Protective effect of antioxidants against para-nonylphenol-induced inhibition of cell growth in Saccharomyces cerevisiae

The cell growth-modulating activity of an endocrine disruptor, p-nonylphenol (NP), was estimated using the yeast Saccharomyces cerevisiae as a simple model of eukaryotic cells. NP caused a dose-dependent suppressive effect on cell growth of S. cerevisiae at 10, 25 and 50 microM. The NP-induced cell growth inhibition was restored when concomitantly lipophilic antioxidants such as alpha-tocopherol and beta-carotene were supplied, but not the hydrophilic antioxidants ascorbic acid or (-)epigallocatechin gallate (EGCG). The cellular oxygen consumption of S. cerevisiae was also inhibited in a dose-dependent fashion by the extracellular addition of NP, and pretreatment with alpha-tocopherol and beta-carotene suppressed NP-induced inhibition of cellular oxygen consumption, but ascorbic acid and EGCG were not effective. Furthermore, NP caused a marked generation of radical oxygen species (ROS) in S. cerevisiae, which was suppressed by treatment with alpha-tocopherol and beta-carotene, but not with ascorbic acid and EGCG. However, NP did not show a significant inhibitory effect on cell growth and survival of mitochondria-deficient petite mutant cells and they showed a relatively weak ROS-generating activity compared with parent yeast cells. These results suggest that NP-induced inhibition of cell growth and oxygen consumption in S. cerevisiae might be possibly associated with ROS generation in yeast mitochondria. The significance of this finding is discussed from the viewpoint of NP-induced oxidative stress against eukaryotic cells.     

Lipoxygenase-catalysed degradation of carotenoids from tomato in the presence of antioxidant vitamins

Carotenoid extract from ripe tomato fruit was subjected to a lipoxygenase-catalysed co-oxidation in the presence of vitamin C and vitamin E at different concentrations. Relative retention (%) of major carotenoids by the experimental mixture was used as an index of their degradation and interaction with the antioxidants. Oxidation-prevention activity of each antioxidant against pigment co-oxidation as impacted by their molar concentration was studied. beta-Carotene was found to be the most sensitive pigment, followed by lycoxanthin and lycopene. Ascorbic acid when added in the range of 0-1.8 mM interacted with the different carotenoids by different modes. Evidence was given on regeneration, by ascorbic acid, of lycopene during the course of co-oxidation. The concentration required for alpha-tocopherol acetate to exhibit antioxidative effect was 10 times higher than that of ascorbic acid. beta-Carotene was prevented, by alpha-tocopherol acetate, faster than lycoxanthin and lycopene. The latter carotenoids differed substantially in their interaction with the lipophilic antioxidant at only the lowest concentration (0.3 mM). It was established that under the given conditions there is no synergism between vitamin C and vitamin E that improves their oxidation prevention against co-oxidation of carotenoids. Moreover, the combined use of antioxidants caused more oxidative degradation of beta-carotene.     

Antioxidant modulation of oxidant-stimulated uptake and release of arachidonic acid in eicosapentaenoic acid-supplemented human lymphoma U937 cells

Omega-3 polyunsaturated fatty acids (PUFA) are increasingly finding use as treatments for a variety of medical conditions. PUFA supplementation can, however, result in increased oxidative stress causing elevated turnover rate of membrane phospholipids, impairment of membrane integrity and increased formation of inflammatory mediators. The aim of this study was to determine which antioxidant compounds were most effective in ameliorating the stimulation of phospholipid turnover by oxidative stress. U937 cells were supplemented with eicosapentaenoic acid and either ascorbic acid, alpha-tocopherol, beta-carotene or astaxanthin prior to being challenged with oxidant. Although all antioxidants were found to be effective in decreasing oxidant-stimulated peroxide formation, only alpha-tocopherol significantly decreased oxidant-stimulated release of 3H-labeled arachidonic acid (AA), while ascorbic acid markedly increased release. All antioxidants except alpha-tocopherol decreased oxidant-stimulated 3H-AA uptake. Our data suggest that antioxidants are not equally effective in combating the effects of oxidative stress upon membrane phospholipid turnover, and that optimal protection will require mixtures of antioxidants.     

