Influence of dicarbonyls on kinetic characteristics of glutathione peroxidase

Se-containing glutathione peroxidase (GSH-Px) is one of the key enzymes of the body’s antioxidant system. The kinetic characteristics of GSH-Px (substrate is tert-butyl hydroperoxide) after modification of the enzyme by various concentrations of natural dicarbonyls (glyoxal, methylglyoxal, malonic dialdehyde) were studied. It was shown that dicarbonyls affected both K _m and V _max for GSH-Px. It is suggested that the effect of various dicarbonyls on GSH-Px depends on the molecular mechanisms of their interaction with the amino acid residues of the enzyme.     

Methylglyoxal as a scavenger for superoxide anion-radical

Methylglyoxal at a concentration of 5 mM caused a significant inhibition of superoxide anion radical (O₂^·-) comparable to the effect of Tirone. In the process of O₂^·- generation in the system of egg phosphatidylcholine liposome peroxidation induced by the azo-initiator AIBN, a marked inhibition of chemiluminescence in the presence of 100 mM methylglyoxal was found. At the same time, methylglyoxal did not inhibit free radical peroxidation of low-density lipoprotein particles, which indicates the absence of interaction with methylglyoxal alkoxyl and peroxyl polyenoic lipid radicals. These findings deepen information about the role of methylglyoxal in the regulation of free radical processes.     

The initiation of free radical peroxidation of low-density lipoproteins by glucose and its metabolite methylglyoxal: a common molecular mechanism of vascular wall injure in atherosclerosis and diabetes

It was found that glucose in the range of concentrations 12.5–100 mM stimulated Cu²⁺–mediated free radical peroxidation of low-density lipoproteins (LDL) from human blood plasma. Considering the kinetic parameters of LDL peroxidation we proposed that intensification of this process may be caused by formation of free radical intermediates of glucose auto-oxidation. Addition of SOD to the medium inhibited LDL oxidation, indicating the formation of superoxide anion-radicals under autoxidation of glucose. Similarly, SOD inhibited free radical peroxidation of liposomes from egg lecithin in the presence of glucose that confirms the generation of superoxide radicals under co-oxidation of unsaturated lipids and glucose. Normalization of glucose level in the blood of patients with type 2 diabetes mellitus during therapy was accompanied by a significant decrease in LDL oxidation in vivo (the decrease in primary and secondary lipoperoxidation products). The formation of superoxide anion-radicals was observed during interaction of aminoacid l-lysine with a product of glucose oxidative metabolism–methylglyoxal, but not with a product of lipoperoxidation malonyldialdehyde. In accordance with the foregoing the administration of sugar-lowering drug metformin, which binds and utilizes methylglyoxal, caused a stronger inhibition of LDL peroxidation in the blood of patients with diabetes mellitus, probably due to decrease in methylglyoxal-dependent generation of superoxide anion-radicals. Based on the results we set out the hypothesis about autocatalytic mechanism of free radical reactions involving natural dicarbonyls and suppose the common molecular mechanism of vascular wall injury in atherosclerosis and diabetes.     

Impact of SOD in eNOS uncoupling: a two-edged sword between hydrogen peroxide and peroxynitrite

In endothelial cell dysfunction, the uncoupling of eNOS results in higher superoxide (O₂^??) and lower NO production and a reduction in NO availability. Superoxide reacts with NO to form a potent oxidizing agent peroxynitrite (ONOO^?) resulting in nitrosative and nitroxidative stresses and dismutates to form hydrogen peroxide. Studies have shown superoxide dismutase (SOD) plays an important role in reduction of O₂^?? and ONOO^? during eNOS uncoupling. However, the administration or over-expression of SOD was ineffective or displayed deleterious effects in some cases. An understanding of interactions of the two enzyme systems eNOS and SOD is important in determining endothelial cell function. We analyzed complex biochemical interactions involving eNOS and SOD in eNOS uncoupling. A computational model of biochemical pathway of the eNOS-related NO and O₂^?? production and downstream reactions involving NO, O₂^??, ONOO^?, H₂O₂ and SOD was developed. The effects of SOD concentration on the concentration profiles of NO, O₂^??, ONOO^? and H₂O₂ in eNOS coupling/uncoupling were investigated. The results include (i) SOD moderately improves NO production and concentration during eNOS uncoupling, (ii) O₂^?? production rate is independent of SOD concentration, (iii) Increase in SOD concentration from 0.1 to 100 μM reduces O₂^?? concentration by 90% at all [BH₄]/[TBP] ratios, (iv) SOD reduces ONOO^? concentration and increases H₂O₂ concentration during eNOS uncoupling, (v) Catalase can reduce H₂O₂ concentration and (vi) Dismutation rate by SOD is the most sensitive parameter during eNOS uncoupling. Thus, SOD plays a dual role in eNOS uncoupling as an attenuator of nitrosative/nitroxidative stress and an augmenter of oxidative stress.     

