Inhibition of human low density lipoprotein oxidation by flavonols and their glycosides

Antioxidative effects of the flavonols and their glycosides, i.e., quercetin (Q), quercetin galactopyranoside (QG), quercetin rhamnolpyranoside (QR), rutin (R), morin (MO), myrecetin (MY), kaempferol (K) and kaempferol glucoside (KG), against free radical initiated peroxidation of human low density lipoprotein (LDL) were studied. The peroxidation was initiated either by a water-soluble initiator 2,2'-azobis(2-amidino propane hydrochloride) (AAPH), or by cupric ion (Cu2+). The reaction kinetics were monitored either by the uptake of oxygen and the depletion of alpha-tocopherol (TOH) presented in the native LDL, or by the formation of thiobarbituric acid reactive substances (TBARS). Kinetic analysis of the antioxidation process demonstrates that these flavonols and their glycosides are effective antioxidants against AAPH- and Cu(2+)-initiated LDL peroxidation, the flavonols bearing ortho-dihydroxyl groups possess significantly higher antioxidant activity than those bearing no such functionalities, and the glycosides are less active than their parent aglycones.     

Antioxidant activity of isocoumarins isolated from Paepalanthus bromelioides on mitochondria

The isocoumarins (1-50 microM) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin isolated from Paepalanthus bromelioides, were assessed for antioxidant activity using isolated rat liver mitochondria and non-mitochondrial systems, and compared with the flavonoid quercetin. The paepalantine and paepalantine dimers, but not vioxanthin, were effective at scavenging both 1,1-diphenyl-2-picrylhydrazyl (DPPH(*)) and superoxide (O(2)(-)) radicals in non-mitochondrial systems, and protected mitochondria from tert-butylhydroperoxide-induced H(2)O(2) accumulation and Fe(2+)-citrate-mediated mitochondrial membrane lipid peroxidation, with almost the same potency as quercetin. These results point towards paepalantine, followed by paepalantine dimer, as being a powerful agent affording protection, apparently via O(2)(-) scavenging, from oxidative stress conditions imposed on mitochondria, the main intracellular source and target of those reactive oxygen species. This strong antioxidant action of paepalantine was reproduced in HepG2 cells exposed to oxidative stress condition induced by H(2)O(2).     

Oxidative Inactivation of Brain Ecto-5′-Nucleotidase by Thiols/Fe2+ System

5-Nucleotidase, responsible for the conversion of adenosine-5-monophosphate into adenosine, was purified from bovine brain membranes, and subjected to oxidative inactivation. The 5-nucleotidase activity decreased slightly after the exposure to either glutathione or Fe²⁺. The glutathione-mediated inactivation of 5-nucleotidase was potentiated remarkably by Fe²⁺, but not Cu²⁺, in a concentration-dependent manner. Similarly, glutathione exhibited a concentration-dependent enhancement of the Fe²⁺-mediated inactivation. In comparison, the glutathione/Fe²⁺ system was much more effective than the ascorbate/Fe²⁺ system in inactivating the enzyme. In support of an intermediary role of superoxide ions or H₂O₂ in the action of glutathione/Fe²⁺ system, superoxide dismutase and catalase expressed a substantial protection against the inactivation by the glutathione/Fe²⁺ system. Meanwhile, hydroxyl radical scavangers such as mannitol, benzoate or ethanol were incapable of preventing the inactivation, excluding the participation of extraneous hydroxyl radicals. Whereas adenosine 5-monophosphate as substrate exhibited a modest protection against the glutathione/Fe²⁺ action, a remarkable protection was expressed by divalent metal ions such as Zn²⁺ or Mn²⁺. Structure-activity study with a variety of thiols indicates that the inactivating action of thiols in combination with Fe²⁺ resides in the free sulfhydyl group and amino group of thiols. Overall, thiols, expressing more inhibitory effect on the activity of 5-nucleotidase, were found to be more effective in potentiating the Fe²⁺- mediated inactivation. Further, kinetic analyses indicate that Fe²⁺ and thiols inhibit the 5-nucleotidase in a competitive or uncompetitive manner, respectively. These results suggest that ecto-5-nucleotidase from brain membrane is one of proteins susceptible to thiols/ Fe²⁺-catalyzed oxidation, and the oxidative inactivation may be related to the selective association of Fe²⁺ and thiols to the enzyme molecule.     