Protection by quercetin and quercetin 3-O-beta-D-glucuronide of peroxynitrite-induced antioxidant consumption in human plasma low-density lipoprotein.

Effect of quercetin and its conjugated metabolite quercetin 3-O-beta-D-glucuronide (Q3GA), on peroxynitrite-induced consumption of lipophilic antioxidants in human plasma low-density lipoprotein (LDL) was measured to estimate the role of dietary flavonoids in the defense system against oxidative modification of LDL based on the reaction of nitric oxide and superoxide anion. Synthesized peroxynitrite-induced consumption of endogenous lycopene beta-carotene and alpha-tocopherol was effectively suppressed by adding quercetin aglycone into LDL solution. Q3GA also inhibited the consumption of these antioxidants effectively. These results indicate that dietary quercetin is capable of inhibiting peroxynitrite-induced oxidative modification of LDL in association with lipophilic antioxidants present within this lipoprotein particle.     

Chemiluminescence evaluation of oxidative damage to biomolecules induced by singlet oxygen and the protective effects of antioxidants

A chemiluminescence (CL) method was developed for the evaluation of oxidative damage to biomolecules induced by singlet oxygen ((1)O(2)) and for the evaluation of the protective effects of antioxidants. The (1)O(2) was generated from the reaction of H(2)O(2)+OCl(-). Results showed that the CL signal from the reaction of H(2)O(2)+OCl(-) was weak, however, it was enhanced dose-dependently with the addition of DNA and unsaturated fatty acid, respectively. Spectra analysis indicated that the enhanced CL could be ascribed to the decay of triplet-excited carbonyl compounds, which were generated from the reaction of (1)O(2) plus the biomolecules. On the other hand, the enhanced CL produced in the above systems could be effectively inhibited by lycopene, beta-carotene, VC, and VE, but could not be inhibited by mannitol, SOD, and NaN(3). The mechanism therein was discussed.     

beta-Carotene: interactions with alpha-tocopherol and ascorbic acid in microsomal lipid peroxidation

beta-Carotene, alpha-tocopherol, and ascorbic acid were tested for their ability to inhibit, enhance, or react synergistically with O(2) (15, 150, 760 torr) and, 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH) or 1,1'-azobis (cyclohexane-carbonitrile) (ACCN) in isolated rat liver microsomes. beta-Carotene did not protect against lipid peroxidation, i.e., malondialdehyde (MDA) formation, in microsomal samples incubated at 37 degrees C with aqueous soluble AAPH at all added beta-carotene concentrations and oxygen tensions. More MDA (16%, p < 0.001) was produced at 15 torr of O(2,) and 160 nmol/mg protein of beta-carotene compared to respective vehicle control. Individually, alpha-tocopherol and ascorbic acid exhibited antioxidant protection (ascorbic acid &z.Gt; alpha-tocopherol); however, a mixture of both compounds was no more protective than ascorbic acid alone. beta-Carotene demonstrated a concentration-dependent antioxidant affect at 15 torr O(2) (p < 0.01); but a prooxidant effect at higher O(2) at 150 and 760 torr (>57%, p < 0.001) by lipid-soluble ACCN. alpha-Tocopherol exhibited concentration-dependent inhibitory effects on microsomal MDA formation at all oxygen tensions, but was most effective under 150 torr. Ascorbic acid demonstrated a concentration-dependent antioxidant effect only at 150 torr. ACCN-induced lipid peroxidation was no greater for the combination of the three compounds than ascorbic acid added alone. Thus, antioxidant or prooxidant activities for beta-carotene, alpha-tocopherol, and ascorbic acid in microsomal suspensions are related to O(2) tension, solubility, antioxidant concentrations and are governed by complex interactions. Differences between AAPH- and ACCN-induced lipid peroxidation are related to differences in lipid solubility.     

