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.     

Dietary antioxidants provide differential subcellular protection in epithelial cells

Abstract

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 μM) of ascorbic acid, α-tocopherol and β-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.     

Green tea metabolite EGCG protects membranes against oxidative damage in vitro

Green tea polyphenols like epigallocatechin gallate (EGCG) have been proposed as a cancer chemopreventative. Several studies have shown that EGCG can act as an antioxidant by trapping proxyl radicals and inhibiting lipid peroxidation. The main propose of this study is to investigate the antioxidant capacity of EGCG using erythrocyte membrane-bound ATPases as a model. The effects of EGCG on t-butylhydroperoxide-induced lipid peroxidation and the activity of membrane-bound ATPases in human erythrocyte membranes were studied. The extent of oxidative damage in membranes was assessed by measuring lipid peroxidation, (TBARS, thiobarbituric acid reactive substances formation) and the activity of ATPases (Na(+)/K(+), Ca(2+), and CaM-activated Ca(2+) pump ATPases). EGCG blocked t-BHP induced lipid peroxidation in erythrocyte membranes, significantly (0.45 +/- 0.02 vs 0.20 +/- 0.01; t-BHP vs t-BHP + EGCG respectively, microm/L TBARS) (p < 0.05). EGCG also protected ATPases against t-BHP induced damage; for Na/K ATPase (2.4 +/- 0.2 vs 1.6 +/- 0.1 vs 2.44 +/- 0.2, nmol Pi/min/mg protein, control vs t-BHP vs t-BHP and EGCG respectively), for Ca ATPase (5.8 +/- 0.4 vs 3.9 +/- 0.3 vs 5.6 +/- 0.34, nmol Pi/min/mg protein, control vs t-BHP vs t-BHP and EGCG respectively) and for CaM-Ca ATPase (14.7 +/- 0.7 vs 7.3 +/- 0.4 vs 11.6 +/- 0.55, nmol Pi/min/mg protein, control vs t-BHP vs t-BHP and EGCG respectively) (p < 0.05). In conclusion our results indicate that EGCG is a powerful antioxidant that is capable protecting erythrocyte membrane-bound ATPases against oxidative stress.     

Polyhydroxylated 4-thiaflavans as multipotent antioxidants: protective effect on oxidative DNA damage in vitro

Hydroxylated 4-thiaflavans, possessing the antioxidant features of catechol containing flavonoids and/or tocopherols, were evaluated as protective agents against oxidation damage induced in herring sperm DNA by cumene hydroperoxide (CumOOH) or by the glutathione/ferric ion (GSH/Fe(3+)) system. Our data indicate that the effective protection exerted by some of the tested compounds is overall higher than those provided by catechin and alpha-tocopherol, which might be attributed both to the scavenging properties and chelation of Fe(2+) ions.     

Thioredoxin-1 contributes to protection against DON-induced oxidative damage in HepG2 cells

Leucocytes are susceptible to the toxic effects of deoxynivalenol (DON), which is a trichothecene mycotoxin produced by a number of fungi including Fusarium species. One mechanism of action is mediated by reactive oxygen species (ROS). The liver is an important target for toxicity caused by foreign compounds including mycotoxins. On the other hand, little is known about the influence of the redox state on hepatocytes treated with DON. The present study investigated the effect of DON on the cytosolic redox state and antioxidative system in the human hepatoma cell line HepG2. The cell viability of human monocyte cell line THP-1 or leukemia cell line KU812 treated with 2.5 and 5???mol/l DON were significantly reduced. However, HepG2 cells showed no toxic effects under the same conditions and did not exhibit an increased oxidative state. Further experiments showed that thioredoxin-1 (Trx-1) protein levels but not glutathione increased in the cells treated with 10???mol/l DON. In addition, the enhancement of Trx-1 content was repressed by antioxidants. These results suggest that DON-induced accumulation of Trx-1 in HepG2 cells plays one of the key roles in protection against cytotoxicity caused by DON and that the mechanism may be mediated by the antioxidant properties of Trx-1.     

