Growth inhibition of tobacco tissue cultures with 6-azauracil, 6-azauridine and maleic hydrazide

Tissue cultures of Nicotiana tabacum, Nicotiana suaveolens and Nicotiana suaveolens × Nicotiana langsdorffii were cultured in the presence of the growth inhibitors maleic hydrazide and 6-azauracil as well as 6-azauridine. Inhibition of growth was complete at 10⁻⁴ molar concentrations in all 3 genotypes of cells. The inhibition due to 6-azauracil could be completely relieved with uridine and partially relieved with uracil. The effect with maleic hydrazide was irreversible, a fact which indicates a different mechanism or level of inhibition. This study supports the concept that derivatives of 6-azauracil inhibit the synthesis of uridine derivatives required for ribose nucleic acid synthesis and growth.     

Inhibitory effect of 6-azauracil on purified rabbit liver 4-aminobutyrate aminotransferase

Among uracil derivatives investigated, 6-azauracil, 6-azathymine, and 5-iodouracil were found to be potent inhibitors of purified rabbit liver 4-aminobutyrate aminotransferase while 6-azauridine and 6-azauridine 5'-phosphate were not. The enzyme inhibited by 6-azauracil was reactivated by dialysis but not by addition of pyridoxal 5'-phosphate. 6-Azauracil acted as a non-competitive inhibitor with respect to beta-alanine as well as 2-oxoglutaric acid, and had a K1 of approximately 0.7 mM at pH 7.3. The kinetic data suggested that 2-oxoglutaric acid acted as an inhibitor as well as an amino acceptor for the enzyme; a catalytic site was associated with an apparent Km of 0.15 mM for 2-oxoglutaric acid and a low affinity site was associated with an I50 of approximately 5 mM for the 2-oxo acid. With inhibitory concentrations of 2-oxoglutaric acid as substrate the inhibitory effect of 6-azauracil was considerably diminished. From these findings, the inhibitory effect of 6-azauracil was revealed to be different from that of structural analogs of 4-aminobutyric acid showing that 6-azauracil is a new type of 4-aminobutyrate aminotransferase inhibitor.     

Exogenous ascorbic acid (vitamin C) increases resistance to salt stress and reduces lipid peroxidation

The transition from reversible to permanent wilting, in whole tomato seedlings (Lycopersicon esculentum Mill. cv. M82) following severe salt-stress by root exposure to 300 mM NaCl, was investigated. Salinized seedlings wilted rapidly but recovered if returned to non-saline nutrient solution within 6 h. However, after 9 h of salt-treatment 100% of the seedlings remained wilted and died. Remarkably, the addition of an anti-oxidant (0.5 mM ascorbic acid) to the root medium, prior to and during salt-treatment for 9 h, facilitated the subsequent recovery and long-term survival of c. 50% of the wilted seedlings. Other organic solutes without known anti-oxidant activity were not effective. Salt-stress increased the accumulation in roots, stems and leaves, of lipid peroxidation products produced by interactions with damaging active oxygen species. Additional ascorbic acid partially inhibited this response but did not significantly reduce sodium uptake or plasma membrane leakiness.     

Altered production of the active oxygen species is involved in enhanced cytotoxic action of acylated derivatives of ascorbate to tumor cells