Ability of antioxidants to prevent oxidative mutations in Salmonella typhimurium TA102

An assay for the ability of antioxidants to prevent mutations induced by various oxidants in Salmonella typhimurium TA102 cells was developed. Protection against hydrogen-peroxide-induced mutagenicity was observed for quercetin, caffeic acid, ascorbic acid and dimethyl sulfoxide (used as a solvent for water-insoluble antioxidants). No protective effect was observed for green tea extract (weakly pro-oxidative), catechin, rutin, sinigrin, ferulic acid and alpha-tocopherol. Mutagenicity caused by tert-butyl hydroperoxide (tBOOH) was prevented most effectively by quercetin and ascorbic acid, whereas weaker effects were observed for green tea extract and for rutin, and no effect being observed for the other antioxidants tested. The results for hydrogen peroxide indicate iron chelation to be the most important protective mechanism. Radical scavenging appeared to be effective only with dimethyl sulfoxide and ascorbic acid, which are effective scavengers of hydroxyl radicals and were used here in high concentrations. It is proposed that the hydrogen-peroxide-induced mutations in the Salmonella cells are caused by hydroxyl radicals generated by iron ions closely associated with DNA. Protection against mutagenicity caused by tert-butyl hydroperoxide appears to occur mainly through the scavenging of alkoxyl and possibly of alkyl radicals.     

Design,synthesis, and antioxidant activity of FeAOX-6, a novel agent deriving from a molecular combination of the chromanyl and polyisoprenyl moieties

Several lines of evidence suggest potential benefits by a combination of carotenoids and tocopherols in chronic diseases. Therefore, we have designed FeAOX-6, a novel antioxidant that combines into a single molecule the chroman head of tocopherols and a fragment of lycopene, consisting of a polyisoprenyl sequence of four conjugated double bonds. The ability of FeAOX-6 in inhibiting lipid peroxidation and reactive oxygen species (ROS) production induced by different sources of free radicals (t-BOOH, AAPH, and H2O2) in arachidonic acid solution and in isolated thymocytes was investigated. Its antioxidant efficiency was also compared with that of alpha-tocopherol, lycopene, and a mixture of the two antioxidants. The results strongly suggest that FeAOX-6 can act as a potent antioxidant in our models, by inhibiting malondialdehyde production and ROS generation in a dose- and a time-dependent manner. In the cell model, the compound also provides a higher antioxidant capacity than alpha-tocopherol and lycopene, alone or in combination, suggesting the possibility of an oxidative intramolecular cooperation.     

Protective effects of alpha-tocopherol and beta-carotene on para-nonylphenol-induced inhibition of cell growth, cellular respiration and glucose-induced proton extrusion of bacteria

para-Nonylphenol (NP) showed a dose-dependent inhibition against the cell growth of Bacillus subtilis, Micrococcus luteus, Pseudomonas aeruginosa and Staphylococcus aureus at 5-100 microM. However, other typical plastic-derived endocrine disruptors such as bisphenol A and di-2-ethylhexyl phthalate (DEHP) did not significantly affect the cell growth of these bacteria at 5-100 microM. The NP-induced cell growth inhibition was restored when concomitantly supplemented with lipophilic antioxidants such as alpha-tocopherol and beta-carotene, but not with hydrophilic antioxidants, ascorbic acid and (-)-epigallocatechin gallate (EGCG). NP also suppressed in a dose-dependent manner cellular oxygen consumption and glucose-induced proton extrusion of these bacteria at 10-100 microM. Both effects were prevented when added with alpha-tocopherol and beta-carotene, but not with ascorbic acid and EGCG. The significance of these results is discussed from the viewpoint of environmental microbiology and a possible biochemical mechanism of the inhibitory effect of NP is suggested.     

Reduced levels of rat lens antioxidant vitamins upon in vitro UVB irradiation

Ultraviolet (UV) radiation is one of the major risk factors of cataractogenesis. UV radiation induced damage to the eye lens is believed to be mediated through reactive oxygen species. Antioxidant defense systems, enzymatic and non-enzymatic, resist this damage. In the present study, the levels of rat lens endogenous antioxidants, L-ascorbic acid, alpha-tocopherol and beta-carotene, have been determined by HPLC upon in vitro UVB irradiation. UVB irradiation for 24 h (300 nm; 100 μW/cm(2)) of three months old rat lens suspended in RPMI medium, leads to 69-89% decrease in endogenous levels of these antioxidants. The addition of ascorbic acid (2 mM), alpha-tocopherol (2.5 μM) or beta-carotene (10 μM), separately to the medium during irradiation significantly prevented the decrease in their endogenous levels, thereby suggesting a protective role for these antioxidant micronutrients against photodamage to the eye lens.     

