Antioxidant activities of the flaxseed lignan secoisolariciresinol diglucoside, its aglycone secoisolariciresinol and the mammalian lignans enterodiol and enterolactone in vitro.

The flaxseed lignan secoisolariciresinol diglucoside (SDG) and mammalian lignans enterodiol (ED) and enterolactone (EL) were previously shown to be effective antioxidants against DNA damage and lipid peroxidation. Others reported inhibition of activated cell chemiluminescence by supra-physiological concentrations of secoisolariciresinol (SECO), ED and EL. Thus, we evaluated the antioxidant efficacy of potential physiological concentrations of SDG, SECO, ED and EL against 1,1-diphenyl-2-picrylhydrazyl (DPPH()), and 2,2'-azo-bis(2-amidinopropane) dihydrochloride (AAPH)-initiated peroxyl radical plasmid DNA damage and phosphatidylcholine liposome lipid peroxidation. SDG and SECO were effective (p<0.01) antioxidants against DPPH() at 25-200muM; whereas, ED and EL were inactive. Efficacy of lignans and controls against AAPH peroxyl radical-induced DNA damage was: SDG>SECO=17alpha-estradiol>ED=EL>genistein>daidzein. Lignan efficacy against AAPH-induced liposome lipid peroxidation was: SDG>SECO=ED=EL. Plant lignan antioxidant activity was attributed to the 3-methoxy-4-hydroxyl substituents of SDG and SECO, versus the meta mono-phenol structures of ED and EL. Benzylic hydrogen abstraction and potential resonance stabilization of phenoxyl radicals in an aqueous environment likely contributed to the antioxidant activity of the mammalian lignans. These represent likely extra- and intracellular antioxidant activities of flax-derived lignans at concentrations potentially achievable in vivo.     

Protective effect of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on copper-induced hydroxyl radical generation in the rat heart

The present study was examined the effect of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on Cu(II)-induced hydroxyl radical generation (OH) in the extracellular fluid of rat myocardium. Rats were anesthetized and sodium salicylate in Ringer's solution (0.5 nmol/microl/min) was infused through a microdialysis probe to detect the generation of OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the myocardium. When Cu(II) was infused through the microdialysis probe, Cu(II) increased in OH formation trapped as 2,3-DHBA in the dialysate. When fluvastatin (100 microM) was administered to Cu(II) (50 microM)-pretreated animals, the levels of 2,3-DHBA at 300 min after administration of fluvastatin significantly decreased. In cumulative dose dependent experiments, three concentrations of Cu(II), 10, 25 and 50 microM, were infused through the microdialysis probe in the rat myocardium. A positive linear correlation between Cu(II) and the formation of 2,3-DHBA (R(2)=0.980) was observed. However, when corresponding experiments were performed with fluvastatin (100 microM) pretreated animals, the level of 2,3-DHBA decreased. These results suggest that blocking LDL oxidation by fluvastatin may attenuate Cu(II)-induced OH formation in the rat heart.     

Antioxidant, prooxidant and cytotoxic activity of hydroxylated resveratrol analogues: structure-activity relationship

Resveratrol (trans-3,4',5-trihydroxystilbene), a naturally occurring hydroxystilbene, is considered an essential antioxidative constituent of red wine possessing chemopreventive properties. However, resveratrol and even more its metabolite piceatannol were reported to have also cytostatic activities. In order to find out whether this is related to antioxidative properties of those compounds, we synthesized five other polyhydroxylated resveratrol analogues and studied structure-activity relationships between pro-/antioxidant properties and cytotoxicity. Radical scavenging experiments with O(2)(*-) (5,5-dimethyl-1-pyrroline-N-oxide/electron spin resonance (DMPO/ESR)) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (photometry) revealed that 3,3',4',5-tetrahydroxystilbene (IC(50): 2.69microM; k(9): 443000M(-1)s(-1)), 3,4,4',5-tetrahydroxystilbene (IC(50): 41.5microM; k(9): 882000M(-1)s(-1)) and 3,3',4,4',5,5'-hexahydroxystilbene (IC(50): 5.02microM), exerted a more than 6600-fold higher antiradical activity than resveratrol and its two other analogues. Furthermore, in HL-60 leukemic cells hydroxystilbenes with ortho-hydroxyl groups exhibited a more than three-fold higher cytostatic activity compared to hydroxystilbenes with other substitution patterns. Oxidation of ortho-hydroxystilbenes in a microsomal model system resulted in the existence of ortho-semiquinones, which were observed by ESR spectroscopy. Further experiments revealed that these intermediates undergo redox-cycling thereby consuming additional oxygen and forming cytotoxic oxygen radicals. In contrast to compounds with other substitution patterns hydroxystilbenes with one or two resorcinol groups (compounds 1 and 3) did not show an additional oxygen consumption or semiquinone formation. These findings suggest that the increased cytotoxicity of ortho-hydroxystilbenes is related to the presence of ortho-semiquinones formed during metabolism or autoxidation.     

