Two-electron electrochemical oxidation of quercetin and kaempferol changes only the flavonoid C-ring

Bulk electrolysis of the antioxidant flavonoids quercetin and kaempferol in acetonitrile both yield a single oxidation product in two-electron processes. The oxidation products are more polar than their parent compounds, with an increased molecular weight of 16 g/mol, and were identified as 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone and 2-(4-hydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone for quercetin and kaempferol, respectively. Two-electron oxidation of the parent flavonoid is suggested to yield a 3,4-flavandione with unchanged substitution pattern in the A- and B-ring, which may rearrange to form the substituted 3(2H)-benzofuranone through the chalcan-trione ring-chain tautomer. The acidity of the 3-OH group is suggested to determine the fate of the flavonoid phenoxyl radical, originally formed by one-electron oxidation, as no well-defined oxidation product of luteolin (lacking the 3-OH group) could be isolated despite rather similar half-peak potentials: E_P/2 = 0.97 V, 0.98 V and 1.17 V vs. NHE for quercetin, kaempferol and luteolin, respectively, as measured by cyclic voltammetry in acetonitrile.     

Mitochondria superoxide dismutase mimetic inhibits peroxide-induced oxidative damage and apoptosis: role of mitochondrial superoxide

The purpose of this study was to test the hypothesis whether Mito-carboxy proxyl (Mito-CP), a mitochondria-targeted nitroxide, inhibits peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC). Glucose/glucose oxidase (Glu/GO)-induced oxidative stress was monitored by dichlorodihydrofluorescein oxidation catalyzed by intracellular H(2)O(2) and transferrin receptor-mediated iron transported into cells. Pretreatment of BAECs with Mito-CP significantly diminished H(2)O(2)- and lipid peroxide-induced intracellular formation of dichlorofluorescene and protein oxidation. Electron paramagnetic resonance (EPR) studies confirmed the selective accumulation of Mito-CP into the mitochondria. Mito-CP inhibited the cytochrome c release and caspase-3 activation in cells treated with peroxides. Mito-CP inhibited both H(2)O(2)- and lipid peroxide-induced inactivation of complex I and aconitase, overexpression of transferrin receptor (TfR), and mitochondrial uptake of (55)Fe, while restoring the mitochondrial membrane potential and proteasomal activity. In contrast, the "untargeted" carboxy proxyl (CP) nitroxide probe did not protect the cells from peroxide-induced oxidative stress and apoptosis. However, both CP and Mito-CP inhibited superoxide-induced cytochrome c reduction to the same extent in a xanthine/xanthine oxidase system. We conclude that selective uptake of Mito-CP into the mitochondria is responsible for inhibiting peroxide-mediated Tf-Fe uptake and apoptosis and restoration of the proteasomal function.     

Tetraena mongolica Maxim can accumulate large amounts of triacylglycerol in phloem cells and xylem parenchyma of stems

Tetraena mongolica Maxim is a narrowly monotypic genus of Zygophyllaceae found in a very limited area in the western part of Inner Mongolia, China. The plant is called "oil firewood" and its stems and branches are used as fuelwood. As triacylglycerol (TAG) is the main component of the plant oil, the TAG content was analyzed, as were the distribution of oleosomes in different tissues of the stem. This was in order to ascertain whether the term "oil firewood" referred to this storage lipid. Stems of T. mongolica indeed contained high levels of TAG (approximately 46 mg/g of dry matter or DM). The concentration of TAG in phloem (90 mg/g of DM) was much higher than that in xylem (20mg/g of DM), and semi-thin sections stained by Sudan Black B showed that almost all cells in the phloem contained oleosomes whereas in the xylem, oleosomes were found only in parenchymatous cells. These results suggest that T. mongolica has a high capacity to accumulate TAG in its stem cells.     

Antioxidative flavonoid quercetin: implication of its intestinal absorption and metabolism

Quercetin is a typical flavonoid ubiquitously present in fruits and vegetables, and its antioxidant effect is implied to be helpful for human health. The bioavailability of quercetin glycosides should be clarified, because dietary quercetin is mostly present as its glycoside form. Although quercetin glycosides are subject to deglycosidation by enterobacteria for the absorption at large intestine, small intestine acts as an effective absorption site for glucose-bound glycosides (quercertin glucosides). This is because small intestinal cells possess a glucoside-hydrolyzing activity and their glucose transport system is capable of participating in the glucoside absorption. A study using a cultured cell model for intestinal absorption explains that the hydrolysis of the glucosides accelerates their absorption in the small intestine. Small intestine is also recognized as the site for metabolic conversion of quercetin and other flavonoids as it possesses enzymatic activity of glucuronidation and sulfation. Modulation of the intestinal absorption and metabolism may be beneficial for regulating the biological effects of dietary quercetin.     