Our previous study shows that 6-O-acyl derivatives of L-ascorbic acid inhibits more markedly cell growth of mouse Ehrlich carcinoma than ascorbic acid. The present study shows that 6-O-palmitoyl ascorbic acid but not ascorbic acid prolongs the lifespan of mice into which tumors such as Meth A fibrosarcoma, MM46 mammary carcinoma, Ehrlich carcinoma and sarcoma 180 are implanted. The potentiated cytotoxicity of 6-O-palmitoyl ascorbic acid is not due to an increase in duration time of the cytotoxic action, because 6-O-palmitoyl ascorbic acid is gradually inactivated during contact with tumor cells and exhibits a similar action time curve to that of ascorbic acid as shown by clonal growth assay. Cytotoxicity of 6-O-palmitoyl ascorbic acid is markedly diminished by combined addition of catalase and superoxide dismutase (SOD), as shown by dye exclusion assay, whereas the cytotoxicity was slightly reduced by either enzyme alone but not by the specifically inactivated or heat-denatured enzymes. In contrast, cytotoxicity of ascorbic acid is abolished by catalyse but not SOD. Autooxidation of 6-O-palmitoyl ascorbic acid was not inhibited by catalase plus SOD. The results indicate that cytotoxicity of 6-O-palmitoyl ascorbic acid is attributed at least partly to both hydrogen peroxide (H2O2) and superoxide (O2-.) generated at the early stage. Cytotoxicity of 6-O-palmitoyl ascorbic acid is also appreciably attenuated by singlet oxygen (1O2) scavengers such as hydroquinone, 1,4-diazobicyclo-2,2,2-octane or sodium azide, but not by hydroxyl radical scavengers including butylated hydroxytoluene, D-mannitol, benzoic acid and ethanol. Thus, in contrast to cytotoxicity of ascorbic acid mediated entirely by H2O2 initially generated, acylated ascorbic acid produces a diversity of active oxygen species including H2O2, O2-. and other species secondarily generated via disproportion, which may be additively involved in the enhanced cytotoxic action.     

Oxygen-dependent antagonism of lipid peroxidation

Measurements of the rates for formation of conjugated dienes, malonylaldehyde, and lipid hydroperoxides show that increasing the concentration of O2 from 0.11 mM to 0.35 mM or 0.69 mM can slow the rate of linoleic acid peroxidation in a xanthine oxidase/hypoxanthine system. This effect is seen at pH 7.0 but not 7.4 and depends on the presence of monounsaturated fatty acids (oleic, cis, or trans vaccenic acid). Oxygen antagonism of ascorbic acid-iron-EDTA mediated lipid peroxidation is similarly dependent on fatty acid mixtures and occurs at pH 5.0 and 6.0 but not 7.0. The efficiency of initiation of peroxidation in the xanthine oxidase system is unaffected by monounsaturated fatty acids and O2 concentration. Increasing the O2 concentration increases the rate of superoxide radical production, but there is no change in salicylate hydroxylation (e.g., OH. production) or ferrous ion concentration. Oxygen-mediated slower rates of lipid peroxidation are associated with either increased H2O2 production or, based on an indirect assay, singlet O2 production. Increased O2 concentrations increase the rate of azobisisobutyronitrile-initiated lipid peroxidation as expected but addition of exogenous superoxide radicals slows the rate. Under similar conditions superoxide reacts with fatty acids to produce singlet O2. Overall, the data suggest that O2-mediated antagonism occurs because of termination reactions between hydroperoxyl (HO2.) and organic radicals, and singlet O2 or H2O2 are products of these reactions.     

Contribution of cytochromes and proteins to the effect of ascorbic acid on artificial and microsomal hydroxylation systems containing oxygen and hydrogen peroxide.

Hydroxylation systems containing cytochromes, proteins and ascorbic acid were studied at physiological pH (7.4) under O2 or N2 with added H2O2. Proteins inhibited aromatic hydroxylation of p-nitrophenol or oxidative demethylation of ethylmorphine in ascorbic acid-containing systems incubated under O2, but strongly activated the systems containing H2O2. Cytochrome c and partially purified cytochrome P-450 from rat liver microsomal preparations activated the system in either O2 or H2O2. The systems needed ascorbic acid (or other enol structures) for activation. Cytochrome iron participated probably in the activation of O2, whereas cytochrome protein participated in a free radical activation of H2O2 (or of O2).     

Counteraction of Salinity Stress on Wheat Plants by Grain Soaking in Ascorbic Acid, Thiamin or Sodium Salicylate

The interactive effects of salinity stress (40, 80, 120 and 160 mM NaCl) and ascorbic acid (0.6 mM), thiamin (0.3 mM) or sodium salicylate (0.6 mM) were studied in wheat (Triticum aestivum L.). The contents of cellulose, lignin of either shoots or roots, pectin of root and soluble sugars of shoots were lowered with the rise of NaCl concentration. On the other hand, the contents of hemicellulose and soluble sugars of roots, starch and soluble proteins of shoots, proline of either shoots or roots, and amino acids of roots were raised. Also, increasing NaCl concentration in the culture media increased Na⁺ and Ca²⁺ accumulation and gradually lowered K⁺ and Mg²⁺ concentration in different organs of wheat plant. Grain soaking in ascorbic acid, thiamin or sodium salicylate could counteract the adverse effects of NaCl salinity on the seedlings of wheat plant by suppression of salt stress induced accumulation of proline.     