Ferricytochrome c Directly Oxidizes Aminoacetone to Methylglyoxal,a Catabolite Accumulated in Carbonyl Stress

Age-related diseases are associated with increased production of reactive oxygen and carbonyl species such as methylglyoxal. Aminoacetone, a putative threonine catabolite, is reportedly known to undergo metal-catalyzed oxidation to methylglyoxal, NH₄ ⁺ ion, and H₂O₂ coupled with (i) permeabilization of rat liver mitochondria, and (ii) apoptosis of insulin-producing cells. Oxidation of aminoacetone to methylglyoxal is now shown to be accelerated by ferricytochrome c, a reaction initiated by one-electron reduction of ferricytochrome c by aminoacetone without amino acid modifications. The participation of O₂ ^•− and HO^• radical intermediates is demonstrated by the inhibitory effect of added superoxide dismutase and Electron Paramagnetic Resonance spin-trapping experiments with 5,5′-dimethyl-1-pyrroline-N-oxide. We hypothesize that two consecutive one-electron transfers from aminoacetone (E₀ values = −0.51 and −1.0 V) to ferricytochrome c (E₀ = 0.26 V) may lead to aminoacetone enoyl radical and, subsequently, imine aminoacetone, whose hydrolysis yields methylglyoxal and NH₄ ⁺ ion. In the presence of oxygen, aminoacetone enoyl and O₂ ^•− radicals propagate aminoacetone oxidation to methylglyoxal and H₂O₂. These data endorse the hypothesis that aminoacetone, putatively accumulated in diabetes, may directly reduce ferricyt c yielding methylglyoxal and free radicals, thereby triggering redox imbalance and adverse mitochondrial responses.     

Hypoosmotic hemolysis of erythrocytes by active carbonyl forms

Low-molecular-weight dicarbonyls formed during free radical peroxidation of polyene lipids (malondialdehyde) and autooxidation (glyoxal) or other oxidative transformations of glucose (methylglyoxal) are able to modify the structure of lipid-protein supramolecular complexes of cells. We investigated changes in the erythrocyte membrane structure after an 18-h exposure of human red blood cells in the presence of various natural dicarbonyls. The changes in the mechanical properties of the membrane after membrane modification by carbonyls were evaluated by the susceptibility of erythrocytes to hypoosmotic hemolysis. It has been shown that treatment of red blood cells with malondialdehyde increases the resistance of these cells to hypoosmotic hemolysis, whereas the malondialdehyde isomer, methylglyoxal, in contrast, makes red blood cells more sensitive to the action of hypoosmotic solutions. Paradoxically, a homologue of malondialdehyde, glyoxal, has no effect on hemolysis of red blood cells in hypoosmotic solutions. The findings point to the possibility of the multidirectional effect of low-molecular-weight dicarbonyls with similar structures on the structure and function of biological membranes.     

Hydrogen peroxide is critical for UV-induced apoptosis inhibition

Abstract

Apoptosis is an important cell death system that deletes damaged and mutated cells, preventing the induction of cancer. We previously have reported that UV irradiation inhibited the apoptosis induced by serum starvation and cell detachment. This phenomenon is suitable for clarifying the relationship between cancer and the dysregulation of apoptosis by UV irradiation. Here, we have studied the factors responsible for this inhibition of apoptosis, focusing on reactive oxygen species (ROS) and DNA damage. Treatment with xanthine oxidase in the presence of hypoxanthine, which is known to produce superoxide anion (O₂^??) and hydrogen peroxide (H₂O₂), inhibited the induction of apoptosis. The xanthine oxidase-induced anti-apoptotic effect was suppressed in the presence of an H₂O₂-eliminating enzyme, catalase, but not in the presence of an O₂^??-eliminating enzyme, superoxide dismutase. Treatment with H₂O₂ itself significantly inhibited the induction of apoptosis. Furthermore, the effect of the inhibition of cell death by UVB irradiation and by H₂O₂ treatment decreased in H₂O₂-resistant cells. Although both UVB and H₂O₂ are known to induce DNA damage, other DNA damaging agents, like γ-irradiation and treatment with cisplatin and bleomycin, showed no inhibition of apoptosis. These findings suggested that H₂O₂ was essential to the inhibition of apoptosis, in which DNA damage had no role.     