Comparative study on antioxidant activity of different varieties of commonly consumed legumes in India

Legumes are rich source of proteins, dietary fiber, micronutrients and bioactive phytochemicals. Thirty different varieties of commonly consumed legumes in India, were screened for phenolic content and antioxidant activity using, radical scavenging [(1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid, (ABTS⁺)], Ferric Reducing Antioxidant Power (FRAP) and metal ion (Fe²⁺) chelation assays. Legumes varied largely in their antioxidant activity. Horse gram, common beans, cowpea (brown and red) and fenugreek showed high DPPH radical scavenging activity (>400 units/g), while lablab bean (cream and white), chickpea (cream and green), butter bean and pea (white and green) showed low antioxidant activity (<125 units/g). Green gram, black gram, pigeon pea, lentils, cowpea (white) and common bean (maroon) showed intermediate activity. Similar trend was observed when the activity was assessed with ABTS⁺ and FRAP assays. Thus most of the varieties having light color seed coat, except soybean exhibited low antioxidant activity. While legumes having dark color seed coat did not always possessed high antioxidant activity (e.g. moth bean, black pea, black gram, lentils). Antioxidant activity showed positive correlation (r² > 0.95) with phenolic contents, in DPPH, ABTS⁺ and FRAP assays, whereas poor correlation (r² = 0.297) was observed between Fe²⁺ chelating activity of the legumes and phenolic contents.     

Selective iNOS inhibition reduces renal damage induced by cisplatin

Cisplatin is a chemotherapeutic agent used in the treatment of several cancer tumors; however, nephrotoxicity has restricted its use. Reactive oxygen species and peroxynitrite, which is formed by the reaction between superoxide anion and nitric oxide (NO*), are implicated in cisplatin-induced nephrotoxicity. In contrast, both toxic and beneficial effects of NO* have been suggested in cisplatin-induced nephrotoxicity. Therefore, nowadays the role of NO* in this experimental model remains controversial. The aim of the present work was to elucidate the role of NO* in cisplatin-induced renal damage using N-[3-(aminomethyl)benzyl]acetamidine (1400W), a selective and irreversible inhibitor of iNOS. The mRNA levels of iNOS were increased in cisplatin-treated rats. The administration of 1400W reduced the cisplatin induced histological damage, renal dysfunction (increase in proteinuria and kidney injury molecule expression and decrease in creatinine clearance), tubulointerstitial infiltration, oxidative stress (increase in renal malondialdehyde and inmmunostaining for 4-hydroxy-2-nonenal) and nitrosative stress (immunostaining for 3-nitrotyrosine). In addition, the administration of 1400W was unable to modify systolic blood pressure in control rats. Our data demonstrate that selective iNOS inhibition reduces the cisplatin-induced nephrotoxicity and nitrosative stress which strongly suggest that in this experimental model (1) the NO* production is toxic and (2) iNOS is the main source of NO*.     