Dietary antioxidants provide differential subcellular protection in epithelial cells

This study aimed to evaluate the organelle-specific antioxidant/pro-oxidant actions of clinically important dietary antioxidants against oxidative stress. An in vitro cellular model was employed to investigate the antioxidant/pro-oxidant effects of various concentrations (1, 10 and 100 microM) of ascorbic acid, alpha-tocopherol and beta-carotene during H2O2-induced oxidative stress. Damage to nuclear and mitochondrial genomes was analyzed by quantitative polymerase chain reaction and oxidation of membrane lipids was measured via colorimetric assays. The key findings were: (i) dietary antioxidants conferred a dose-dependent protective effect (with a pro-oxidant shift at higher concentrations); (ii) the protection conferred to different sub-cellular organelles is highly specific to the dietary antioxidant; (iii) the mtDNA is highly sensitive to oxidative attack compared to nDNA (P < 0.05); and (iv) mtDNA protection conferred by dietary antioxidants was required to improve protection against oxidative-induced cell death. This study shows that antioxidant-induced protection of mtDNA is an important target for future oxidative stress therapies.     

Effects of dietary antioxidants on human DNA ex vivo

The protective effect of fruits and vegetables against cancer is well established. It is believed that this effect is mediated by antioxidants and decreased oxidative damage to DNA. However, the identity of the antioxidant(s) responsible is not clear. Moreover, a potentially damaging pro-oxidant effect of some antioxidants has been reported. In this study the ex vivo effects of several dietary antioxidants, including quercetin, various catechins, ascorbic acid and alpha-tocopherol, were investigated, at concentrations up to 200 microM, using the single cell gel electrophoresis (comet) assay for DNA damage. Lymphocytes from three healthy subjects were pre-incubated with these antioxidants, and the comet assay was performed on treated, untreated, challenged and unchallenged cells in parallel, oxidant challenge being induced by 5 min exposure to hydrogen peroxide (final concentrations H2O2: 30, 45, or 60 microM). Results using this ex vivo cellular assay showed protection by some antioxidants (quercetin, caffeic acid), no effect by some (catechin, epicatechin, catechin gallate, epicatechin gallate) and an apparently damaging effect by others (epigallocatechin, epigallocatechin gallate). Damage may have been caused by production of H2O2 from these polyphenolics. Neither ascorbic acid nor alpha-tocopherol protected or damaged DNA. Further study of the role of quercetin and caffeic acid in DNA protection is needed.     

Antioxidant and free radical scavenging properties of N-acetylcysteine amide (NACA) and comparison with N-acetylcysteine (NAC)

The antioxidant potential of N-acetylcysteine amide (NACA), also known as AD4, was assessed by employing different in vitro assays. These included reducing power, free radical scavenging capacities, peroxidation inhibiting activity through linoleic acid emulsion system and metal chelating capacity, as compared to NAC and three widely used antioxidants, alpha-tocopherol, ascorbic acid and butylated hydroxytoluene (BHT). Of the antioxidant properties that were investigated, NACA was shown to possess higher 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging ability and reducing power than NAC, at all the concentrations, whereas the scavenging ability of H(2)O(2) differed with concentration. While NACA had greater H(2)O(2) scavenging capacity at the highest concentration, NAC was better than NACA at lower concentrations. NAC and NACA had a 60% and 55% higher ability to prevent beta-carotene bleaching, respectively, as compared to control. The chelating activity of NACA was more than 50% that of the metal chelating capacity of EDTA and four and nine times that of BHT and alpha-tocopherol, respectively. When compared to NACA and NAC; alpha-tocopherol had higher DPPH scavenging abilities and BHT and alpha-tocopherol had better beta-carotene bleaching power. These findings provide evidence that the novel antioxidant, NACA, has indeed enhanced the antioxidant properties of NAC.     

Effect of antioxidants on regeneration of protoplasts in the mycelial fungus Trichoderma reesei 6/16

The relative content of antioxidants in the mycelium of Trichoderma reesei 6/16 obtained by propagation of fungal protoplasts was shown to decrease (as compared to the initial culture taken for preparation of protoplasts) and restored only in the second generation of regenerated mycelium. In this respect, the effects of various antioxidants (beta-carotene, ascorbic acid, alpha-tocopherol, and ionol) on the frequency of regeneration of T. reesei 6/16 protoplasts were studied. beta-Carotene increased the viability of fungal protoplasts to the greatest extent. The effect of ascorbic acid depended on the presence of Fe ions. Ionol did not cause any measurable protective effect.     