Oxidized quercetin reacts with thiols rather than with ascorbate: implication for quercetin supplementation

When an antioxidant scavenges a reactive species, i.e., when it exerts its antioxidant activity, the antioxidant is converted into potentially harmful oxidation products. In this way, the antioxidant quercetin might yield an ortho-quinone, denoted as QQ, which has four tautomeric forms, i.e., the ortho-quinone and three quinonmethides. We evaluated the interaction of QQ with ascorbate or glutathione (GSH). Ascorbate recycles QQ to the parent compound quercetin, while GSH forms two adducts with QQ, i.e., 6-GSQ and 8-GSQ. When both GSH and ascorbate are present, QQ is converted exclusively into GSQ. In the absence of GSH, protein thiols will be arylated by QQ. This protein arylation is not prevented by ascorbate. Thiol arylation by quinones and quinonmethides can impair several vital enzymes. This implies that the product formed when quercetin displays its antioxidant scavenging effect is toxic in the absence of GSH. Therefore, an adequate GSH level should be maintained when quercetin is supplemented.     

Membrane cholesterol contents influence the protective effects of quercetin and rutin in erythrocytes damaged by oxidative stress

Flavonoids are potent scavengers of reactive oxygen species (ROS) that effectively prevent erythrocyte oxidation. Their antioxidant activities are governed by their structural characteristics and their ability to interact with and penetrate lipid bilayers. In order to gain a better understanding of the relationship between cholesterol contents and the antioxidant effectiveness of flavonoids against oxidative damage induced by ROS in cells, here we analyzed the integrity and structural stability of cholesterol-modified (enriched or depleted) and control erythrocytes exposed to tert-butyl hydroperoxide in the presence of quercetin or rutin. In control and cholesterol-enriched erythrocytes, quercetin provided greater protection against lipid peroxidation, ROS formation, and it preserved better cellular integrity than rutin. Both antioxidants suppressed the alterations in membrane fluidity and lipid losses with similar efficiency, reducing hemoglobin oxidation by 30% and GSH losses by 60% in the above-mentioned erythrocytes. Cholesterol depletion reduced the efficiency of the antioxidant power of both flavonoids against oxidative damage induced in the erythrocyte membrane, while a stronger degree of protection of GSH and hemoglobin contents was observed, mainly in the presence of rutin. These findings suggest a preferential incorporation of the antioxidants into the membranes from erythrocytes with normal and high cholesterol contents, whereas they would mainly be located in the cytoplasm of cholesterol-depleted erythrocytes.     

Tetrahydropteridine deficiency impairs mitochondrial function in Dictyostelium discoideum Ax2

A putative cellular function of tetrahydropteridines (l-erythro-tetrahydrobiopterin and d-threo-tetrahydrobiopterin) was investigated in Dictyostelium discoideum Ax2 using a mutant disrupted in the gene encoding sepiapterin reductase (SR). The SR mutant, which produces about 3% of tetrahydropteridines if compared to wild-type, was elucidated to have several functional defects related to mitochondria and oxidative stress: retarded growth, poor spore viability, impaired mitochondrial function, and increased susceptibility to oxidative stress induced by hydroxylamine or cumene-hydroperoxide. However, the physiological defects were almost completely rescued by extrachromosomal expression of Dictyostelium SR. The results strongly suggested that tetrahydropteridines in Dictyostelium are associated with mitochondrial function, probably via direct protection against oxidative stress.     

The pur3 gene from the pur cluster encodes a monophosphatase essential for puromycin biosynthesis in Streptomyces

The pur3 gene of the puromycin (pur) cluster from Streptomyces alboniger is essential for the biosynthesis of this antibiotic. Cell extracts from Streptomyces lividans containing pur3 had monophosphatase activity versus a variety of mononucleotides including 3'-amino-3'-dAMP (3'-N-3'-dAMP), (N6,N6)-dimethyl-3'-amino-3'-dAMP (PAN-5'-P) and AMP. This is in accordance with the high similarity of this protein to inositol monophosphatases from different sources. Pur3 was expressed in Escherichia coli as a recombinant protein and purified to apparent homogeneity. Similar to the intact protein in S. lividans, this recombinant enzyme dephosphorylated a wide variety of substrates for which the lowest Km values were obtained for the putative intermediates of the puromycin biosynthetic pathway 3'-N-3'-dAMP (Km = 1.37 mM) and PAN-5'-P (Km = 1.40 mM). The identification of this activity has allowed the revision of a previous proposal for the puromycin biosynthetic pathway.     