NADPH oxidase-mitochondria axis-derived ROS mediate arsenite-induced HIF-1α stabilization by inhibiting prolyl hydroxylases activity

Arsenic exposure has been shown to induce hypoxia inducible factor 1α (HIF-1α) accumulation, however the underlying mechanism remains unknown. In the present study, we tested the hypothesis that arsenic exposure triggered the interaction between NADPH oxidase and mitochondria to promote reactive oxygen species (ROS) production, which inactivate prolyl hydroxylases (PHDs) activity, leading to the stabilization of HIF-1α protein. Exposure of human immortalized liver cell line HL-7702 cells to arsenite induced HIF-1α accumulation in a dose-dependent manner, which was abolished by SOD mimetic MnTMPyP. Inhibition of NADPH oxidase with diphenyleneiodonium chloride (DPI) or inhibition of mitochondrial respiratory chain with rotenone significantly blocked arsenite-induced ROS production, and the mitochondria appeared to be the major source of ROS production. Arsenite treatment inhibited HIF-1α hydroxylation by prolyl hydroxylases (PHDs) and increased HIF-1α stabilization, but did not affect HIF-1α mRNA expression and Akt activation. Supplementation of ascorbate or Fe(II) completely abolished arsenite-induced PHDs inhibition and HIF-1α stabilization. In conclusion, these results define a unique mechanism of HIF-1α accumulation following arsenic exposure, that is, arsenic activates NADPH oxidase–mitochondria axis to produce ROS, which deplete intracellular ascorbate and Fe(II) to inactivate PHDs, leading to HIF-1α stabilization.     

New advances in molecular mechanisms and the prevention of adriamycin toxicity by antioxidant nutrients

Anthracyclines (doxorubicin, daunorubicin, epirubicin, and idarubicin) are currently the most effective group of anti-neoplastic drugs used in clinical practice. Of these, doxorubicin (also called adriamycin) is a key chemotherapeutic agent in cancer treatment, although its use is limited as a consequence of the chronic and acute toxicity associated with this drug. The molecular mechanisms of doxorubicin account for both the anti-cancer and the toxic side effects. Many antioxidants have been assayed, with positive or negative results, to prevent the toxicity of doxorubicin. The present review has two main goals: (1) to report the latest findings regarding the molecular mechanisms of doxorubicin toxicity; (2) to update our understanding of the role of natural antioxidants in preventive therapy against doxorubicin-induced toxicity. This review provides new evidence for the chemoprevention of doxorubicin toxicity, making use of natural antioxidants – in particular vitamin E, vitamin C, coenzyme Q, carotenoids, vitamin A, flavonoids, polyphenol, resveratrol, antioxidant from virgin olive oil and selenium – and offers new insights into the molecular mechanisms of doxorubicin toxicity with respect to DNA damage, free radicals and other parameters.     

Protective effect of diphlorethohydroxycarmalol isolated from Ishige okamurae against high glucose-induced-oxidative stress in human umbilical vein endothelial cells

In the present study, the protective effect of diphlorethohydroxycarmalol (DPHC) isolated from Ishige okamurae, a brown algae, on high glucose-induced-oxidative stress was investigated using human umbilical vein endothelial cells (HUVECs). High concentration of glucose (30 mM) treatment induced cytotoxicity whereas DPHC prevented cells from high glucose-induced damage; restoring cell viability was significantly increased. In addition, the lipid peroxidation, intracellular reactive oxygen species (ROS), and nitric oxide (NO) levels induced by high glucose treatment were effectively inhibited by addition of DPHC in a dose-dependent manner. DPHC also suppressed the over-expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins as well as nuclear factor-kappa B (NF-κB) activation induced by high glucose in HUVECs. These finding indicate that DPHC might be used as potential pharmaceutical agent which will reduce the damage caused by high glucose-induced-oxidative stress associated with diabetes.     

Antioxidant and antigenotoxic activities in Acacia salicina extracts and its protective role against DNA strand scission induced by hydroxyl radical

The antioxidant potency of Acacia salicina extracts was investigated. Total antioxidant capacity was determined using an ABTS⁺ assay. Superoxide radical scavenging was measured using riboflavin-light-nitro blue tetrazolium (NBT) assay. In addition, the content of phenols, total flavonoids and sterols were measured in the tested extracts. The petroleum ether exhibited a potent scavenging activity toward ABTS radical cations. Whereas, chloroform extract showed the highest activity against superoxides radicals and was also able to protect pKS plasmid DNA against hydroxyl radicals induced DNA damages. The antimutagenicity of these extracts was assayed using the Ames assay against Salmonella typhimurium TA98 and S. typhimurium TA 1535 tester strains at different concentrations. These extracts decreased significantly the mutagenecity induced by sodium azide (SA) and 4-nitro-o-phenylenediamine (NOP). The antioxidant and antimutagenecity activities exhibited by A. salicina depended on the chemical composition of the tested extracts.     