The spectral and paramagnetic properties of oxyhemoglobin solutions UV-irradiated in the presence of ascorbic acid]

Absorption spectra and ESR of aqueous and aqueous/glyceric solutions of oxyhemoglobin exposed to UV radiation (250-400 nm) at 293 and 77 K in the presence of ascorbic acid have been analyzed. Vitamin C (5 x 10(-5) M) has been shown to exert a photoprotective effect with regard to oxyhemoglobin (2 x 10(-6) M) UV-irradiated with a dose of 0.86 x 10(5) J/m2 at 293 K. The photoprotective effect of ascorbic acid is also displayed after UV irradiation of frozen (77 K) aqueous/glyceric oxyhemoglobin solutions (2.53 x 10(-5) M). It is concluded that ascorbic acid can be a scavenger with respect to active UV-induced particles in protein systems, including O2-. and OH. Proposed is a mode of processes leading to UV inactivation of hemoprotein molecules.     

Chloroperoxidase-catalyzed enantioselective oxidation of methyl phenyl sulfide with dihydroxyfumaric acid/oxygen or ascorbic acid/oxygen as oxidants

The chloroperoxidase catalyzed oxidation of methyl phenyl sulfide to (R)-methyl phenyl sulfoxide was investigated, both in batch and membrane reactors, using as oxidant H2O2, or O2 in the presence of either dihydroxyfumaric acid or ascorbic acid. The effects of pH and nature and concentration of the oxidants on the selectivity, stability, and productivity of the enzyme were evaluated. The highest selectivity was displayed by ascorbic acid/O2, even though the activity of chloroperoxidase with this system was lower than that obtained with the others. When the reaction was carried out in a membrane reactor, it was possible to reuse the enzyme for several conversion cycles. The results obtained with ascorbic acid/O2 and dihydroxyfumaric acid/O2 as oxidants do not seem to be compatible with either a mechanism involving hydroxyl radicals as the active species or with the hypothesis that oxidation occurs through the initial formation of H2O2. Copyright 1999 John Wiley & Sons, Inc.     

On the application of the Clark oxygen electrode to the study of enzyme kinetics in apolar solvents: the catalase reaction.

A method for recording O2 concentrations in nonconducting organic media with the Clark oxygen electrode was developed. Spontaneous oxidation of Na2S2O4 and the enzymatic reduction of NaBO3 or H2O2 by bovine liver catalase trapped in hydrated micelles of dioctylsulfosuccinate (AOT)/toluene were used as model systems. O2 titration with the above systems showed that air-saturated 1.6 M H2O/0.2 M AOT/toluene media contain seven times more O2 (1.4 mM) than aqueous solutions (0.2 mM). The measured Km values of catalase for NaBO3 and H2O2 in organic media were Kmov = 15 and 17 mM, respectively, whereas in aqueous buffer the values were 45 and 54 mM. In the toluene media, catalase activity increased with the W0 (H2O/AOT molar ratio) of the micellar preparation, reaching maximal activity at W0 = 10-12; under this condition, the catalytic center activity (Kp) of H2O2 was 7 x 10(6) min-1, similar to that obtained in the aqueous buffer (H2O2 = 7 x 10(6) min-1). It was found that the optimal pH for catalase in toluene media (pH 8.0) was shifted 1.0 unit compared to that in the aqueous buffer (pH 7.0). On the other hand, catalase was severely inhibited by NaN3 in both media. Thus, polarography based on the Clark oxygen electrode seems to be an easy, rapid, and sensitive technique for studying enzyme reactions consuming or evolving O2 in apolar media.     