Evaluation of the degree of medical radiation damage with a highly sensitive chemiluminescence method.

A highly sensitive chemiluminescence (CL) method for evaluation of medical radiation damage degree is presented. According to the principle of cell stress response to ionizing radiation, lymphocytes will produce reactive oxygen species (ROS) after irradiation. The ROS produced can react with 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha] pyrazin-3-one (MCLA), a specific CL probe for superoxide anion (O(.-) (2)) and singlet oxygen ((1)O(2)), to emit light at 465 nm. The CL intensity is positively related to the amount of generated ROS detected 30 min after irradiation. Cell viability, which is inversely related to cell mortality, was determined by MTT assay after 3 days' culture. The results show that both CL intensity and cell mortality of lymphocytes increase with the increase of the radiation dose when the dosage is no more than 3 Gy, suggesting a positive relationship between the degree of lymphocyte cell damage and the amount of ROS generated. In addition, the effects of catalase, Cu-Zn superoxide dismutase (SOD), mannitol, sodium azide (NaN(3)), and D(2)O on MCLA-dependent CL of lymphocytes are discussed. We believe that the MCLA-dependent CL method would potentially provide an easy way for evaluating the degree of lymphocyte damage induced by radiation.     

A method to measure the oxidizability of both the aqueous and lipid compartments of plasma.

The lipophilic radical initiator (MeO-AMVN) and the fluorescent probe C11BODIPY581/591 (BODIPY) were used to measure the lipid compartment oxidizability of human plasma. Aqueous plasma oxidizability was initiated by the aqueous peroxyl radical generator, AAPH, and 2',7'-dichlorodihydrofluorescein (DCFH) was employed as the marker of the oxidative reaction. The distribution in aqueous and lipid compartments of the two radical initiators was determined by measuring the rate of consumption of the plasma hydrophilic and lipophilic endogenous antioxidants. In the presence of AAPH (20 mM), the order of consumption was: ascorbic acid > alpha-tocopherol > uric acid > beta-carotene, indicating a gradient of peroxyl radicals from the aqueous to the lipid phase. When MeO-AMVN was used (2mM), beta-carotene was consumed earlier than uric acid and almost at the same time as alpha-tocopherol, reflecting the diffusion and activation of MeO-AMVN in the lipophilic phase. The rate of BODIPY oxidation (increase in green fluorescence) significantly increased after the depletion of endogenous alpha-tocopherol and beta-carotene, whereas it was delayed for 180 min when AAPH was used instead of MeO-AMVN. The measurement of lipid oxidation in plasma was validated by adding to plasma the two lipophilic antioxidants, alpha-tocopherol and beta-carotene, whose inhibitory effects on BODIPY oxidation were dependent on the duration of the preincubation period and hence to their lipid diffusion. DCFH oxidation induced by AAPH only began after uric acid, the main hydrophilic plasma antioxidant, was consumed. In contrast, when MeO-AMVN was used, DCFH oxidation was delayed for 120 min, indicating its localization in the aqueous domain. In summary, the selective fluorescence method reported here is capable of distinguishing the lipophilic and hydrophilic components of the total antioxidant capacity of plasma.     

alpha-Tocopherol increases caspase-3 up-regulation and apoptosis by beta-carotene cleavage products in human neutrophils

It has been found that beta-carotene cleavage products (CarCP), besides having mutagenic and toxic effects on mitochondria due to their prooxidative properties, also initiate spontaneous apoptosis of human neutrophils. Therefore, it was expected that antioxidants such as alpha-tocopherol would inhibit the stimulation of apoptosis and caspase-3 activity by CarCP. However, we found that alpha-tocopherol increases caspase-3 up-regulation and stimulation of apoptosis of human neutrophils by CarCP. Ascorbic acid does not alter this caspase-3 up-regulating and proapoptotic effect exerted by alpha-tocopherol. Both alpha-tocopherol and ascorbic acid, in the absence of CarCP, decrease intracellular caspase-3 activity and spontaneous apoptosis of neutrophils. Uric acid alone or in combination with CarCP does not exert apparent effects on caspase-3 activity and apoptosis. Up-regulating effect of alpha-tocopherol is not observed in the presence of retinol that markedly stimulates apoptosis by itself, whereas increase of caspase-3 activity is induced by concomitant addition of alpha-tocopherol and beta-ionone, a cyclohexenyl degradation product of beta-carotene with shorter aliphatic chain.     