BCNU-induced gR2 DEFECT mediates S-glutathionylation of Complex I and respiratory uncoupling in myocardium

A deficiency of mitochondrial glutathione reductase (or GR2) is capable of adversely affecting the reduction of GSSG and increasing mitochondrial oxidative stress. BCNU [1,3-bis (2-chloroethyl)-1-nitrosourea] is an anticancer agent and known inhibitor of cytosolic GR ex vivo and in vivo. Here we tested the hypothesis that a BCNU-induced GR2 defect contributes to mitochondrial dysfunction and subsequent impairment of heart function. Intraperitoneal administration of BCNU (40 mg/kg) specifically inhibited GR2 activity by 79.8 ± 2.7% in the mitochondria of rat heart. However, BCNU treatment modestly enhanced the activities of mitochondrial Complex I and other ETC components. The cardiac function of BCNU-treated rats was analyzed by echocardiography, revealing a systolic dysfunction associated with decreased ejection fraction, decreased cardiac output, and an increase in left ventricular internal dimension and left ventricular volume in systole. The respiratory control index of isolated mitochondria from the myocardium was moderately decreased after BCNU treatment, whereas NADH-linked uncoupling of oxygen consumption was significantly enhanced. Extracellular flux analysis to measure the fatty acid oxidation of myocytes indicated a 20% enhancement after BCNU treatment. When the mitochondria were immunoblotted with antibodies against GSH and UCP3, both protein S-glutathionylation of Complex I and expression of UCP3 were significantly up-regulated. Overexpression of SOD2 in the myocardium significantly reversed BCNU-induced GR2 inhibition and mitochondrial impairment. In conclusion, BCNU-mediated cardiotoxicity is characterized by the GR2 deficiency that negatively regulates heart function by impairing mitochondrial integrity, increasing oxidative stress with Complex I S-glutathionylation, and enhancing uncoupling of mitochondrial respiration.     

Rescuing hepatocytes from iron-catalyzed oxidative stress using vitamins B1 and B6

In the following rescue experiments, iron-mediated hepatocyte oxidative stress cytotoxicity was found to be prevented if vitamin B1 or B6 was added 1 h after treatment with iron. The role of iron in catalyzing Fenton-mediated oxidative damage has been implicated in iron overload genetic diseases, carcinogenesis (colon cancer), Alzheimer’s disease and complications associated with the metabolic syndrome through the generation of reactive oxygen species (ROS). The objectives of this study were to interpret the cytotoxic mechanisms and intracellular targets of oxidative stress using “accelerated cytotoxicity mechanism screening” techniques (ACMS) and to evaluate the rescue strategies of vitamins B1 and B6. Significant cytoprotection by antioxidants or ROS scavengers indicated that iron-mediated cytotoxicity could be attributed to reactive oxygen species. Of the B6 vitamers, pyridoxal was best at rescuing hepatocytes from iron-catalyzed lipid peroxidation (LPO), protein oxidation, and DNA damage, while pyridoxamine manifested greatest protection against ROS-mediated damage. Thiamin (B1) decreased LPO, mitochondrial and protein damage and DNA oxidation. Together, these results indicate that added B1 and B6 vitamins protect against the multiple targets of iron-catalyzed oxidative damage in hepatocytes. This study provides insight into the search for multi-targeted natural therapies to slow or retard the progression of diseases associated with Fenton-mediated oxidative damage.     