The glutathione thiyl radical does not react with nitrogen monoxide

Laser flash photolysis experiments shows that the rate constant for the reaction of the glutathione thiyl radical with nitrogen monoxide to give S-nitrosoglutathione is lower than 2.8+/-0.6 x 10(7)M(-1)s(-1). The conversion of the thiyl radical to its carbon-centred form at 10(3)s(-1) exceeds the formation of S-nitrosoglutathione when physiological concentrations of nitrogen monoxide are taken into account.     

Inhibition of autophagy attenuates pancreatic cancer growth independent of TP53/TRP53 status

Basal levels of autophagy are elevated in most pancreatic ductal adenocarcinomas (PDAC). Suppressing autophagy pharmacologically using chloroquine (CQ) or genetically with RNAi to essential autophagy genes inhibits human pancreatic cancer growth in vitro and in vivo, which presents possible treatment opportunities for PDAC patients using the CQ-derivative hydroxychloroquine (HCQ). Indeed, such clinical trials are ongoing. However, autophagy is a complex cellular mechanism to maintain cell homeostasis under stress. Based on its biological role, a dual role of autophagy in tumorigenesis has been proposed: at tumor initiation, autophagy helps maintain genomic stability and prevent tumor initiation; while in advanced disease, autophagy degrades and recycles cellular components to meet the metabolic needs for rapid growth. This model was proven to be the case in mouse lung tumor models. However, in contrast to prior work in various PDAC model systems, loss of autophagy in PDAC mouse models with embryonic homozygous Trp53 deletion does not inhibit tumor growth and paradoxically increases progression. This raised concerns whether there may be a genotype-dependent reliance of PDAC on autophagy. In a recent study, our group used a Trp53 heterozygous mouse PDAC model and human PDX xenografts to address the question. Our results demonstrate that autophagy inhibition was effective against PDAC tumors irrespective of TP53/TRP53 status.     

High affinity selenium uptake in a keratinocyte model

The distribution of selenium in mammals has been recently shown to be mediated primarily by selenoprotein P. Even in the absence of selenoprotein P, selenium is distributed from the liver into all organs and tissues when supplemented in the diet. The form of selenium that is actively taken up by mammalian cells at trace concentrations has yet to be determined. We used a human keratinocyte model to determine whether reduction of the oxyanion selenite (SeO(3)(2-)) to the more reduced form of selenide (HSe(-)) would affect uptake. Indeed a reduced form of selenium, presumably selenide, was actively transported into keratinocytes and displayed saturation kinetics with an apparent K(m) of 279 nM. ATPase inhibitors blocked the uptake of selenide, as did the competing anions molybdate and chromate, but not sulfate. These results suggest that the small molecule form of selenium that is distributed in tissues is hydrogen selenide, despite its sensitivity to oxygen and reactivity to thiols.     

Discrimination against the cytosine analog tC by Escherichia coli DNA polymerase IV DinB

The cytosine analog 1,3-diaza-2-oxophenothiazine (tC) is a fluorescent nucleotide that forms Watson-Crick base pairs with dG. The Klenow fragment of DNA polymerase I (an A-family polymerase) can efficiently bypass tC on the template strand and incorporate deoxyribose-triphosphate-tC into the growing primer terminus. Y-family DNA polymerases are known for their ability to accommodate bulky lesions and modified bases and to replicate beyond such nonstandard DNA structures in a process known as translesion synthesis. We probed the ability of the Escherichia coli Y-family DNA polymerase DinB (Pol IV) to copy DNA containing tC and to incorporate tC into a growing DNA strand. DinB selectively adds dGTP across from tC in template DNA but cannot extend beyond the newly formed G:tC base pair. However, we find that DinB incorporates the tC deoxyribonucleotide triphosphate opposite template G and extends from tC. Therefore, DinB displays asymmetry in terms of its ability to discriminate against the modification of the DNA template compared to the incoming nucleotide. In addition, our finding that DinB (a lesion-bypass DNA polymerase) specifically discriminates against tC in the template strand may suggest that DinB discriminates against template modifications in the major groove of DNA.     

