Miloš Mojović

Generation of hydroxyl radical in isolated pea root cell wall, and the role of cell wall-bound peroxidase, Mn-SOD and phenolics in their production.

The hydroxyl radical produced in the apoplast has been demonstrated to facilitate cell wall loosening during cell elongation. Cell wall-bound peroxidases (PODs) have been implicated in hydroxyl radical formation. For this mechanism, the apoplast or cell walls should contain the electron donors for (i) H(2)O(2) formation from dioxygen; and (ii) the POD-catalyzed reduction of H(2)O(2) to the hydroxyl radical. The aim of the work was to identify the electron donors in these reactions. In this report, hydroxyl radical (.OH) generation in the cell wall isolated from pea roots was detected in the absence of any exogenous reductants, suggesting that the plant cell wall possesses the capacity to generate .OH in situ. Distinct POD and Mn-superoxide dismutase (Mn-SOD) isoforms different from other cellular isoforms were shown by native gel electropho-resis to be preferably bound to the cell walls. Electron paramagnetic resonance (EPR) spectroscopy of cell wall isolates containing the spin-trapping reagent, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), was used for detection of and differentiation between .OH and the superoxide radical (O(2)(-).). The data obtained using POD inhibitors confirmed that tightly bound cell wall PODs are involved in DEPMPO/OH adduct formation. A decrease in DEPMPO/OH adduct formation in the presence of H(2)O(2) scavengers demonstrated that this hydroxyl radical was derived from H(2)O(2). During the generation of .OH, the concentration of quinhydrone structures (as detected by EPR spectroscopy) increased, suggesting that the H(2)O(2) required for the formation of .OH in isolated cell walls is produced during the reduction of O(2) by hydroxycinnamic acids. Cell wall isolates in which the proteins have been denaturated (including the endogenous POD and SOD) did not produce .OH. Addition of exogenous H(2)O(2) again induced the production of .OH, and these were shown to originate from the Fenton reaction with tightly bound metal ions. However, the appearance of the DEPMPO/OOH adduct could also be observed, due to the production of O(2)(-). when endogenous SOD has been inactivated. Also, O(2)(-). was converted to .OH in an in vitro horseradish peroxidase (HRP)/H(2)O(2) system to which exogenous SOD has been added. Taken together with the discovery of the cell wall-bound Mn-SOD isoform, these results support the role of such a cell wall-bound SOD in the formation of .OH jointly with the cell wall-bound POD. According to the above findings, it seems that the hydroxycinnamic acids from the cell wall, acting as reductants, contribute to the formation of H(2)O(2) in the presence of O(2) in an autocatalytic manner, and that POD and Mn-SOD coupled together generate .OH from such H(2)O(2).

Silicon ameliorates manganese toxicity in cucumber by decreasing hydroxyl radical accumulation in the leaf apoplast

This work was focused on the role of silicon (Si) in amelioration of manganese (Mn) toxicity caused by elevated production of hydroxyl radicals (·OH) in the leaf apoplast of cucumber (Cucumis sativus L.). The plants were grown in nutrient solutions with adequate (0.5 μM) or excessive (100 μM) Mn concentrations with or without Si being supplied. The symptoms of Mn toxicity were absent in the leaves of Si-treated plants subjected to excess Mn, although the leaf Mn concentration remained extremely high. The apoplastic concentration of free Mn(2+) and H(2)O(2) of high Mn-treated plants was significantly decreased by Si treatment. Si supply suppressed the Mn-induced increased abundance of peroxidase (POD) isoforms in the leaf apoplastic fluid, and led to a rapid suppression of guaiacol-POD activity under excess Mn. The spin-trapping reagent 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide was used to detect ·OH by electron paramagnetic resonance spectroscopy. Although supplying Si markedly decreased the accumulation of ·OH in the leaf apoplast with excess Mn, adding monosilicic acid to the Mn(2+)/H(2)O(2) reaction mixture did not directly affect the Fenton reaction in vitro. The results indicate that Si contributes indirectly to a decrease in ·OH in the leaf apoplast by decreasing the free apoplastic Mn(2+), thus regulating the Fenton reaction. A direct inhibitory effect of Si on guaiacol-POD activity (demonstrated in vitro) may also contribute to decreasing the POD-mediated generation of ·OH.