A mitochondria-targeted mass spectrometry probe to detect glyoxals: implications for diabetes

The glycation of protein and nucleic acids that occurs as a consequence of hyperglycemia disrupts cell function and contributes to many pathologies, including those associated with diabetes and aging. Intracellular glycation occurs after the generation of the reactive 1,2-dicarbonyls methylglyoxal and glyoxal, and disruption of mitochondrial function is associated with hyperglycemia. However, the contribution of these reactive dicarbonyls to mitochondrial damage in pathology is unclear owing to uncertainties about their levels within mitochondria in cells and in vivo. To address this we have developed a mitochondria-targeted reagent (MitoG) designed to assess the levels of mitochondrial dicarbonyls within cells. MitoG comprises a lipophilic triphenylphosphonium cationic function, which directs the molecules to mitochondria within cells, and an o-phenylenediamine moiety that reacts with dicarbonyls to give distinctive and stable products. The extent of accumulation of these diagnostic heterocyclic products can be readily and sensitively quantified by liquid chromatography–tandem mass spectrometry, enabling changes to be determined. Using the MitoG-based analysis we assessed the formation of methylglyoxal and glyoxal in response to hyperglycemia in cells in culture and in the Akita mouse model of diabetes in vivo. These findings indicated that the levels of methylglyoxal and glyoxal within mitochondria increase during hyperglycemia both in cells and in vivo, suggesting that they can contribute to the pathological mitochondrial dysfunction that occurs in diabetes and aging.     

Puerarin inhibits the retinal pericyte apoptosis induced by advanced glycation end products in vitro and in vivo by inhibiting NADPH oxidase-related oxidative stress

Activation of NAD(P)H oxidase has been reported to produce superoxide (O₂^??) extracellularly as an autocrine/paracrine regulator or intracellularly as a signaling messenger in a variety of mammalian cells. However, it remains unknown how the activity of NAD(P)H oxidase is regulated in arterial myocytes. Recently, CD38-associated ADP-ribosylcyclase has been reported to use an NAD(P)H oxidase product, NAD⁺ or NADP⁺, to produce cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate, which mediates intracellular Ca^2 + signaling. This study was designed to test a hypothesis that the CD38/cADPR pathway as a downstream event exerts feedback regulatory action on the NAD(P)H oxidase activity in production of extra- or intracellular O₂^?? in mouse coronary arterial myocytes (CAMs). By fluorescence microscopic imaging, we simultaneously monitored extra- and intracellular O₂^?? production in wild-type (CD38^+/+) and CD38 knockout (CD38^?/?) CAMs in response to oxotremorine (OXO), a muscarinic type 1 receptor agonist. It was found that CD38 deficiency prevented OXO-induced intracellular but not extracellular O₂^?? production in CAMs. Consistently, the OXO-induced intracellular O₂^?? production was markedly inhibited by CD38 shRNA or the CD38 inhibitor nicotinamide in CD38^+/+ CAMs. Further, Nox4 siRNA inhibited OXO-induced intracellular but not extracellular O₂^?? production, whereas Nox1 siRNA attenuated both intracellular and extracellular O₂^?? production in CD38^+/+ CAMs. Direct delivery of exogenous cADPR into CAMs markedly elevated intracellular Ca^2 + and O₂^?? production in CD38^?/? CAMs. Functionally, CD38 deficiency or Nox1 siRNA and Nox4 siRNA prevented OXO-induced contraction in isolated perfused coronary arteries in CD38 WT mice. These results provide direct evidence that the CD38/cADPR pathway is an important controller of Nox4-mediated intracellular O₂^?? production and that CD38-dependent intracellular O₂^?? production is augmented in an autocrine manner by CD38-independent Nox1-derived extracellular O₂^?? production in CAMs.     