Kinetics of the plastoquinone pool oxidation following illumination Oxygen incorporation into photosynthetic electron transport chain

The oxidation of the PQ-pool after illumination with 50 or 500 micromol quantam(-2)s(-1) was measured in isolated thylakoids as the increase in DeltaA(263), i.e., as the appearance of PQ. While it was not observed under anaerobic conditions, under aerobic conditions it was biphasic. The first faster phase constituted 26% or 44% of total reappearance of PQ, after weak or strong light respectively. The dependence on oxygen presence as well as the correlation with the rate of oxygen consumption led to conclusion that this phase represents the appearance of PQ from PQ(*-) produced in the course of PQH(2) oxidation by superoxide accumulated in the light within the membrane.     

The reversibility of the glutathionyl-quercetin adduct spreads oxidized quercetin-induced toxicity

Quercetin is one of the most prominent dietary antioxidants. During its antioxidant activity, quercetin becomes oxidized into its o-quinone/quinone methide QQ. QQ is toxic since it instantaneously reacts with thiols of, e.g., proteins. In cells, QQ will initially form an adduct with glutathione (GSH), giving GSQ. We have found that GSQ is not stable; it dissociates continuously into GSH and QQ with a half life of 2min. Surprisingly, GSQ incubated with 2-mercapto-ethanol (MSH), a far less reactive thiol, results in the conversion of GSQ into the MSH-adduct MSQ. A similar conversion of GSQ into relatively stable protein thiol-quercetin adducts is expected. With the dithiol dihydrolipoic acid (L(SH)(2)), quercetin is formed out of GSQ. These results indicate that GSQ acts as transport and storage of QQ. In that way, the initially highly focussed toxicity of QQ is dispersed by the formation of GSQ that finally spreads QQ-induced toxicity, probably even over cells.     

Tetramethylphenylenediamine-induced hepatocyte cytotoxicity caused by lysosomal labilisation and redox cycling with oxygen activation

It has already been reported that in vivo muscle necrosis induced by various phenylenediamine derivatives correlated with their in vitro autoxidation rate [9]. Now in a more detailed investigation of the cytotoxic mechanism of a ring-methylated phenylenediamine known as tetramethylphenylenediamine or durenediamine (DD) towards isolated rat hepatocytes has been carried out. Cytotoxicity was preceded by ROS formation which was markedly increased by inactivating DT-diaphorase or catalase but were prevented by a subtoxic concentration of the mitochondrial respiratory inhibitor cyanide. This suggests that ROS generation could be attributed to a futile two-electron redox cycle involving oxidation of phenylenediamine to the corresponding diimine by the mitochondrial electron transfer chain and re-reduction by the DT-diaphorase. Endocytosis inhibitors, lysosomotropic agents or lysosomal protease inhibitors also prevented DD-induced cytotoxicity suggesting that DD-induced ROS caused lysosomal damage and protease activation in hepatocytes. Furthermore preincubation with deferoxamine (a ferric iron chelator) or addition of antioxidants, catalase or ROS scavengers (mannitol, tempol or dimethylsulfoxide) prevented DD cytotoxicity. These results suggest that H(2)O(2) reacts with lysosomal Fe(2+) to form "ROS" which causes lysosomal lipid peroxidation, membrane disruption, protease release and cell death.     

The single chlorophyll a molecule in the cytochrome b6f complex: unusual optical properties protect the complex against singlet oxygen

