to the diacylhydrazine complex which then diffuses from the cell. Other diacylhydrazine analogues that were charge neutral at physiological pH demonstrated high Fe chelation efficacy. Thus, for this class of ligands, the charge of the chelator appears to be an important factor for determining their ability to access intracellular Fe. The results of this study are significant for understanding the biological activity of 2-pyridylcarbaldehyde isonicotinoylhydrazone and for the design of novel diacylhydrazine chelators for clinical use.     

Effect of pH and oxalate on hydroquinone-derived hydroxyl radical formation during brown rot wood degradation

The redox cycle of 2,5-dimethoxybenzoquinone (2,5-DMBQ) is proposed as a source of reducing equivalent for the regeneration of Fe2+ and H2O2 in brown rot fungal decay of wood. Oxalate has also been proposed to be the physiological iron reductant. We characterized the effect of pH and oxalate on the 2,5-DMBQ-driven Fenton chemistry and on Fe3+ reduction and oxidation. Hydroxyl radical formation was assessed by lipid peroxidation. We found that hydroquinone (2,5-DMHQ) is very stable in the absence of iron at pH 2 to 4, the pH of degraded wood. 2,5-DMHQ readily reduces Fe3+ at a rate constant of 4.5 x 10(3) M(-1)s(-1) at pH 4.0. Fe2+ is also very stable at a low pH. H2O2 generation results from the autoxidation of the semiquinone radical and was observed only when 2,5-DMHQ was incubated with Fe3+. Consistent with this conclusion, lipid peroxidation occurred only in incubation mixtures containing both 2,5-DMHQ and Fe3+. Catalase and hydroxyl radical scavengers were effective inhibitors of lipid peroxidation, whereas superoxide dismutase caused no inhibition. At a low concentration of oxalate (50 micro M), ferric ion reduction and lipid peroxidation are enhanced. Thus, the enhancement of both ferric ion reduction and lipid peroxidation may be due to oxalate increasing the solubility of the ferric ion. Increasing the oxalate concentration such that the oxalate/ferric ion ratio favored formation of the 2:1 and 3:1 complexes resulted in inhibition of iron reduction and lipid peroxidation. Our results confirm that hydroxyl radical formation occurs via the 2,5-DMBQ redox cycle.     

Pyridoxal isonicotinoyl hydrazone inhibits iron-induced ascorbate oxidation and ascorbyl radical formation

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.     

Ferrous ion autoxidation and its chelation in iron-loaded human liver HepG2 cells.

Ferrous ion (Fe(2+)) is long thought to be the most likely active species, producing oxidants through interaction of Fe(2+) with oxygen (O(2)). Because current iron overload therapy uses only Fe(3+) chelators, such as desferrioxamine (DFO), we have tested a hypothesis that addition of a Fe(2+) chelator, 2,2'-dipyridyl (DP), may be more efficient and effective in preventing iron-induced oxidative damage in human liver HepG2 cells than DFO alone. Using ferrozine as an assay for iron measurement, levels of cellular iron in HepG2 cells treated with iron compounds correlated well with the extent of lipid peroxidation (r = 0.99 after log transformation). DP or DFO alone decreased levels of iron and lipid peroxidation in cells treated with iron. DFO + DP together had the most significant effect in preventing cells from lipid peroxidation but not as effective in decreasing overall iron levels in the cells. Using ESR spin trapping technique, we further tested factors that can affect oxidant-producing activity of Fe(2+) with dissolved O(2) in a cell-free system. Oxidant formation enhanced with increasing Fe(2+) concentrations and reached a maximum at 5 mM of Fe(2+). When the concentration of Fe(2+) was increased to 50 mM, the oxidant-producing activity of Fe(2+) sharply decreased to zero. The initial ratio of Fe(3+):Fe(2+) did not affect the oxidant producing activity of Fe(2+). However, an acidic pH (< 3.5) significantly slowed down the rate of the reaction. Our results suggest that reaction of Fe(2+) with O(2) is an important one for oxidant formation in biological system, and therefore, drugs capable of inhibiting redox activity of Fe(2+) should be considered in combination with a Fe(3+) chelator for iron overload chelation therapy.     

Factors affecting the free radical scavenging behavior of chitosan sulfate

Scavenging activity of hydroxyethyl chitosan sulfate (HCS) against 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl and carbon-centered radical species were studied using electron spin resonance (ESR) spectroscopy. In addition, its antioxidant activity to retard lipid peroxidation was also evaluated in a linoleic acid model system. HCS could scavenge DPPH (33.78%, 2.5 mg/mL) and carbon-centered radicals (67.74%, 0.25 mg/mL) effectively. However, chitosan sulfate did not exhibit any scavenging activity against hydroxyl radicals, but increased its generation. This was different from the published literature and was presumed due to the loss of chelating ability on Fe2+. This assumption could further confirm from the results obtained for Fe2+-ferrozine method that upon sulfation chitooligosaccharides lost its chelation properties. Therefore, HCS can be identified as antioxidant that effectively scavenges carbon centered radicals to retard lipid peroxidation.