The role of selenium peroxidases in the protection against oxidative damage of membranes

The present review deals with the chemical properties of selenium in relation to its antioxidant properties and its reactivity in biological systems. The interaction of selenite with thiols and glutathione and the reactivity of selenocompounds with hydroperoxides are described. After a short survey on distribution, metabolism and organification of selenium, the role of this element as a component of the two seleno-dependent glutathione peroxidases is described. The main features of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are also reviewed. Both enzymes reduce different hydroperoxides to the corresponding alcohols and the major difference is the reduction of lipid hydroperoxides in membrane matrix catalyzed only by the phospholipid hydroperoxide glutathione peroxidase. However, in spite of the different specificity for the peroxidic substrates, the kinetic mechanism of both glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase seems identical and proceeds through a tert-uni ping pong mechanism. In the reaction cycle, indeed, as supported by the kinetic data, the oxidation of the ionized selenol by the hydroperoxide yields a selenenic acid that in turn is reduced back by two reactions with reduced glutathione. Special emphasis has been given to the role of selenium-dependent glutathione peroxidases in the prevention of membrane lipid peroxidation. While glutathione peroxidase is able to reduce hydrogen peroxide and other hydroperoxides possibly present in the soluble compartment of the cell, this enzyme fails to inhibit microsomal lipid peroxidation induced by NADPH or ascorbate and iron complexes. On the other hand, phospholipid hydroperoxide glutathione peroxidase, by reducing the phospholipid hydroperoxides in the membranes, actively prevents lipid peroxidation, provided a normal content of vitamin E is present in the membranes. In fact, by preventing the free radical generation from lipid hydroperoxides, phospholipid hydroperoxide glutathione peroxidase decreases the vitamin E requirement necessary to inhibit lipid peroxidation. Finally, the possible regulatory role of the selenoperoxidases on the arachidonic acid cascade enzymes (cyclooxygenase and lipoxygenase) is discussed.     

Cooperation of antioxidants in protection against photosensitized oxidation

The aim of this study was to determine whether alpha-tocopherol and zeaxanthin offer synergistic protection against photosensitized lipid peroxidation mediated by singlet oxygen and free radicals. The antioxidant action of zeaxanthin and alpha-tocopherol was studied in liposomes made of phosphatidylcholine and cholesterol. Progress of lipid peroxidation, induced by aerobic photoexcitation of rose bengal, was monitored by the detection of lipid hydroperoxides and by electron spin resonance oximetry. In addition, cholesterol was employed as a mechanistic reporter molecule, which forms characteristic products of the interaction with singlet oxygen or free radicals. Cholesterol hydroperoxides were quantitatively determined by HPLC/electrochemical detection. HPLC/ultraviolet-visible (UV-VIS) absorption detection was used to measure concentrations of zeaxanthin and alpha-tocopherol. Zeaxanthin, even at concentrations of 2.5 microM, effectively protected against singlet oxygen-mediated lipid peroxidation but was rapidly consumed due to interaction with free radicals. alpha-Tocopherol alone was not effective in protecting against lipid peroxidation, even at concentration of 0.1 mM. Combinations of zeaxanthin and alpha-tocopherol exerted a synergistic protection against lipid peroxidation. The synergistic effect may be explained in terms of prevention of carotenoid consumption by effective scavenging of free radicals by alpha-tocopherol therefore allowing zeaxanthing to quench the primary oxidant-singlet oxygen effectively.     

Chemical induction of cellular antioxidants affords marked protection against oxidative injury in vascular smooth muscle cells

Extensive evidence suggests that reactive oxygen species are critically involved in the pathogenesis of cardiovascular diseases, such as atherosclerosis and myocardial ischemia-reperfusion injury. Consistent with this concept, administration of exogenous antioxidants has been shown to be protective against oxidative cardiovascular injury. However, whether induction of endogenous antioxidants by chemical inducers in vasculature also affords protection against oxidative vascular cell injury has not been extensively investigated. In this study, using rat aortic smooth muscle A10 cells as an in vitro system, we have studied the induction of cellular antioxidants by the unique chemoprotector, 3H-1,2-dithiole-3-thione [corrected] (D3T) and the protective effects of the D3T-induced cellular antioxidants against oxidative cell injury. Incubation of A10 cells with micromolar concentrations of D3T for 24 h resulted in a significant induction of a battery of cellular antioxidants in a concentration-dependent manner. These included reduced glutathione (GSH), GSH peroxidase, GSSG reductase, GSH S-transferase, superoxide dismutase, and catalase. To further examine the protective effects of the induced endogenous antioxidants against oxidative cell injury, A10 cells were pretreated with D3T and then exposed to either xanthine oxidase (XO)/xanthine, 4-hydroxynonenal, or cadmium. We observed that D3T pretreatment of A10 cells led to significant protection against the cytotoxicity induced by XO/xanthine, 4-hydroxynonenal or cadmium, as determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium reduction assay. Taken together, this study demonstrates for the first time that a number of endogenous antioxidants in vascular smooth muscle cells can be induced by exposure to D3T, and that this chemical induction of cellular antioxidants is accompanied by markedly increased resistance to oxidative vascular cell injury.     