Effect of the flavonoid quercetin on culture and isolation ofFrankia fromCasuarina root nodules

The growth of threeFrankia strains (HFPCcl3, ORS021001, ORS020607), isolated from nodules ofCasuarina, was unaffected by 5–15 μmol/L quercetin. Re-isolation ofFrankia strain HFPCc13 fromCasuarina root nodules was carried out with the addition of 15 μmol/L quercetin to three different media. The flavonoid significantly reduced fungal contaminants in the isolation plates and increased the number ofFrankia colonies per plate compared to controls without the flavonoid.     

Effect of quercetin and its glucuronide metabolite upon 6-hydroxydopamine-induced oxidative damage in Neuro-2a cells

Quercetin is ubiquitously distributed in plant foods. This antioxidative polyphenol is mostly converted to conjugated metabolites in the body. Parkinson disease (PD) has been suggested to be related to oxidative stress derived from abnormal dopaminergic activity. We evaluated if dietary quercetin contributes to the antioxidant network in the central nervous system from the viewpoint of PD prevention. A neurotoxin, 6-hydroxydopamine (6-OHDA), was used as a model of PD. 6-OHDA-induced H?O? production and cell death in mouse neuroblastoma, Neuro-2a. Quercetin aglycone suppressed 6-OHDA-induced H?O? production and cell death, although aglycone itself reduced cell viability at higher concentration. Quercetin 3-O-β-D-glucuronide (Q3GA), which is an antioxidative metabolite of dietary quercetin, was little incorporated into the cell resulting in neither suppression of 6-OHDA-induced cell death nor reduction of cell viability. Q3GA was found to be deconjugated to quercetin by microglial MG-6 cells. These results indicate that quercetin metabolites should be converted to their aglycone to exert preventive effect on damage to neuronal cells.     

Effect of quercetin and its glucuronide metabolite upon 6-hydorxydopamine-induced oxidative damage in Neuro-2a cells

Abstract

Quercetin is ubiquitously distributed in plant foods. This antioxidative polyphenol is mostly converted to conjugated metabolites in the body. Parkinson disease (PD) has been suggested to be related to oxidative stress derived from abnormal dopaminergic activity. We evaluated if dietary quercetin contributes to the antioxidant network in the central nervous system from the viewpoint of PD prevention. A neurotoxin, 6-hydroxydopamine (6-OHDA), was used as a model of PD. 6-OHDA-induced H₂O₂ production and cell death in mouse neuroblastoma, Neuro-2a. Quercetin aglycone suppressed 6-OHDA-induced H₂O₂ production and cell death, although aglycone itself reduced cell viability at higher concentration. Quercetin 3-O-β-d-glucuronide (Q3GA), which is an antioxidative metabolite of dietary quercetin, was little incorporated into the cell resulting in neither suppression of 6-OHDA-induced cell death nor reduction of cell viability. Q3GA was found to be deconjugated to quercetin by microglial MG-6 cells. These results indicate that quercetin metabolites should be converted to their aglycone to exert preventive effect on damage to neuronal cells.     

Dendritic chain reaction: Responsive release of hydrogen peroxide upon generation and enzymatic oxidation of methanol

Signal amplification dramatically increases the sensitivity of diagnostic methods. Recently, we introduced a new technique for signal amplification that uses a distinctive dendritic chain reaction (DCR) to generate exponential evolution of a diagnostic signal. In this report, we demonstrate how the modular design of our DCR probe can be used to improve the detection sensitivity. We synthesized a new probe based on a methyl carbonate linkage, which has superior stability in aqueous media. Triggered release of methanol, which was oxidized by alcohol oxidase present in the solution, produced hydrogen peroxide that used as a reagent in the DCR amplification technique. The new probe exhibited higher sensitivity in detection of hydrogen peroxide than our previously reported probe.     