Increased beta-aminoisobutyric acid in rat liver with 6-azauracil and its enantiomer

When 6-azauracil was subcutaneously injected, beta-aminoisobutyric acid and beta-alanine contents were increased 22 and 61-fold, respectively, in rat liver. Incorporation of [methyl-14C]thymine into beta-aminoisobutyric acid was increased to 42-fold by 6-azauracil treatment. The absolute configuration of this amino acid was proved to be the (R)-form by means of a gas-chromatographic technique. 6-Azauracil inhibited beta-alanine-pyruvate aminotransferase activity with an I50 of approx. 2.5 mM.     

Interactive effects of gibberellin A<Subscript>3</Subscript> and ascorbic acid on lipid peroxidation and antioxidant enzyme activities in <Emphasis Type="Italic">Glycine max</Emphasis> seedlings under nickel stress

The effects of nickel in combination with ascorbic acid (AsA) and gibberellin on 7-day-old soybean seedlings were examined. Exposure to 0.25 mM NiCl₂ × 6H₂O for 5 days resulted in toxicity symptoms, such as formation of reddish-brown mottled spots on the leaf blade. Addition of 0.05 mM GA₃ or 1 mM AsA reduced toxic effects of nickel. After their simultaneous application, these symptoms did not appear. Ni decreased dry weights of roots and shoots and reduced chlorophyll content in leaves. An enhanced level of lipoxygenase activity and malondialdehyde, and changes in the activities of the antioxidant enzymes, catalase, guaiacol peroxidase and ascorbate peroxidase, in both roots and leaves indicated that Ni caused an oxidative stress in soybean plants. The Ni-stressed seedlings exposed to AsA or GA₃, especially to GA₃ plus AsA, exhibited an improved growth as compared to Ni-treated plants. GA₃ decreased Ni uptake by roots, while ascorbic acid considerably reduced root-to-shoot translocation of Ni. Interaction of AsA plus GA₃ prevented the decrease in chlorophyll content and lipid peroxidation as well as increased the activities of the antioxidant enzymes. These results suggest that GA₃ plus AsA treatment counteracts the negative effects of Ni on soybean seedlings. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 1, pp. 85–91. The text was submitted by the authors in English.     

The induction of systemic resistance in barley to powdery mildew infection using salicylates and various phenolic acids

Treatment of the first leaves of barley seedlings with either 5, 10, 15 or 20 mM salicylic acid, sodium salicylate or acetylsalicylic acid resulted in significant reductions in powdery mildew infection on the upper, second leaves. In general, the greatest reduction in mildew infection on the second leaves was obtained by spraying the first leaves with a 15 mM concentration of the compounds. Although the largest reduction in mildew infection of the upper leaves was obtained when the compounds were applied to the first leaves 1–2 days before inoculation, very substantial reductions in infection were still obtained if the first leaves were treated 12 days before inoculation. The three compounds had little direct effect on mildew infection. When ¹⁴C-salicylic acid was fed to first leaves of barley seedlings, uptake was rapid and increased with time. Within 6 h, 0.2% of the salicylic acid appeared in the second leaf and by 24 h after feeding, this had increased to 1.4% (1.1 μmol salicylic acid g^-1 fresh wt). The application of various phenolic acids to first leaves also led to reductions in mildew infection on the second leaves. In particular, treatment of the first leaves with 1 mM vanillic acid, isovanillic acid or syringic acid, reduced mildew infection of the second leaves by 81–87%.     

Reaction Properties of the Ascorbic Acid Oxidase from Myrothecium verrucaria

Ascorbic acid oxidase activity in Myrothecium verrucaria extracts resulted in O(2) uptake exceeding 0.5 mole per mole of ascorbic acid and in CO(2) evolution. Measurement of oxidized ascorbic acid at completion of the reaction demonstrated that an average of 10% of the oxidized product disappeared. A comparison of the gas exchange data with the amount of ascorbic acid not accounted for indicated that the reaction could not be explained by independent oxidase and oxygenase systems. Chromatographic examination of the reaction mixtures identified l-threonic acid. Experiments with ascorbic acid-1-(14)C showed that C-1 was partially decarboxylated during the oxidation. Test of the fungal extracts for enzymes that might explain the deviation from expected stoichiometry showed that phenolase, glutathione reductase, cytochrome oxidase, peroxidase and oxalic decarboxylase were not involved. Addition of azide in concentrations sufficient to block catalase increased excess O(2) consumption about 65%. No enzymes were found that could directly attack oxidized ascorbic acid. H(2)O(2) accumulated during oxidation in azide-blocked systems.The O(2) excess could be explained by assuming the enzyme had peroxidative capacity on a reductant other than ascorbic acid. An intermediate of ascorbic acid oxidation appeared to function as the substrate yielding CO(2) and l-threonic acid on degradation. The increase in excess O(2) utilized in azide-blocked systems and the H(2)O(2) accumulation also were explained by the proposed scheme.Another interpretation would involve production of free radicals during ascorbic acid oxidation. Evidence for this was the ability of extracts to oxidize DPNH in the presence of ascorbic acid. Oxygen radicals formed in such reactions were considered possible agents of degradation of ascorbic acid.     

Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents

The C-8 position of deoxyguanosine (dGuo) was hydroxylated by ascorbic acid in the presence of oxygen (O2) in 0.1 M phosphate buffer (pH 6.8) at 37 degrees C. Addition of hydrogen peroxide (H2O2) remarkably enhanced this hydroxylation. The Udenfriend system [ascorbic acid, FeII, ethylenediaminetetraacetic acid (EDTA) and O2] was also effective for hydroxylation of dGuo in high yield. Guanine residues in DNA were also hydroxylated by ascorbic acid. Other reducing agents, such as hydroxylamine, hydrazine, dihydroxymaleic acid, sodium bisulfite and acetol, were also effective for the hydroxylation reaction, as were metal-EDTA complexes (FeII-, SnII-, TiIII-, CuI-EDTA). An OH radical seemed to be involved in this hydroxylation reaction in most of the above hydroxylating systems, but another reaction mechanism may also be involved, particularly when dGuo is hydroxylated by ascorbic acid alone or ascorbic acid plus H2O2. The possible biological significance of the hydroxylation of guanine residues in DNA in relation to mutagenesis and carcinogenesis is discussed.     

A study of the supposed hydroxylation of tyrosine catalysed by peroxidase.

The claim that peroxidase (rather than tyrosinase) is the enzyme responsible for the conversion of tyrosine into dopa (3,4-dihydroxyphenylalanine) in melanogenesis was investigated. The spectral changes that occurred during the action of horseradish peroxidase in the presence of H2O2 on dopa, tyrosine and mixtures of dopa with tyrosine or other phenolic compounds were studied. The effect of ascorbic acid or dihydroxyfumaric acid on some of these changes was also investigated. No evidence was found that tyrosine was hydroxylated by peroxidase in the presence of H2O2 and dopa as cofactor, although tyrosine or other phenolic compounds increased the rate of oxidation of dopa to dopachrome (indoline-5,6-quinone-2-carboxylic acid). Peroxidase was, however, effective in oxidizing tyrosine to dopa in the presence of dihydroxyfumaric acid and oxygen.     

Ascorbate activates soluble guanylate cyclase via H2O2-metabolism by catalase

Conditions necessary for the activation by ascorbic acid of soluble guanylate cyclase purified from bovine lung have been examined. Ascorbic acid (0.1-10 mM) did not directly activate the enzyme, nonetheless, pronounced activation by ascorbate (3-10 mM) was observed in incubation mixtures containing 1 microM bovine liver catalase. Superoxide dismutase (SOD) and mannitol did not affect the catalase-dependent activation of guanylate cyclase elicited by ascorbate, suggesting that superoxide anion and hydroxyl radical were not mediating the activation of the enzyme. However, SOD enhanced the relatively low level activation of the enzyme elicited by catalase in the absence of added ascorbate. Pronounced inhibition (both with and without added ascorbate) was observed of catalase-dependent activation of guanylate cyclase by either ethanol (100 mM) or a fungal catalase preparation. Neither ethanol nor fungal catalase inhibited activation of guanylate cyclase by S-nitrosyl-N-acetyl-penicillamine (SNAP), a source of the nitric oxide free radical. These observations indicate that autoxidation of ascorbic acid or thiols present with the guanylate cyclase preparation leads to generation of H2O2, and its metabolism by bovine liver catalase mediates the concomitant activation of guanylate cyclase. The mechanism of activation appears to be associated with the presence of Compound I of catalase and to be inhibited by superoxide anion.     