Spermine and spermidine mediate protection against oxidative damage caused by hydrogen peroxide

Summary. The polyamines spermidine and spermine have been hypothesized to possess different functions in the protection of DNA from reactive oxygen species. The growth and survival of mouse fibroblasts unable to synthesize spermine were compared to their normal counterparts in their native and polyamine-depleted states in response to oxidative stress. The results of these studies suggest that when present at normal or supraphysiological concentrations, either spermidine or spermine can protect cells from reactive oxygen species. However, when polyamine pools are pharmacologically manipulated to produce cells with low levels of predominately spermine or spermidine, spermine appears to be more effective. Importantly, when cells are depleted of both glutathione and endogenous polyamines, they exhibit increased sensitivity to hydrogen peroxide as compared to glutathione depletion alone, suggesting that polyamines not only play a role in protecting cells from oxidative stress but this role is distinct from that played by glutathione.     

Involvement of DNA polymerase beta in protection against the cytotoxicity of oxidative DNA damage

We had shown previously that DNA polymerase beta (beta-pol) null mouse fibroblasts, deficient in base excision repair (BER), are hypersensitive to monofunctional methylating agents but not to hydrogen peroxide (H2O2). This is surprising because beta-pol is thought to be involved in BER of oxidative as well as methylated DNA damage. We confirm these findings here in early-passage cells. However, with time in culture, beta-pol null cells become hypersensitive to H2O2 and other reactive oxygen species-generating agents. Analysis of in vitro BER reveals a strong deficiency in single-nucleotide BER of 8-oxoguanine (8-oxoG) by both early- and late-passage beta-pol null cell extracts. Therefore, in early-passage wild-type and beta-pol null cells, the capacity for single-nucleotide BER of 8-oxoG does not correlate with cellular sensitivity to H2O2. Expression of beta-pol protein in the late-passage null cells almost completely reverses the H2O2-hypersensitivity phenotype. Methoxyamine (MX) treatment sensitizes late-passage wild-type cells to H2O2 as expected for beta-pol-mediated single-nucleotide BER; however in beta-pol null cells, MX has no effect. The data indicate a role(s) of beta-pol-dependent repair in protection against the cytotoxicity of oxidative DNA damage in wild-type cells.     

Anticarcinogenic antioxidants as inhibitors against intracellular oxidative stress

Oxidative stress has been implicated in the pathogenesis of numerous diseases, including cancer. In the present study, the protective effect of natural antioxidants, such as quercetin and tea polyphenols, on intracellular oxidative stress was studied. Here we report a novel function of quercetin and tea polyphenols, as potential inhibitors of 4-hydroxy-2-nonenal (HNE)-induced intracellular oxidative stress and cytotoxicity. In rat liver epithelial RL34 cells, a potent electrophile HNE dramatically induced the productions of reactive oxygen species (ROS), which correlated well with the reduction in cell viability. We found that quercetin and tea polyphenols, such as epigallocatechin gallate and theaflavins and their gallate esters, significantly inhibited the HNE-induced ROS production and cytotoxicity. In addition, HNE induced a transient decrease in the mitochondrial membrane potential (Δψ), which was also retarded by the antioxidants. These data suggest that the antioxidants, such as quercetin and tea polyphenols, are inhibitors against mitochondrial ROS production.     

Anticarcinogenic antioxidants as inhibitors against intracellular oxidative stress

Oxidative stress has been implicated in the pathogenesis of numerous diseases, including cancer. In the present study, the protective effect of natural antioxidants, such as quercetin and tea polyphenols, on intracellular oxidative stress was studied. Here we report a novel function of quercetin and tea polyphenols, as potential inhibitors of 4-hydroxy-2-nonenal (HNE)-induced intracellular oxidative stress and cytotoxicity. In rat liver epithelial RL34 cells, a potent electrophile HNE dramatically induced the productions of reactive oxygen species (ROS), which correlated well with the reduction in cell viability. We found that quercetin and tea polyphenols, such as epigallocatechin gallate and theaflavins and their gallate esters, significantly inhibited the HNE-induced ROS production and cytotoxicity. In addition, HNE induced a transient decrease in the mitochondrial membrane potential (Δψ), which was also retarded by the antioxidants. These data suggest that the antioxidants, such as quercetin and tea polyphenols, are inhibitors against mitochondrial ROS production.     