Superoxide Generation in Chemically Activated Resistance of Barley in Response to Inoculation with the Powdery Mildew Fungus

In a previous work, a phenotype-specific accumulation of superoxide radical anions (O^??₂) after attack of the powdery mildew fungus (Blumeria [syn. Erysiphe] graminis f.sp. hordei) in near-isogenic barley (Hordeum vulgare L.) lines bearing different Mlx genes for resistance was described (Hückelhoven and Kogel, 1998). We have now a histochemical study of the pathogenesis-related O^??₂ generation in the systemic activated resistance (SAR) response induced in barley cv Pallas by the plant activator 2,6-dichloroisonicotinic acid (DCINA). SAR-specific defence was conducted prevalently characterized by penetration resistance. Fungal arrest was observed before haustorium formation by a highly localized cell wall reinforcement (effective papillae) and, in most cases, by a subsequent hypersensitive cell death (HR). No O^??₂ generation was found in association with these plant defence responses. However, a strong O^??₂ burst in the attacked epidermal cells was detected in the control plants which were not activated by DCINA. This burst coincided with cell wall penetration and subsequent contact of the pathogen with the host plasma membrane. A strong SAR-related O^??₂ burst was induced in the mesophyll tissue beneath the attacked and hypersensitively reacting epidermal cells in plants treated with DCINA. The accumulation of O^??₂ was confined to chloroplasts. The remarkable burst in mesophyll tissue was not followed by mesophyll-HR indicating that chloroplastic O^??₂ generation is not sufficient for the hypersensitive cell death. Since the same pattern of pathogenesis-related O^??₂ accumulation was identified for race-specific response mediated by the Mlg gene for powdery mildew resistance, the present data are consistent with the hypothesis that the SAR phenotype is a phenocopy of the Mlg-type resistance (Kogel et al., 1994).     

Monoamine-dependent Production of Reactive Oxygen Species Catalyzed by Pseudoperoxidase Activity of Human Hemoglobin

Hemoglobin (Hb) solution-based blood substitutes are being developed as oxygen-carrying agents for the prevention of ischemic tissue damage and low blood volume-shock. However, the cell-free Hb molecule has intrinsic toxicity to the tissue since harmful reactive oxygen species (ROS) are readily produced during autoxidation of Hb from the ferrous state to the ferric state, and the cell-free Hb also causes distortion in the oxidant/antioxidant balance in the tissues. There may be further hindering dangers in the use of free Hb as a blood substitute. It has been reported that Hb has peroxidase-like activity oxidizing peroxidase substrates such as aromatic amines. Here we observed the Hb-catalyzed ROS production coupled to oxidation of a neurotransmitter precursor, β-phenylethylamine (PEA). Addition of PEA to Hb solution resulted in generation of superoxide anion (O₂^??). We also observed that PEA increases the Hb-catalyzed monovalent oxidation of ascorbate to ascorbate free radicals (Asc^?). The O₂^?? generation and Asc^? formation were detected by O₂^??-specific chemiluminescence of the Cypridina lucigenin analog and electron spin resonance spectroscopy, respectively. PEA-dependent O₂^?? production and monovalent oxidation of ascorbate in the Hb solution occurred without addition of H₂O₂, but a trace of H₂O₂ added to the system greatly increased the production of both O₂^?? and Asc^?. Addition of GSH completely inhibited the PEA-dependent production of O₂^?? and Asc^? in Hb solution. We propose that the O₂^?? generation and Asc^? formation in the Hb solution are due to the pseudoperoxidase activity-dependent oxidation of PEA and resultant ROS may damage tissues rich in monoamines, if the Hb-based blood substitutes were circulated without addition of ROS scavengers such as thiols.     

Induction of Nitric Oxide Synthase Inhibits Gap Junction Permeability in Cultured Rat Astrocytes