Anti-oxidant activity of spermine and spermidine re-evaluated with oxidizing systems involving iron and copper ions

This study was designed to investigate the direction of redox reactions of spermine and spermidine in the presence of iron and copper. The redox activity of spermine and spermidine was assessed using a variety of methods, including their ability to: (1) reduce Fe(3+) to Fe(2+) ions; (2) protect deoxyribose from oxidation by Fe(2+)-ethylene diaminetetraacetic acid, Fe(3+)-ethylene diaminetetraacetic acid systems with and without H(2)O(2); (3) protect DNA from damage caused by Cu(2+)-H(2)O(2), and Fe(2+)-H(2)O(2) with and without ascorbic acid; (4) inhibit H(2)O(2)-peroxidase-induced luminol dependent chemiluminescence; (5) scavenge diphenyl-picryl-hydrazyl radical. Spermine and spermidine at concentration 1mM reduced 1.8+/-0.3 and 2.5+/-0.1 nmol of Fe(3+) ions during 20 min incubation. Both polyamines enhanced deoxyribose oxidation. The highest enhancement of 7.6-fold in deoxyribose degradation was found for combination of spermine with Fe(3+)-ethylene diaminetetraacetic acid. An 10mM spermine and spermidine decreased CuSO(4)-H(2)O(2)-ascorbic acid- and FeSO(4)-H(2)O(2)-ascorbic-induced DNA damage by 73+/-6, 69+/-4% and 90+/-5, 53+/-4%, respectively. They did not protect DNA from CuSO(4)-H(2)O(2) and FeSO(4)-H(2)O(2). Spermine apparently increased the CuSO(4)-H(2)O(2)-dependent injury to DNA. Polyamines attenuated H(2)O(2)-peroxidase-induced luminol dependent chemiluminescence. Total light emission from specimens containing 10mM spermine or spermidine was attenuated by 85.3+/-1.5 and 87+/-3.6%. During 20 min incubation 1mM spermine or spermidine decomposed 8.1+/-1.4 and 9.2+/-1.8% of diphenyl-picryl-hydrazyl radical. These results demonstrate that polyamines of well known anti-oxidant properties may act as pro-oxidants and enhance oxidative damage to DNA components in the presence of free iron ions and H(2)O(2).     

Diethylmaleate decreased ascorbic acid release induced by cerebral ischemia in cerebral cortex of the anesthetized rat

The effect of diethylmaleate administration on ascorbic acid release following cerebral ischemia was investigated in anesthetized rat brain cortex. Cerebral ischemia, induced by ligating bilateral common carotid arteries and unilateral middle cerebral artery, significantly increased the extracellular ascorbic acid levels. Diethylmaleate (4 mmoles/kg, i.p.), which has been shown in earlier studies to decrease the ischemia-induced glutamate release, significantly reduced the ischemia-induced ascorbic acid release. The ischemia-induced ascorbic acid release was unaffected by perfusing NMDA receptor antagonist MK 801 (75 microM). Additionally, elevated extracellular glutamate levels, achieved by either externally applied glutamate solutions or by perfusing L-trans-pyrrolidine-2,4-dicarboxylate (PDC) (31.4 mM and 15.7 mM) to inhibit the glutamate uptake transporter, also significantly increased the extracellular ascorbic acid levels. These results suggested that ascorbic acid release in cerebral ischemia might be related to the elevated extracellular glutamate levels, which occurs following cerebral ischemia.     

Inactivation of alpha 1-antiproteinase by hydroxyl radicals. The effect of uric acid

The elastase-inhibitory activity of alpha 1-antiproteinase is inactivated by hydroxyl radicals (.OH) generated by pulse radiolysis or by reaction of iron ions with H2O2 in the presence of superoxide or ascorbate. Uric acid did not protect alpha 1-antiproteinase against inactivation by .OH in pulse radiolysis experiments or in the superoxide/iron/H2O2 system, whereas it did in systems containing ascorbic acid. We propose that radicals formed by attack of .OH on uric acid are themselves able to inactivate alpha 1-antiproteinase, but that these uric acid radicals can be 'repaired' by ascorbic acid.