Intracellular generation of reactive oxygen species by mitochondria

Mitochondria have bioenergetic properties that strongly suggest their involvement in the cellular formation of reactive oxygen species (ROS). Apparent confirmation of this process has come from work with isolated mitochondria, which have been shown to produce H(2)O(2) from dismutating superoxide radicals. Two different sites were reported to shuttle single electrons to oxygen out of the normal respiratory sequence. However, the mechanisms for ROS formation at these two sites are controversial. Arguments against mitochondrial ROS formation in the living cell are based on the fact that bioenergetic alterations may result from the mechanical removal of mitochondria from their natural environment. Furthermore, the invasive detection methods that are generally used may be inappropriate because of the possible interaction of the detection system with mitochondrial constituents. The use of non-invasive detection methods has proved that ROS formation does not occur unless changes in the physical state of the membrane are established. The aim of this commentary is to discuss critically the arguments in favor of mitochondria as the main intracellular source of ROS. The pros and cons of working with isolated mitochondria, as well as the detection methodology are carefully analyzed to judge whether or not the above assumption is correct. The conclusion that mitochondria are the main ROS generators in the cell contradicts the fact that ROS release was not observed. However, if electron flow from ubiquinol to the bc(1) complex is hindered due to changes in lipid fluidity, single electrons may transfer to dioxygen and produce H(2)O(2) via superoxide radicals.     

The effect of lipid peroxidation products on reactive oxygen species formation and nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophages

Lipid peroxidation induced by oxidants leads to the formation of highly reactive metabolites. These can affect various immune functions, including reactive oxygen species (ROS) and nitric oxide (NO) production.The aim of the present study was to investigate the effects of lipid peroxidation products (LPPs) - acrolein, 4-hydroxynonenal, and malondialdehyde - on ROS and NO production in RAW 264.7 macrophages and to compare these effects with the cytotoxic properties of LPPs. Macrophages were stimulated with lipopolysaccharide (0.1 μg/ml) and treated with selected LPPs (concentration range: 0.1-100 μM). ATP test, luminol-enhanced chemiluminescence, Griess reaction, Western blotting analysis, amperometric and total peroxyl radical-trapping antioxidant parameter assay were used for determining the LPPs cytotoxicity, ROS and NO production, inducible nitric oxide synthase expression, NO scavenging, and antioxidant properties of LPPs, respectively.Our study shows that the cytotoxic action of acrolein and 4-hydroxynonenal works in a dose- and time-dependent manner. Further, our results imply that acrolein, 4-hydroxynonenal, and malondialdehyde can inhibit, to a different degree, ROS and NO production in stimulated macrophages, partially independently of their toxic effect. Also, changes in enzymatic pathways (especially NADPH-oxidase and nitric oxide synthase inhibition) and NO scavenging properties are included in the downregulation of reactive species formation.     

Exposure to ZnO nanoparticles induces oxidative stress and cytotoxicity in human colon carcinoma cells

Engineered nanoparticles offer great promise in many industrial and biomedical applications, however little information is available about gastrointestinal toxicity. The purpose of this study was to assess the cytotoxicity, oxidative stress, apoptosis and proinflammatory mediator release induced by ZnO nanoparticles on human colon carcinoma LoVo cells. The biological activity of these particles was related to their physico-chemical characteristics. The physico-chemical characteristics were evaluated by analytical electron microscopy. The cytotoxicity was determined by growth curves and water-soluble tetrazolium assay. The reactive oxygen species production, cellular glutathione content, changes of mitochondrial membrane potential and apoptosis cell death were quantified by flow cytometry. The inflammatory cytokines were evaluated by enzyme-linked immunoadsorbent assay. Treatment with ZnO (5 μg/cm² corresponding to 11.5 μg/ml) for 24 h induced on LoVo cells a significant decrease of cell viability, H₂O₂/OH increase, O2⁻ and GSH decrease, depolarization of inner mitochondrial membranes, apoptosis and IL-8 release. Higher doses induced about 98% of cytotoxicity already after 24 h of treatment. The experimental data show that oxidative stress may be a key route in inducing the cytotoxicity of ZnO nanoparticles in colon carcinoma cells. Moreover, the study of the relationship between toxicological effects and physico-chemical characteristics of particles suggests that surface area does not play a primary role in the cytotoxicity.     