Synthesis and characterization of self-assembled polyaniline nanotubes/silica nanocomposites.

Self-assembled semiconducting, paramagnetic polyaniline nanotubes have been synthesized by the oxidative polymerization of aniline with ammonium peroxydisulfate in aqueous medium in the presence of colloidal silica particles of an average diameter approximately 12 nm, without added acid. The electrical conductivity of polyaniline nanotubes/silica nanocomposites is in the range (3.3-4.0)x10(-3) S cm(-1). The presence of paramagnetic polaronic emeraldine salt form of polyaniline and phenazine units in nanocomposites was proved by FTIR, Raman, and EPR spectroscopies. The influence of the initial silica/aniline weight ratio on the morphology of polyaniline/silica nanocomposites was studied by scanning and transmission electron microscopies. Nanocomposites synthesized by using the initial weight ratio silica/aniline<or=0.2 contain polyaniline nanotubes which have a typical outer diameter of 100-250 nm and an inner diameter of 10-80 nm, and nanorods with a diameter of 60-100 nm, accompanied with polyaniline/silica nanogranules, while the nanocomposite synthesized at weight ratio silica/aniline approximately 2 contains polyaniline/silica nanogranules with an average diameter of 35-70 nm. The evolution of molecular and supramolecular structure of polyaniline in the presence of colloidal silica is discussed.

Synthesis and Characterization of Conducting Self-Assembled Polyaniline Nanotubes/Zeolite Nanocomposite

Self-assembled conducting, paramagnetic polyaniline nanotubes have been synthesized by the oxidative polymerization of aniline with ammonium peroxydisulfate in aqueous medium in the presence of zeolite HZSM-5, without added acid. The influence of initial zeolite/aniline weight ratio on the conductivity, molecular and supramolecular structure, paramagnetic characteristics, thermal stability, and specific surface area of polyaniline/zeolite composites was studied. The conducting (approximately 10(-2) S cm(-1)), semiconducting (3 x 10(-5) S cm(-1)), and nonconducting (5 x 10(-9) S cm(-1)) composites are produced using the zeolite/aniline weight ratios 1, 5, and 10, respectively. The coexistence of polyaniline nanotubes, which have a typical outer diameter of 70-170 nm and an inner diameter of 5-50 nm, accompanied by nanorods with a diameter of 60-100 nm and polyaniline/zeolite mesoporous aggregates, distinct from the morphology of microporous zeolite HZSM-5, was proved in the conducting nanocomposite by scanning and transmission electron microscopies. FTIR spectroscopy confirmed the presence of polyaniline in the form of conducting emeraldine salt and suggested significant interaction of polyaniline with zeolite. The evolution of molecular and supramolecular structure of polyaniline in the presence of zeolite was discussed.

Relevance of the capacity of phosphorylated fructose to scavenge the hydroxyl radical

The hydroxyl radical (*OH) has detrimental biological activity due to its very high reactivity. Our experiments were designed to determine the effects of equimolar concentrations of glucose, fructose and mannitol and three phosphorylated forms of fructose (fructose-1-phosphate (F1P); fructose-6-phosphate (F6P); and fructose-1,6-bis(phosphate) (F16BP)) on *OH radical production via the Fenton reaction. EPR spectroscopy using spin-trap DEPMPO was applied to detect radical production. We found that the percentage inhibition of *OH radical formation decreased in the order F16BP>F1P>F6P>fructose>mannitol=glucose. As ketoses can sequester redox-active iron thus preventing the Fenton reaction, the Haber-Weiss-like system was also employed to generate *OH, so that the effect of iron sequestration could be distinguished from direct *OH radical scavenging. In the latter system, the rank order of *OH scavenging activity was F16BP>F1P>F6P>fructose=mannitol=glucose. Our results clearly demonstrate that intracellular phosphorylated forms of fructose have more scavenging properties than fructose or glucose, leading us to conclude that the acute administration of fructose could overcome the bodys reaction to exogenous antioxidants during appropriate therapy in certain pathophysiological conditions related to oxidative stress, such as sepsis, neurodegenerative diseases, atherosclerosis, malignancy, and some complications of pregnancy.