Effect of chloramphenicol on mitochondrial and extramitochondrial systems in the isolated perfused rat heart

The isolated, perfused working rat heart was used as a model for investigating the effects of chloramphenicol on mitochondrial amino acid incorporation in an intact organ. The most obvious inhibitory effects of chloramphenicol were extramitochondrial: decreased mechanical performance of the heart and marked reduction in glucose uptake and lactate production. The ATP levels of the perfused heart were significantly increased at high levels of chloramphenicol. Chloramphenicol (50 to 500 μg/ml perfusate) did not inhibit the incorporation into the mitochondria or other subcellular fractions. A specific inhibitory effect on mitochondrial protein synthesis could only be observed when the cytoplasmic protein synthetizing system had been inhibited by cycloheximide. Under these conditions it could be demonstrated that the chloramphenicol sensitivity was greater for the synthesis of the insoluble proteins than for that of the soluble proteins of the mitochondria The chloramphenicol inhibition of mitochondrial protein synthesis which could be obtained in the isolated heart was approx. 70% which was twice as high as could be achieved when isolated mitochondria were incorporating amino acids.     

Import of proteins into mitochondria. Yeast cells grown in the presence of carbonyl cyanide m-chlorophenylhydrazone accumulate massive amounts of some mitochondrial precursor polypeptides

Cytoplasmically synthesized precursors of mitochondrial polypeptides have previously been observed in trace amounts after pulse labeling of yeast spheroplasts or after in vitro translation of yeast mRNA (Maccecchini, M. L., Rudin, Y., Blobel, G., and Schatz, G. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 343-347). Some of these precursors are shown here to accumulate in large amounts (up to 150 micrograms/g of cell protein) during growth of a cytoplasmic petite (rho-) mutant in the presence of carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation. Cytochrome c1 precursor accumulated under these conditions is unstable; it is degraded with a half-life of about 10 min. In contrast, the F1-ATPase beta-subunit precursor is degraded considerably more slowly and, following removal of the uncoupler, can be post-translationally imported into mitochondria where it is processed to the mature polypeptide.     

Oxidative damage during chagasic cardiomyopathy development: role of mitochondrial oxidant release and inefficient antioxidant defense

In this study, we evaluated the oxidant status and antioxidant defense capabilities of the heart during the course of Trypanosoma cruzi infection and disease development in a murine model system. Our data show that the extent of protein carbonylation and lipid peroxidation is increased in the heart, but not the skeletal muscle, of infected mice. The level of oxidative injury biomarkers in the myocardium consistently increased with chronic disease severity. The antioxidant defense constituted by catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GSR), and reduced glutathione was increased in murine heart and skeletal tissue in response to the stress of T. cruzi infection. After the initial burst, CAT, GPx, and GSR remained unresponsive to the severity of chronic tissue damage in chagasic hearts. The cardiac level of Mn(2+) superoxide dismutase (MnSOD) was diminished in chagasic mice. Our data suggest that the host responds to acute injuries by activating antioxidant defenses that are of sufficient magnitude to scavenge the reactive oxidants in skeletal tissue. The myocardia of infected mice, however, sustain increased oxidative injuries with disease progression. We surmise that MnSOD deficiencies, resulting in the increased release of mitochondrial free radicals, lead to sustained oxidative stress that exceeds the cardiac antioxidant defense capacity and contribute to persistent oxidative damage in chagasic myocardium.     

Cytotoxic phospholipid oxidation products. Cell death from mitochondrial damage and the intrinsic caspase cascade

Phospholipid oxidation products accumulate in the necrotic core of atherosclerotic lesions, in apoptotic cells, and circulate in oxidized low density lipoprotein. Phospholipid oxidation generates toxic products, but little is known about which specific products are cytotoxic, their receptors, or the mechanism(s) that induces cell death. We find the most common phospholipid oxidation product of oxidized low density lipoprotein, phosphatidylcholine with esterified sn-2-azelaic acid, induced apoptosis at low micromolar concentrations. The synthetic ether phospholipid hexadecyl azelaoyl phosphatidylcholine (HAzPC) was rapidly internalized, and overexpression of PLA2g7 (PAF acetylhydrolase) that specifically hydrolyzes such oxidized phospholipids suppressed apoptosis. Internalized HAzPC associated with mitochondria, and cytochrome c, and apoptosis-inducing factor escaped from mitochondria to the cytoplasm and nucleus, respectively, in cells exposed to HAzPC. Isolated mitochondria exposed to HAzPC rapidly swelled and released cytochrome c and apoptosis-inducing factor. Other phospholipid oxidation products induced swelling, but HAzPC was the most effective and was twice as effective as its diacyl homolog. Cytoplasmic cytochrome c completes the apoptosome, and activated caspase 9 and 3 were present in cells exposed to HAzPC. Irreversible inhibition of caspase 9 blocked downstream caspase 3 activation and prevented apoptosis. Mitochondrial damage initiated this apoptotic cascade, because overexpression of Bcl-X(L), an anti-apoptotic protein localized to mitochondria, blocked cytochrome c escape and apoptosis. Thus, exogenous phospholipid oxidation products target intracellular mitochondria to activate the intrinsic apoptotic cascade.     