The cytochrome b(6)f complex of oxygenic photosynthesis mediates electron transfer between the reaction centers of photosystems I and II and facilitates coupled proton translocation across the membrane. High-resolution x-ray crystallographic structures (Kurisu et al., 2003; Stroebel et al., 2003) of the cytochrome b(6)f complex unambiguously show that a Chl a molecule is an intrinsic component of the cytochrome b(6)f complex. Although the functional role of this Chl a is presently unclear (Kuhlbrandt, 2003), an excited Chl a molecule is known to produce toxic singlet oxygen as the result of energy transfer from the excited triplet state of the Chl a to oxygen molecules. To prevent singlet oxygen formation in light-harvesting complexes, a carotenoid is typically positioned within approximately 4 A of the Chl a molecule, effectively quenching the triplet excited state of the Chl a. However, in the cytochrome b(6)f complex, the beta-carotene is too far (> or =14 Angstroms) from the Chl a for effective quenching of the Chl a triplet excited state. In this study, we propose that in this complex, the protection is at least partly realized through special arrangement of the local protein structure, which shortens the singlet excited state lifetime of the Chl a by a factor of 20-25 and thus significantly reduces the formation of the Chl a triplet state. Based on optical ultrafast absorption difference experiments and structure-based calculations, it is proposed that the Chl a singlet excited state lifetime is shortened due to electron exchange transfer with the nearby tyrosine residue. To our knowledge, this kind of protection mechanism against singlet oxygen has not yet been reported for any other chlorophyll-containing protein complex. It is also reported that the Chl a molecule in the cytochrome b(6)f complex does not change orientation in its excited state.     

Relationship between the action of reactive oxygen and nitrogen species on bilayer membranes and antioxidants

Membrane lipid peroxidation (LPO) induced by hydroxyl (*OH) and ascorbyl (*Asc) radicals and by peroxynitrite (ONOO-) was investigated in asolectin (ASO), egg phosphatidylcholine (PC) and PC/phosphatidic acid mixtures (PC:PA) liposomes and rat liver microsomes (MC). Enthalpy variation (DeltaH) of PC:PA at different molar ratios were obtained by differential scanning calorimetry. It was also evaluated the LPO inhibition by quercetin, melatonin and Vitamin B6. The oxidant effect power follows the order *OH approximately *Asc > ONOO- on PC and MC; whilst on ASO liposomes, it follows *Asc > *OH approximately ONOO-. Increasing amounts of PA in PC liposomes resulted in lower levels of LPO. The DeltaH values indicate a more ordered membrane arrangement as a function of PA amount. The results were discussed in order to provide a complete view involving the influence of membranes, oxidants and antioxidants intrinsic behavior on the LPO dynamics.     

Protective effect of fosfomycin on gentamicin-induced lipid peroxidation of rat renal tissue

Fosfomycin is clinically recognized to reduce the aminoglycoside antibiotics-induced nephrotoxicity. However, little has been clarified why fosfomycin protects the kidney from the aminoglycosides-induced nephrotoxicity. Gentamicin, a typical aminoglycoside, is reported to cause lipid peroxidation. We focused on lipid peroxidation induced by gentamicin as a mechanism for the aminoglycosides-induced nephrotoxicity. The aim of this study is to investigate the effect of fosfomycin on the gentamicin-induced lipid peroxidation. In rat renal cortex mitochondria, fosfomycin was shown to depress the gentamicin-induced lipid peroxidation, which was evaluated by formation of thiobarbituric acid reactive substances (TBARS). Interestingly, this effect was observed in rat renal cortex mitochondria, but not in rat liver microsomes. However, fosfomycin did not affect lipid peroxidation of arachidonic acid caused by gentamicin with iron. Fosfomycin inhibited the gentamicin-induced iron release from rat renal cortex mitochondria. These results indicated that fosfomycin inhibited the gentamicin-induced lipid peroxidation by depressing the iron release from mitochondria. This may possibly be one mechanism for the protection of fosfomycin against the gentamicin-induced nephrotoxicity.     

Resistance to Ditylenchus africanus present in peanut breeding lines

Peanut is an important cash crop both for commercial and small-scale farmers in South Africa. The effect of Ditylenchus africanus on peanut is mainly qualitative, leading to downgrading of consignments. This nematode is difficult to control because of its high reproductive and damage potential. The objective of this study was to identify peanut genotypes with resistance to D. africanus that would also be sustainable under field conditions. Selected peanut genotypes were evaluated against D. africanus in microplot and field trials. The inbred lines PC254K1 and CG7 were confirmed to be resistant to D. africanus. The resistance expressed by these two genotypes was sustainable under field conditions. The breeding line PC287K5 maintained low nematode numbers in some trials, but its level of resistance was not as strong or as sustainable as that of PC254K1 or CG7. However, PC287K5 could still play an important role in the peanut industry where lower D. africanus populations occur.