Zinc-induced oxidative stress in Verbascum thapsus is caused by an accumulation of reactive oxygen species and quinhydrone in the cell wall

Oxidative stress is one aspect of metal toxicity. Zinc, although unable to perform univalent oxido-reduction reactions, can induce the oxidative damage of cellular components and alter antioxidative systems. Verbascum thapsus L. plants that were grown hydroponically were exposed to 1 and 5 mM Zn²+. Reactive oxygen species (ROS) accumulation was demonstrated by the fluorescent probe H₂ DCFDA and EPR measurements. The extent of zinc-induced oxidative damage was assessed by measuring the level of protein carbonylation. Activities and isoform profiles of some antioxidant enzymes and the changes in ascorbate and total phenolic contents of leaves and roots were determined. Stunted growth because of zinc accumulation, preferentially in the roots, was accompanied by H₂O₂ production in the leaf and root apoplasts. Increased EPR signals of the endogenous oxidant quinhydrone, •CH₃ and •OH, were found in the cell walls of zinc-treated plants. The activities of the antioxidative enzymes ascorbate peroxidase (APX) (EC 1.11.1.11), soluble superoxide dismutase (SOD) (EC 1.15.1.1), peroxidase (POD), (EC 1.11.1.7) and monodehydroascorbate reductase (EC 1.6.5.4) were increased; those of glutathione reductase (EC 1.6.4.2), dehydroascorbate reductase (EC 1.8.5.1) and ascorbate oxidase (AAO) (EC 1.10.3.3) were decreased with zinc treatment. Zinc induced a cell-wall-bound SOD isoform in both organs. Leaves accumulated more ascorbate and phenolics in comparison to roots. We propose a mechanism for zinc-promoted oxidative stress in V. thapsus L. through the generation of charge transfer complexes and quinhydrone because of phenoxyl radical stabilisation by Zn²+ in the cell wall. Our results suggest that the SOD and APX responses are mediated by ROS accumulation in the apoplast. The importance of the POD/Phe/AA (ascorbic acid) scavenging system in the apoplast is also discussed.

Flavonolignan 2,3-dehydroderivatives: Preparation, antiradical and cytoprotective activity

The protective constituents of silymarin, an extract from Silybum marianum fruits, have been extensively studied in terms of their antioxidant and hepatoprotective activities. Here, we explore the electron-donor properties of the major silymarin flavonolignans. Silybin (SB), silychristin (SCH), silydianin (SD) and their respective 2,3-dehydroderivatives (DHSB, DHSCH and DHSD) were oxidized electrochemically and their antiradical/antioxidant properties were investigated. Namely, Folin-Ciocalteau reduction, DPPH and ABTS(+) radical scavenging, inhibition of microsomal lipid peroxidation and cytoprotective effects against tert-butyl hydroperoxide-induced damage to a human hepatocellular carcinoma HepG2 cell line were evaluated. Due to the presence of the highly reactive C3-OH group and the C-2,3 double bond (ring C) allowing electron delocalization across the whole structure in the 2,3-dehydroderivatives, these compounds are much more easily oxidized than the corresponding flavonolignans SB, SCH and SD. This finding was unequivocally confirmed not only by experimental approaches, but also by density functional theory (DFT) calculations. The hierarchy in terms of ability to undergo electrochemical oxidation (DHSCH~DHSD>DHSB>>SCH/SD>SB) was consistent with their antiradical activities, mainly DPPH scavenging, as well as in vitro cytoprotection of HepG2 cells. The results are discussed in the context of the antioxidant vs. prooxidant activities of flavonolignans and molecular interactions in complex biological systems.

Anti-cancer effects of cerium oxide nanoparticles and its intracellular redox activity