Mitochondria: omega-3 in the route of mitochondrial reactive oxygen species

Mitochondria are the main organelles that produce reactive oxygen species (ROS). Overproduction of ROS induces oxidative damage to macromolecules, including lipids, and can damage cellular membrane structure and functions. Mitochondria, the main target of ROS-induced damage, are equipped with a network of antioxidants that control ROS production. Dietary intake of omega-3 polyunsaturated fatty acids (ω3PUFAs) and consequently the increase in ω3PUFA content of membrane lipids may be disadvantageous to the health because ROS-induced oxidative peroxidation of ω3PUFAs within membrane phospholipids can lead to the formation of toxic products. Mitochondrial control of lipid peroxidation is one of the mechanisms that protect cell against oxidative damage. This review discusses the role of mitochondria in ROS generation and the mechanisms by which it regulates ROS production. The susceptibility to peroxidation of PUFAs by ROS raises the question of the adverse effects of ω3PUFA dietary supplementation on embryonic development and prenatal developmental outcomes.     

Comparison of the prevention of aflatoxin b(1)-induced genotoxicity by quercetin and quercetin pentaacetate

Earlier work carried out in our laboratory highlighted the mode of action of acetoxy 4-methylcoumarins in preventing the genotoxicity of aflatoxin B(1) (AFB(1)). We have in this report extended the observations to quercetin pentaacetate (QPA), which unlike quercetin (Q) has demonstrated time-dependent inhibition of liver microsome catalysed AFB(1) epoxidation as measured by AFB(1) binding to DNA. The action of QPA is similar to that of the acetoxy 4-methylcoumarins in that they are acted upon by microsomal transacetylase leading to modulation of catalytic activities of certain enzymes (such as P-450 enzymes, NADPH cytochrome C reductase and glutathione S-transferase) possibly by way of protein acetylation. In the present work, we have documented the transacetylase-mediated action of QPA in preventing genotoxicity due to AFB(1).     

A Highlights from MBoC Selection: Kinetic control by limiting glutaredoxin amounts enables thiol oxidation in the reducing mitochondrial intermembrane space

The mitochondrial intermembrane space (IMS) harbors an oxidizing machinery that drives import and folding of small cysteine-containing proteins without targeting signals. The main component of this pathway is the oxidoreductase Mia40, which introduces disulfides into its substrates. We recently showed that the IMS glutathione pool is maintained as reducing as that of the cytosol. It thus remained unclear how equilibration of protein disulfides with the IMS glutathione pool is prevented in order to allow oxidation-driven protein import. Here we demonstrate the presence of glutaredoxins in the IMS and show that limiting amounts of these glutaredoxins provide a kinetic barrier to prevent the thermodynamically feasible reduction of Mia40 substrates by the IMS glutathione pool. Moreover, they allow Mia40 to exist in a predominantly oxidized state. Consequently, overexpression of glutaredoxin 2 in the IMS results in a more reduced Mia40 redox state and a delay in oxidative folding and mitochondrial import of different Mia40 substrates. Our findings thus indicate that carefully balanced glutaredoxin amounts in the IMS ensure efficient oxidative folding in the reducing environment of this compartment.     

Mitochondria and the regulation of free radical damage in the eye

Neuronal cell death can be determined by the overall level of reactive oxygen species (ROS) resulting from the combination of extrinsic sources and intrinsic production as a byproduct of oxidative phosphorylation. Key controllers of the intrinsic production of ROS are the mitochondrial uncoupling proteins (UCPs). By allowing a controlled leak of protons across the inner mitochondrial membrane activation of these proteins can decrease ROS and promote cell survival. In both primate models of Parkinson’s disease and mouse models of seizures, increased activity of UCP2 significantly increased neuronal cells survival. In the retina UCP2 is expressed in many neurons and glial cells, but was not detected in rod photoreceptors. Retinal ganglion cell survival following excitotoxic damage was much greater in animals overexpressing UCP2. Traditional Chinese medicines, such as an extract of Cistanche tubulosa, may provide benefit by altering mitochondrial metabolism.     