Rice NTRC is a high-efficiency redox system for chloroplast protection against oxidative damage

One of the mechanisms plants have developed for chloroplast protection against oxidative damage involves a 2-Cys peroxiredoxin, which has been proposed to be reduced by ferredoxin and plastid thioredoxins, Trx x and CDSP32, the FTR/Trx pathway. We show that rice (Oryza sativa) chloroplast NADPH THIOREDOXIN REDUCTASE (NTRC), with a thioredoxin domain, uses NADPH to reduce the chloroplast 2-Cys peroxiredoxin BAS1, which then reduces hydrogen peroxide. The presence of both NTR and Trx-like domains in a single polypeptide is absolutely required for the high catalytic efficiency of NTRC. An Arabidopsis thaliana knockout mutant for NTRC shows irregular mesophyll cell shape, abnormal chloroplast structure, and unbalanced BAS1 redox state, resulting in impaired photosynthesis rate under low light. Constitutive expression of wild-type NTRC in mutant transgenic lines rescued this phenotype. Moreover, prolonged darkness followed by light/dark incubation produced an increase in hydrogen peroxide and lipid peroxidation in leaves and accelerated senescence of NTRC-deficient plants. We propose that NTRC constitutes an alternative system for chloroplast protection against oxidative damage, using NADPH as the source of reducing power. Since no light-driven reduced ferredoxin is produced at night, the NTRC-BAS1 pathway may be a key detoxification system during darkness, with NADPH produced by the oxidative pentose phosphate pathway as the source of reducing power.     

Ozone oxidative preconditioning: a protection against cellular damage by free radicals.

There is some anecdotal evidence that oxygen-ozone therapy may be beneficial in some human diseases. However so far only a few biochemical and pharmacodynamic mechanisms have been elucidated. On the basis of preliminary data we postulated that controlled ozone administration would promote an oxidative preconditioning preventing the hepatocellular damage mediated by free radicals. Six groups of rats were classified as follows: (1) negative control, using intraperitoneal sunflower oil; (2) positive control using carbon tetrachloride (CCl4) as an oxidative challenge; (3) oxygen-ozone, pretreatment via rectal insufflation (15 sessions) and after it, CCl4; (4) oxygen, as group 3 but using oxygen only; (5) control oxygen-ozone, as group 3, but without CCl4; group (6) control oxygen, as group 5, but using oxygen only. We have evaluated critical biochemical parameters such as levels of transaminase, cholinesterase, superoxide dismutase, catalase, phospholipase A, calcium dependent ATPase, reduced glutathione, glucose 6 phosphate dehydrogenase and lipid peroxidation. Interestingly, in spite of CCl4 administration, group 3 did not differ from group 1, while groups 2 and 4 showed significant differences from groups 1 and 3 and displayed hepatic damage. To our knowledge these are the first experimental results showing that repeated administration of ozone in atoxic doses is able to induce an adaptation to oxidative stress thus enabling the animals to maintain hepatocellular integrity after CCl4 poisoning.     

Role of vitamin E in the protection of the resident macrophage membrane against oxidative damage

The variation in tocopherol content of resident peritoneal rat macrophages was investigated during an oxidative stress provided by superoxide anions. Fluorometric measurements showed that phagocytic cells contain 298 +/- 18 ng vit.E/mg prot. The vitamin E level remains nearly constant during 1 h of incubation: 266 +/- 46 ng vit.E/mg prot. HPLC control at 37 degrees C validates our fluorometric measurement. Superoxide anions (O2-.) synthesis was activated by phorbol myristate acetate (PMA) (0.5 microgram/ml), after 1 h of incubation a decrease of 40% of the macrophage tocopherol level was observed and assessed by HLPC control. No tocopherolquinone (TQ) was detected. To clarify this point, tocopherol oxidation was followed spectrophotometrically. Results did not show any appearance of TQ at 265 nm but appearance of a peak at 307 mm. This our results show for the first time that macrophages possess vitamin E which plays a partial role in the protection of their plasma membrane. The lack of detection of TQ is of interest and the study of this unidentified product of oxidation should help us to understand the exact metabolism of vitamin E.     