Data on medical applications of cerium oxide nanoparticles CeO2 (CONP) are promising, yet information regarding their action in cells is incomplete and there are conflicting reports about in vitro toxicity. Herein, we have studied cytotoxic effect of CONP in several cancer and normal cell lines and their potential to change intracellular redox status. The IC50 was achieved only in two of eight tested cell lines, melanoma 518A2 and colorectal adenocarcinoma HT-29. Self-propagating room temperature method was applied to produce CONP with an average crystalline size of 4 nm. The results confirmed presence of Ce(3+) and O(2-) vacancies. The induction of cell death by CONP and the production of reactive oxygen species (ROS) were analyzed by flow-cytometry. Free radicals related antioxidant capacity of the cells was studied by the reduction of stable free radical TEMPONE using electron spin resonance spectroscopy. CONP showed low or moderate cytotoxicity in cancer cell lines: adenocarcinoma DLD1 and multi-drug resistant DLD1-TxR, non-small cell lung carcinoma NCI-H460 and multi-drug resistant NCI-H460/R, while normal cell lines (keratinocytes HaCaT, lung fetal fibroblasts MRC-5) were insensitive. The most sensitive were 518A2 melanoma and HT-29 colorectal adenocarcinoma cell lines, with the IC50 values being between 100 and 200 μM. Decreased rate of TEMPONE reduction and increased production of certain ROS species (peroxynitrite and hydrogen peroxide anion) indicates that free radical metabolism, thus redox status was changed, and antioxidant capacity damaged in the CONP treated 518A2 and HT-29 cells. In conclusion, changes in intracellular redox status induced by CONP are partly attributed to the prooxidant activity of the nanoparticles. Further, ROS induced cell damages might eventually lead to the cell death. However, low inhibitory potential of CONP in the other human cell lines tested indicates that CONP may be safe for human usage in industry and medicine.

Role of fructose in the adaptation of plants to cold-induced oxidative stress.

This work presents findings, which indicate important role of fructose, fructose 6-phosphate (F6P), and fructose 1,6-bisphosphate (FBP) in preservation of homeostasis in plants under low temperature. Cold combined with light is known to incite increased generation of superoxide in chloroplasts leading to photoinhibition, but also an increased level of soluble sugars. In the present study, oxidative stress in pea leaves provoked by cold/light regime was asserted by the observed decrease of the level of oxidized form of PSI pigment P700 (P700+). Alongside, the increased antioxidative status and the accumulation of fructose were observed. The antioxidative properties of fructose and its phosphorylated forms were evaluated to appraise their potential protective role in plants exposed to chilling stress. Fructose, and particularly F6P and FBP exhibited high capacities for scavenging superoxide and showed to be involved in antioxidative protection in pea leaves. These results combined with previously established links implicate that the increase in level of fructose sugars through various pathways intercalated into physiological mechanisms of homeostasis represents important non-enzymatic antioxidative defense in plants under cold-related stress.

NO Dismutase Activity of Seven‐Coordinate Manganese(II) Pentaazamacrocyclic Complexes

Seven-coordinate Mn pentaazamacrocyclic complexes represent the most potent synthetic mimics of native superoxide dismutase (SOD), which catalyze dismutation of superoxide (O2C ) into O2 andH2O2 with efficiency that can exceed that of the mitochondrial MnSOD. A number of studies demonstrated the ability of these SOD mimics to protect cells and tissues from oxidative damage caused by superoxide (and/or the product of its reaction with nitric oxide, peroxynitrite), for example in inflammation and oxidation reperfusion injury. The members of this class of SODmimics have entered Phase II clinical trials in the USA. It is emphasized that the major advantage of Mn pentaazamacrocycles over other SOD mimics is their high selectivity for O2C and lack of reactivity with NO, a key molecule in biological processes. However, direct studies of the reaction between NO and this class of complexes, which would support such claims, have not been reported. Various metal complexes, including manganese complexes, react readily with NO, either to yield metal nitrosyls, or to produce N2O and metal nitrite complexes by NO disproportionation. Coordinated NO in metal nitrosyls can exist in one of its three formal redox states NO, NO, and NO . For a number of NO and NO complexes reactivity towards selected nucleophiles and electrophiles, respectively, has been documented. We demonstrated recently that natural MnSOD enzyme reacts with NO according to distinct catalytic NO disproportionation (dismutation) mechanism which yields both reactive species NO and NO . Collectively these results prompted us to (re)examine the reaction of these complexes with NO. The chosen approach has been 1) to establish the reactivity of complexes with NO, 2) to establish the mechanistic details of the reaction, and 3) to demonstrate the potential validity of the complex reaction with NO in a biological setting. In the present study we used [Mn(pyane)Cl2] (1), [8] as a general representative of this class of SOD mimics, and its