Oxyradicals under UV-b stress and their quenching by antioxidants

Formation of oxyradicals under UV-B stress was investigated using cucumber cotyledons. UV-B radiation induced production of free radicals which were analyzed by ESR spectroscopy. Evidence was obtained for the formation of superoxide and hydroxyl radicals in the tissues by comparing PBN-adducts formed with radicals obtained by chemical autooxidation of KO2 and Fenton's reaction. Addition of superoxide dismutase (SOD) to the reaction mixture partially reduced the intensity of signals confirming the production of superoxide radical as well as hydroxyl radicals. These radicals were quenched in vitro by the natural antioxidants alpha-tocopherol, ascorbic acid and benzoquinone. Changes in the level of antioxidants were also monitored under UV-B stress. The endogenous level of ascorbic acid was enhanced and alpha-tocopherol level was reduced in the tissue after exposure to UV-B radiation. The present report happens to be the first direct evidence obtained for the formation of superoxide and hydroxyl radicals in plant tissues exposed to UV-B radiation.     

Nicotinamide (vitamin B3) as an effective antioxidant against oxidative damage in rat brain mitochondria

Nicotinamide (vitamin B3) an endogenous metabolite, showed significant inhibition of oxidative damage induced by reactive oxygen species (ROS) generated by ascorbate-Fe2+ and photosensitization systems in rat brain mitochondria. It protected against both protein oxidation and lipid peroxidation, at millimolar concentrations. Inhibition was more pronounced against oxidation of proteins than peroxidation of lipids. Chemically related endogenous compounds, tryptophan and isonicotinic acid, showed comparable inhibitory properties. The protective effect observed, at biologically relevant concentrations, with nicotinamide was more than that of the endogenous antioxidants ascorbic acid and alpha-tocopherol. Hence our studies suggest that nicotinamide (vitamin B3) can be considered as a potent antioxidant capable of protecting the cellular membranes in brain, which is highly susceptible to prooxidants, against oxidative damage induced by ROS.     

Inhibitory effect of flavonoids on low-density lipoprotein peroxidation catalyzed by mammalian 15-lipoxygenase

Lipoxygenase-dependent low-density lipoprotein (LDL) oxidation is believed to be involved in atherogenesis. Inhibition of lipoxygenase-induced lipid peroxidation might, therefore, be an important mode to suppress the development of atherosclerosis. Because dietary antioxidants inhibit LDL oxidation in vitro and their intake is inversely associated with coronary heart diseases, we compared the inhibitory effect of three typical flavonoids-quercetin, epicatechin, and flavone-with alpha-tocopherol and ascorbic acid against human LDL oxidation catalyzed by mammalian 15-lipoxygenase. The oxidative modification of LDL was monitored by measurement of cholesteryl ester hydroperoxide (CE-OOH) formation and consumption of antioxidants by using HLPC. Quercetin and epicatechin were the strongest inhibitors of LDL oxidation catalyzed by 15-lipoxygenase; ascorbic acid was an effective inhibitor in the first 3 h of oxidation; and fivefold alpha-tocopherol-enriched LDL showed a partial inhibition of CE-OOH formation only after 4-6 h of incubation. Flavone had no effect. Quercetin, ascorbic acid, and alpha-tocopherol were consumed in the first 3 h of incubation. Consumption of LDL alpha-tocopherol was partially inhibited by ascorbic acid and quercetin, whereas epicatechin and flavone were without effect. These results emphasize the inhibitory effect of the flavonoids quercetin and epicatechin on 15-lipoxygenase-mediated LDL lipid peroxidation. At similar concentrations, they are stronger antioxidants than ascorbic acid, alpha-tocopherol, and flavone.