Abstract: Nitric oxide (^?NO) synthase (NOS) was induced in cultured rat astrocytes by incubation with lipopolysaccharide (LPS) for 18 h and gap junction permeability was assessed by the scrape-loading/Lucifer yellow transfer technique. Induction of NOS was confirmed by determining either the N^G-methyl-l -arginine (NMMA)-inhibitable production of nitrites and nitrates or the conversion of l -[³H]arginine to l -[³H]citrulline. Incubation with LPS dose-dependently inhibited gap junction permeability to 63.3% at 0.05 µg/ml LPS and no further inhibition was observed on increasing the LPS concentration up to 0.5 µg/ml. LPS-mediated gap junction inhibition was irreversible but was prevented by incubation with the NOS inhibitor NMMA and with the superoxide anion (O₂^??) scavenger superoxide dismutase. Incubation of the cells with both the ^?NO donor S-nitroso-N-acetylpenicillamine and the O₂^??-generating system xanthine/xanthine oxidase inhibited gap junction permeability. These results suggest that the in situ reaction between ^?NO and O₂^??, to form the peroxynitrite anion (ONOO^?), may be responsible for the inhibition of gap junction permeability. Scavenging the ONOO^? derivative hydroxyl radical (^?OH) with either dimethyl sulfoxide or mannitol prevented the LPS-mediated inhibition of gap junction permeability. Finally, exposure of astrocytes to authentic ONOO^? caused a dose-dependent inhibition of gap junction permeability (65.7% of inhibition at 0.5 mM ONOO^?). The pathophysiological relevance of ONOO^?-mediated inhibition of gap junctional communication in astrocytes after NOS induction by LPS is discussed, stressing the possible role played by this mechanism in some neurodegenerative diseases.     

ANTIOXIDANT ENZYME RESPONSE AND REACTIVE OXYGEN SPECIES PRODUCTION IN MARINE RAPHIDOPHYTES1

Raphidophytes (class Raphidophyceae) produce high levels of reactive oxygen species (ROS), yet little is known regarding cellular scavenging mechanisms needed for protection against these radicals. Enzymatic activities of the antioxidants superoxide dismutase (SOD) and catalase (CAT) were measured in conjunction with the production of superoxide (O₂^??) and hydrogen peroxide (H₂O₂) in batch cultures of five different raphidophytes species during early exponential, late‐exponential, and stationary growth phases. The greatest concentrations of O₂^?? per cell were detected during exponential growth with reduced levels in stationary phases in raphidophytes Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara, Chattonella marina (Subrahman.) Y. Hara et Chihara, and Chattonella antiqua (Hada) Ono (strain 18). Decreasing trends from exponential to stationary phases for SOD activity and H₂O₂ per cell were observed in all species tested. Significant correlations between O₂^?? per cell and SOD activity per cell over growth phase were only observed in three raphidophytes (Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua strain 18), likely due to different cellular locations of externally released O₂^?? radicals and intracellular SOD enzymes measured in this study. CAT activity was greatest at early exponential phase for several raphidophytes, but correlations between H₂O₂ per cell and CAT activity per cell were only observed for Fibrocapsa japonica Toriumi et Takano, Chattonella antiqua (strain 18), and Chattonella subsalsa Biecheler. Our results suggest that SOD and CAT play important protective roles against ROS during exponential growth of several raphidophytes, while other antioxidant pathways may play a larger role for scavenging ROS during later growth.     

Oxidative stress and nitrite dynamics under maximal load in elite athletes: relation to sport type

Maximal workload in elite athletes induces increased generation of reactive oxygen/nitrogen species (RONS) and oxidative stress, but the dynamics of RONS production are not fully explored. The aim of our study was to examine the effects of long-term engagement in sports with different energy requirements (aerobic, anaerobic, and aerobic/anaerobic) on oxidative stress parameters during progressive exercise test. Concentrations of lactates, nitric oxide (NO) measured through stabile end product-nitrites (NO₂ ^?), superoxide anion radical (O₂ ^??), and thiobarbituric reactive substances (TBARS) as index of lipid peroxidation were determined in rest, after maximal workload, and at 4 and 10th min of recovery in blood plasma of top level competitors in rowing, cycling, and taekwondo. Results showed that sportmen had similar concentrations of lactates and O₂ ^?? in rest. Nitrite concentrations in rest were the lowest in taekwondo fighters, while rowers had the highest levels among examined groups. The order of magnitude for TBARS level in the rest was bicycling > taekwondo > rowing. During exercise at maximal intensity, the concentration of lactate significantly elevated to similar levels in all tested sportsmen and they were persistently elevated during recovery period of 4 and 10 min. There were no significant changes in O₂ ^??, nitrite, and TBARS levels neither at the maximum intensity of exercise nor during the recovery period comparing to the rest period in examined individuals. Our results showed that long term different training strategies establish different basal nitrites and lipid peroxidation levels in sportmen. However, progressive exercise does not influence basal nitrite and oxidative stress parameters level neither at maximal load nor during the first 10 min of recovery in sportmen studied.     