Palladacycles catalyse the oxidation of critical thiols of the mitochondrial membrane proteins and lead to mitochondrial permeabilization and cytochrome c release associated with apoptosis

Permeabilization of the mitochondrial membrane has been extensively associated with necrotic and apoptotic cell death. Similarly to what had been previously observed for B16F10-Nex2 murine melanoma cells, PdC (palladacycle compounds) obtained from the reaction of dmpa (N,N-dimethyl-1-phenethylamine) with the dppe [1,2-ethanebis(diphenylphosphine)] were able to induce apoptosis in HTC (hepatoma, tissue culture) cells, presenting anticancer activity in vitro. To elucidate cell site-specific actions of dmpa:dppe that could respond to the induction of apoptosis in cancer cells in the present study, we investigated the effects of PdC on isolated RLM (rat liver mitochondria). Our results showed that these palladacycles are able to induce a Ca2+-independent mitochondrial swelling that was not inhibited by ADP, Mg2+ and antioxidants. However, the PdC-induced mitochondrial permeabilization was partially prevented by pre-incubation with CsA (cyclosporin A), NEM (N-ethylmaleimide) and bongkreic acid and totally prevented by DTT (dithiothreitol). A decrease in the content of reduced thiol groups of the mitochondrial membrane proteins was also observed, as well as the presence of membrane protein aggregates in SDS/PAGE without lipid and GSH oxidation. FTIR (Fourier-transform IR) analysis of PdC-treated RLM demonstrated the formation of disulfide bonds between critical thiols in mitochondrial membrane proteins. Associated with the mitochondrial permeabilization, PdC also induced the release of cytochrome c, which is sensitive to inhibition by DTT. Besides the contribution to clarify the pro-apoptotic mechanism of PdC, this study shows that the catalysis of specific protein thiol cross-linkage is enough to induce mitochondrial permeabilization and cytochrome c release.     

Oxidative damage to mitochondrial DNA and glutathione oxidation in apoptosis: studies in vivo and in vitro.

Free radicals may be involved in apoptosis although this is the subject of some controversy. Furthermore, the source of free radicals in apoptotic cells is not certain. The aim of this study was to elucidate the role of oxidative stress in the induction of apoptosis in serum-deprived fibroblast cultures and in weaned lactating mammary glands as in vitro and in vivo experimental models, respectively. Oxidative damage to mtDNA is higher in apoptotic cells than in controls. Oxidized glutathione (GSSG) levels in mitochondria from lactating mammary gland are also higher in apoptosis. There is a direct relationship between mtDNA damage and the GSSG/reduced glutathione (GSH) ratio. Furthermore, whole cell GSH is decreased and GSSG is increased in both models of apoptosis. Glutathione oxidation precedes nuclear DNA fragmentation. These signs of oxidative stress are caused, at least in part, by an increase in peroxide production by mitochondria from apoptotic cells. We report a direct relationship between glutathione oxidation and mtDNA damage in apoptosis. Our results support the role of mitochondrial oxidative stress in the induction of apoptosis.     

The cardioprotective effect of urocortin during ischaemia/reperfusion involves the prevention of mitochondrial damage

We have previously shown, using Affymetrix gene chip technology, that urocortin induces the expression of several diverse genes in cardiac myocytes. An ATP sensitive inwardly rectifying potassium channel, Katp (Kir6.1), the enzyme calcium independent phospholipase A2 (iPLA2), and protein kinase C epsilon (PKCepsilon) and that these genes are involved in the cardioprotective mechanism of action of urocortin. Here we demonstrate that these gene products are localized to cardiac myocyte mitochondria and for the first time show that urocortin protects cardiac myocytes from ischaemia/reperfusion induced cell death by preventing mitochondrial damage. Using pharmacological agents to Katp channels and iPLA2 and synthetic peptide inhibitors of PKCepsilon, we go on to demonstrate that these three gene products are involved in the urocortin induced protection of cardiac myocyte mitochondria. These proteins may interact at the mitochondria to produce the protective effect.