Antioxidant and pro-oxidant properties of acylated pelargonidin derivatives extracted from red radish (Raphanus sativus var. niger,Brassicaceae)

The antioxidant and pro-oxidant potential of an extract from red radish, in which the major compounds were acylated pelargonidin derivatives, were assessed with a variety of assays in vitro. The extract appeared to form a complex with Fe³⁺ or Cu²⁺. It displayed a concentration-dependant reducing power (1.16 OD_{700 nm} at a concentration of 4 mM) and scavenging effect against 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radicals (with IC₅₀ = 1.74 ± 0.03 mM). It could promote the cleavage of plasmid DNA with Cu(II)/H₂O₂ or Cu(II) alone. This DNA damage could be inhibited by horseradish peroxidase, catalase, and EDTA, respectively. The extract also showed growth inhibition of Bel-7402 cells at lower concentration. The results suggested that the formation of reactive oxygen species might be involved in the mechanism of DNA damage. The acylated pelargonidin derivatives extracted from red radish could act as antioxidant and pro-oxidant and their antioxidant and pro-oxidant properties were relative to the reaction conditions. It might provide novel antioxidant and anticarcinogenic agents.     

The antioxidant activity of caroverine

Caroverine, 1-(2-diethylaminoethyl)-3-(p-methoxy benzyl)-1,2-dihydro-2-quinoxalin-2-on-hydrochloride, is a class B calcium-channel-blocker and antiglutamatergic agent with significant effects on the brain function. Caroverine exhibits competitive AMPA antagonism, and at higher concentrations, noncompetitive NMDA antagonism. In clinical practice caroverine is used as a spasmolytic and otoneuroprotective agent. Since reactive oxygen species are supposed to be involved in the pathogenesis of inner ear diseases in which caroverine shows beneficial effects, the present study aimed to investigate the antioxidant properties of caroverine. Lipid peroxidation of liposomal membranes was suppressed in the presence of caroverine. In order to understand the mechanism of this antioxidant action of caroverine, we determined the rate constants both for a possible reaction with superoxide (O(2)(.-)) radicals from xanthine/xanthine oxidase and for a possible reaction with hydroxyl (.OH) radicals in Fenton system. Using a defined chemical reaction model O(2)(.-) scavenging was found to occur at a rather low rate constant only (3 x 10(2)M(-1)s(-1)). Thus, a reaction of caroverine with O(2)(.-) radicals is of marginal significance. In contrast, the reaction of caroverine with .OH radicals occurs at an extremely high rate constant (k=1.9 x 10(10)M(-1)s(-1)). The strong antioxidant activity of caroverine is therefore based both on the partial prevention and highly active scavenging of hydroxyl radicals.     

The role of reactive oxygen species in arsenite and monomethylarsonous acid-induced signal transduction in human bladder cells: acute studies

Arsenicals are known to induce ROS, which can lead to DNA damage, oxidative stress, and carcinogenesis. A human urothelial cell line, UROtsa, was used to study the effects of arsenicals on the human bladder. Arsenite [As(III)] and monomethylarsonous acid [MMA(III)] induce oxidative stress in UROtsa cells after exposure to concentrations as low as 1 microM and 50 nM, respectively. Previous research has implicated ROS as signaling molecules in the MAPK signaling pathway. As(III) and MMA(III) have been shown to increase phosphorylation of key proteins in the MAPK signaling cascade downstream of ErbB2. Both Src phosphorylation (p-Src) and cyclooxygenase-2 (COX-2) are induced after exposure to 50 nM MMA(III) and 1 microM As(III). These data suggest that ROS production is a plausible mechanism for the signaling alterations seen in UROtsa cells after acute arsenical exposure. To determine importance of ROS in the MAPK cascade and its downstream induction of p-Src and COX-2, specific ROS antioxidants (both enzymatic and non-enzymatic) were used concomitantly with arsenicals. COX-2 protein and mRNA was shown to be much more influenced by altering the levels of ROS in cells, particularly after MMA(III) treatment. The antioxidant enzyme superoxide dismutase (SOD) effectively blocked both As(III)-and MMA(III)- associated COX-2 induction. The generation of ROS and subsequent altered signaling did lead to changes in protein levels of SOD, which were detected after treatment with either 1 microM As(III) or 50 nM MMA(III). These data suggest that the generation of ROS by arsenicals may be a mechanism leading to the altered cellular signaling seen after low-level arsenical exposure.