Formation of protein S-nitrosylation by reactive oxygen species

Abstract

In the present study, the formation of whole cellular S-nitrosylated proteins (protein-SNOs) by the reactive oxygen species (ROS), hydrogen peroxide (H₂O₂), and superoxide (O₂^??) is demonstrated. A spectrum of protein cysteine oxidative modifications was detected upon incubation of serum-starved mouse embryonic fibroblasts with increasing concentrations of exogenous H₂O₂, ranging from exclusive protein-SNOs at low concentrations to a mixture of protein-SNOs and other protein oxidation at higher concentrations to exclusively non-SNO protein oxidation at the highest concentrations of the oxidant used. Furthermore, formation of protein-SNOs was also detected upon inhibition of the antioxidant protein Cu/Zn superoxide dismutase that results in an increase in intracellular concentration of O₂^??. These results were further validated using the phosphatase and tensin homologue, PTEN, as a model of a protein sensitive to oxidative modifications. The formation of protein-SNOs by H₂O₂ and O₂^?? was prevented by the NO scavenger, c-PTIO, as well as the peroxinitrite decomposition catalyst, FETPPS, and correlated with the production or the consumption of nitric oxide (NO), respectively. These data suggest that the formation of protein-SNOs by H₂O₂ or O₂^?? requires the presence or the production of NO and involves the formation of the nitrosylating intermediate, peroxinitrite.     

Cathodic electrochemiluminescence of the peroxydisulphate–ciprofloxacin system and its analytical applications

The cathodic electrochemiluminescence (ECL) of peroxydisulphate (S₂O₈^2?)–ciprofloxacin (CPF) system at a wax‐impregnated graphite electrode was studied. When CPF was absent, S₂O₈^2? was electrochemically reduced to sulphate free radical (SO₄^??), and dissolved oxygen absorbed on the electrode surface was reduced to protonated superoxide anion radical (HO₂^?). The HO₂^? was oxidized by SO₄^?? to produce molecular oxygen in both singlet and triplet states. Some of the singlet molecular oxygen (¹O₂) further combined through collision to be an energy‐rich precursor singlet molecular oxygen pair (¹O₂)₂. A weak ECL was produced when ¹O₂ or (¹O₂)₂ was converted to ground‐state molecular oxygen (³O₂). When CPF was present, a stronger ECL was produced, which originated from two emitting species. The main emitting species was excited state CPF (CPF*), which was produced by accepting energy from (¹O₂)₂. The other emitting species was excited singlet molecular oxygen pair [(¹O₂)₂*], which originated from the chemical oxidation of CPF by SO₄^?? and dissolved oxygen. Based on the stronger ECL phenomenon, an ECL method for the determination of either S₂O₈^2? or CPF was proposed. The proposed ECL method has been applied to the determination of CPF in pharmaceutical preparations. Copyright © 2011 John Wiley & Sons, Ltd.     

The effect of reactive oxygen species on ethylene production induced by osmotic stress in etiolated mungbean seedling

After 10 h osmotic stress in 25% polyethylene glycol (PEG6000) solution (–1.8 MPa) at 25 °C in darkness, the etiolated mungbean seedlings were transferred to pure water for recovery. The ethylene release rate and the level of reactive oxygen species (ROS), including superoxide radical (O₂^–) and hydrogen peroxide (H₂O₂), were investigated during the recovery process. The results showed that ethylene production rate and amount of ROS increased dramatically after osmotic stress, and a close correlation was observed between ethylene release rate and concentrations of ROS. Inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine (AVG) or aminooxyacetic acid (AOA), could reduce the ethylene release rate, but had no significant influence to the content of O₂^– and H₂O₂. As well as, silver thiosulfate (STS), an inhibitor of ethylene action, exhibited no obvious effect to the concentration of ROS, showing stress-inducible ethylene was not the cause for the increase of stress-inducible ROS. On the other hand, exogenous generator of superoxide radical (methylviologen, MV, or sodium dithionite, Na₂S₂O₄) could enhance the ethylene production evidently, which could be inhibited by exogenous scavenger of superoxide radical (superoxide dismutase, SOD, or 1, 4-diazabicyclo (2,2,2) octane, DABCO). However, either exogenous H₂O₂ or catalase (CAT) had no significant influence on ethylene production. The results suggested that it was superoxide radical but not H₂O₂which was involved directly in osmotic stress-inducible ethylene biosynthesis. The dual-role of superoxide radical on stress ethylene biosynthesis was also discussed.     