Diquat-induced oxidative damage in hepatic microsomes: effects of antioxidants.

The ability of the redox cycling compound, diquat, to induce lipid peroxidation and oxidative damage was investigated using hepatic microsomes. Antioxidants, with demonstrated efficacy in physical models of oxidative stress, were examined in a diquat model. Diquat (10 microM-3 mM) induced lipid peroxidation (TBARS) in hepatic microsomes prepared from Fischer 344 rats. Diquat (1 mM) also increased protein carbonyl formation, NADPH oxidation and superoxide anion radical production (acetylated cytochrome c reduction). The novel antioxidants U-74,006F, U-78,517G and the known antioxidant, DPPD, decreased diquat-induced lipid peroxidation to levels below that of the control. These antioxidants also decreased protein carbonyl formation caused by diquat. U-74,006F and U-78,517G reduced NADPH oxidation slightly; although this inhibition was statistically significant, the biological significance is questionable. DPPD had no effect on this parameter. U-78,517G inhibited the reduction of acetylated cytochrome c slightly, whereas the other antioxidants had little effect. Thus overall, the increase in NADPH oxidation and the production of superoxide anion by redox cycling of diquat were not substantially affected by antioxidants. Neither did the test compounds show evidence of activity as iron chelators. This leads to the suggestion that antioxidants are preventing diquat-induced oxidative damage by scavenging lipid peroxyl radicals and preventing the propagation of the lipid peroxidation process.     

Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae

Glutaredoxins are members of a superfamily of thiol disulfide oxidoreductases involved in maintaining the redox state of target proteins. In Saccharomyces cerevisiae, two glutaredoxins (Grx1 and Grx2) containing a cysteine pair at the active site had been characterized as protecting yeast cells against oxidative damage. In this work, another subfamily of yeast glutaredoxins (Grx3, Grx4, and Grx5) that differs from the first in containing a single cysteine residue at the putative active site is described. This trait is also characteristic for a number of glutaredoxins from bacteria to humans, with which the Grx3/4/5 group has extensive homology over two regions. Mutants lacking Grx5 are partially deficient in growth in rich and minimal media and also highly sensitive to oxidative damage caused by menadione and hydrogen peroxide. A significant increase in total protein carbonyl content is constitutively observed in grx5 cells, and a number of specific proteins, including transketolase, appear to be highly oxidized in this mutant. The synthetic lethality of the grx5 and grx2 mutations on one hand and of grx5 with the grx3 grx4 combination on the other points to a complex functional relationship among yeast glutaredoxins, with Grx5 playing a specially important role in protection against oxidative stress both during ordinary growth conditions and after externally induced damage. Grx5-deficient mutants are also sensitive to osmotic stress, which indicates a relationship between the two types of stress in yeast cells.     

Hydroxy-urea protects erythrocytes against oxidative damage

Hydroxy-urea (OH-U) is used to treat sickle cell anemia by increasing hemoglobin fetal-fraction. It has been suggested that the sickle cell mutations lead to the formation of unstable HbS and release of iron, which can result in lipid peroxidation (LPO), and eventual cell damage. Since oxidative processes might be involved in pathogenesis of sickle cell disease, we investigated the antioxidant property of OH-U in a red blood cell (RBC) model. Intact RBCs or RBC membranes were exposed to t-butyl hydroperoxide (t-BHP, 0.75 mM) or iron (ferrous sulfate; 100 microM) at 37 degrees C for 60 min in the presence or absence of OH-U (1.25 mM). The extent of oxidative damage was measured by LPO (as thiobarbituric acid reactive substances, TBARS), hemoglobin oxidation (as percent of methemoglobin, metHb %), and decrease in the activities of membrane-bound Na+/K+-ATPase and Ca2+-ATPases. Our results show that OH-U inhibited t-BHP-induced LPO in fresh RBC membranes significantly (P <0.01). OH-U significantly inhibited t-BHP-mediated LPO (P <0.01) and metHb formation (P <0.01) in intact RBC. Also, OH-U inhibited iron-induced LPO and metHb formation in intact RBC (P <0.01). In addition, OH-U blocked t-BHP-mediated changes in membrane ATPase activities. Furthermore, OH-U blocked iron-mediated hydroxyl radical generation in a dose-dependent fashion. In conclusion, the observed antioxidant properties of OH-U might contribute to its therapeutic action in sickle cell disease.