Gemcitabine response in pancreatic adenocarcinoma cells is synergistically enhanced by dithiocarbamate derivatives

Pancreatic adenocarcinoma is a common malignancy that remains refractory to all available therapies, including the gold standard drug gemcitabine (GEM). We investigated the effect of the combination of GEM and each of the ionophore compounds pyrrolidine dithiocarbamate (PDTC) and disulfiram [DSF; 1-(diethylthiocarbamoyldisulfanyl)-N,N-diethylmethanethioamide] on p53^?/? pancreatic adenocarcinoma cell growth. PDTC or DSF synergistically inhibited cell proliferation when used in combination with GEM by inducing apoptotic cell death. This effect was associated with an increased mitochondrial O₂^?? production and was further enhanced by zinc ions. Basal levels of mitochondrial O₂^?? or manganese superoxide dismutase (MnSOD) strictly correlated with the IC₅₀ for GEM or the percentage of synergism. Thus, the most relevant values of the antiproliferative synergism were obtained in GEM-resistant pancreatic adenocarcinoma cell lines. Interestingly, the GEM-sensitive T3M4 cells transfected with MnSOD expression vector showed mitochondrial O₂^?? and IC₅₀ for GEM similar to those of resistant cell lines. In vivo experiments performed on nude mice xenotransplanted with the GEM-resistant PaCa44 cell line showed that only the combined treatment with GEM and DSF/Zn completely inhibited the growth of the tumoral masses. These results and the consideration that DSF is already used in clinics strongly support the GEM and DSF/Zn combination as a new approach to overcoming pancreatic cancer resistance to standard chemotherapy.     

Visible-light promoted degradation of the commercial antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT): a kinetic study

Abstract

Visible-light photo-irradiation of the commercial phenolic antioxidants (PhAs) butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), in the presence of vitamin B₂ (riboflavin, Rf), in methanolic solutions and under aerobic conditions, results in the photo-oxidation of the PhAs. The synthetic dye photosensitiser Rose Bengal was also employed for auxiliary experiments. With concentrations of riboflavin and PhAs of ca. 0.02 mM and < 1 mM, respectively, the excited triplet state of the vitamin (³Rf*) is quenched by BHT in a competitive fashion with dissolved ground state triplet oxygen. From the quenching of ³Rf*, the semireduced form of the pigment is generated through an electron transfer process from BHT, with the subsequent production of superoxide anion radical (O₂^??) by reaction with dissolved molecular oxygen. In parallel, the species singlet molecular oxygen, O₂(¹Δ_g), is also generated. Both reactive oxygen species produce the photodegradation of BHT. In the case of BHA, the lack of any effect exerted by superoxide dismutase drives out a significant participation of a O₂^??-mediated mechanism. BHA mainly interacts with O₂(¹Δ_g) and exhibits a desirable property as an antioxidant – a relatively high capacity for O₂(1Δ_g) de-activation and a low photodegradation efficiency by the oxidative species. Electrochemical determinations support the proposed photodegradative mechanism.     

On the decarboxylation of α-keto acids by isolated chloroplasts

The mechanism of the decarboxylation of α-keto acids by isolated chloroplasts has been studied with the aid of superoxide dismutase and catalase. Using photosynthetic and enzymatic systems, which are known to catalyze peroxidic oxidations, we have been able to demonstrate that both the superoxide free radical ion and H₂O₂ are necessary for maximal rates of decarboxylation. In isolated chloroplasts, an auto-oxidizable electron acceptor as well as an electron donor for Photosystem I are absolute requirements for the decarboxylation. H₂O₂ seems to be the primary oxidant in the decarboxylation of pyruvate or glyoxylate by isolated chloroplasts. A secondary rate of decarboxylation is superimposed on the primary one, mediated by superoxide free radical ion. Mn²⁺ stimulates the decarboxylation probably via intermediarily-formed Mn³⁺ in a reaction, which is neither inhibited by catalase nor by superoxide dismutase. A decarboxylation of pyruvate or glyoxylate by isolated chloroplasts in the presence of NADP⁺ is initiated, as soon as the available NADP⁺ is fully reduced. In this case, the open-chain electron transport seems to switch from NADP⁺ to oxygen as the terminal electron acceptor.