Antioxidants protect the yeast Saccharomyces cerevisiae against hypertonic stress

Yeast (Saccharomyces cerevisiae) mutants lacking CuZnSOD have been reported to be hypersensitive to hypertonic media and to show increased oxidative damage. This study demonstrates that hypertonic medium (containing 0.8?M NaCl) increases the generation of superoxide and other reactive species in yeast cells. Other sequelae of exposure to hypertonic medium include oxidation of cellular low-molecular weight thiols and decrease in total antioxidant capacity of cellular extracts. Δsod1 mutant is more sensitive than a wild-type strain to colony growth inhibition on a hypertonic medium. Anaerobic conditions, ascorbate, glutathione, cysteine and dithiothreitol are able to ameliorate this growth inhibition but a range of other antioxidants does not protect. The protective ability of the antioxidants does not correlate with the rate of their reactions with superoxide but seems to be conditioned by low redox potential for one-electron oxidation of free radicals of the antioxidants. It suggests that repair of low-redox potential targets rather than prevention of their damage by superoxide is important in the antioxidant protection against oxidative stress induced by hypertonic conditions.     

Antioxidants protect the yeast Saccharomyces cerevisiae against hypertonic stress

Yeast (Saccharomyces cerevisiae) mutants lacking CuZnSOD have been reported to be hypersensitive to hypertonic media and to show increased oxidative damage. This study demonstrates that hypertonic medium (containing 0.8 M NaCl) increases the generation of superoxide and other reactive species in yeast cells. Other sequelae of exposure to hypertonic medium include oxidation of cellular low-molecular weight thiols and decrease in total antioxidant capacity of cellular extracts. deltasod1 mutant is more sensitive than a wild-type strain to colony growth inhibition on a hypertonic medium. Anaerobic conditions, ascorbate, glutathione, cysteine and dithiothreitol are able to ameliorate this growth inhibition but a range of other antioxidants does not protect. The protective ability of the antioxidants does not correlate with the rate of their reactions with superoxide but seems to be conditioned by low redox potential for one-electron oxidation of free radicals of the antioxidants. It suggests that repair of low-redox potential targets rather than prevention of their damage by superoxide is important in the antioxidant protection against oxidative stress induced by hypertonic conditions.     

Acrolein toxicity involves oxidative stress caused by glutathione depletion in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Exposure of yeast cells to allyl alcohol results in intracellular production of acrolein. The toxicity of so formed acrolein involves oxidative stress, as (1) strains deficient in antioxidant defense are hypersensitive to allyl alcohol, (2) exposure to allyl alcohol increases the level of thiobarbituric-acid-reactive substances and decreases glutathione level in the cells, (3) hypoxic and anoxic atmosphere and antioxidants protect against allyl alcohol toxicity, and (4) allyl alcohol causes activation of Yap1p. No increased formation of reactive oxygen species was detected in cells exposed to allyl alcohol, so oxidative stress is due to depletion of cellular thiols and thus alteration in the redox state of yeast cells.     

Prevention of peroxidation of cardiolipin liposomes by quinol-based antioxidants

In mammalian mitochondria, cardiolipin molecules are the primary targets of oxidation by reactive oxygen species. The interaction of oxidized cardiolipin molecules with the constituents of the apoptotic cascade may lead to cell death. In the present study, we compared the effects of quinol-containing synthetic and natural amphiphilic antioxidants on cardiolipin peroxidation in a model system (liposomes of bovine cardiolipin). We found that both natural ubiquinol and synthetic antioxidants, even being introduced in micro- and submicromolar concentrations, fully protected the liposomal cardiolipin from peroxidation. The duration of their action, however, varied; it increased with the presence of either methoxy groups of ubiquinol or additional reduced redox groups (in the cases of rhodamine and berberine derivates). The concentration of ubiquinol in the mitochondrial membrane substantially exceeds the concentrations of antioxidants we used and would seem to fully prevent peroxidation of membrane cardiolipin. In fact, this does not happen: cardiolipin in mitochondria is oxidized, and this process can be blocked by amphiphilic cationic antioxidants (Y. N. Antonenko et al. (2008) Biochemistry (Moscow), 73, 1273–1287). We suppose that a fraction of mitochondrial cardiolipin could not be protected by natural ubiquinol; in vivo, peroxidation most likely threatens those cardiolipin molecules that, being bound within complexes of membrane proteins, are inaccessible to the bulky hydrophobic ubiquinol molecules diffusing in the lipid bilayer of the inner mitochondrial membrane. The ability to protect these occluded cardiolipin molecules from peroxidation may explain the beneficial therapeutic action of cationic antioxidants, which accumulate electrophoretically within mitochondria under the action of membrane potential.     

Potential anticarcinogenic action of melatonin and other antioxidants mediated by antioxidative mechanisms

The complex process of carcinogenesis is, to a large extent, due to oxidative stress. Numerous indicators of oxidative damage are enhanced in the result of the action of carcinogens. Several antioxidants protect, with different efficacy, against oxidative abuse, exerted by carcinogens. Recently, melatonin (N-acetyl-5-methoxytryptamine) and some other indoleamines have gained particular meaning in the defense against oxidative stress and, consequently, carcinogenesis. Some antioxidants, like ascorbic acid, play a bivalent role in the antioxidative defense, revealing, under specific conditions, prooxidative effects. Among known antioxidants, melatonin is particularly frequently applied in experimental models of anticarcinogenic action. In the numerous studies, examining several parameters of oxidative damage and using several in vitro and in vivo models, this indoleamine has been shown to protect DNA and cellular membranes from the oxidative abuse caused by carcinogens. When either preventing or decreasing the oxidative damage to macromolecules, melatonin also protects against the initiation of cancer. The protection provided by melatonin and some other antioxidants against cellular damage, due to carcinogens, make them potential therapeutic supplements in the conditions of increased cancer risk.     

Plasma membrane coenzyme Q10 and growth control

Summary Coenzyme Q is distributed among cellular membranes and it has a significant concentration at the plasma membrane. The plasma membrane contains a trans-membrane electron transport system, which is centered on coenzyme Q. This molecule is maintained reduced by NAD(P)H-dependent enzymes and can reduce other antioxidants such as tocopheroxyl quinone and ascorbate free radical. Its antioxidant property and its ability to maintain in the reduced state the other antioxidants offers a system to protect membrane components against oxidations and prevents oxidative-stress-dependent cellular damage. Growth factor withdrawal induces cell growth arrest and apoptosis through an oxidative-stress-induced pathway. Coenzyme Q can stimulate growth of different cell lines under serum deficiency, mainly by preventing apoptosis. The protection caused by coenzyme Q is independent of the Bcl-2 protein. Plasma membrane coenzyme Q appears to be essential in the regulation of the redox equilibrium of the cell and redox-dependent pathways.     

Screening of dietary antioxidants against mitochondria-mediated oxidative stress by visualization of intracellular redox state

Mitochondrial impairment and the resulting generation of reactive oxygen species (ROS) have been associated with aging and its related pathological conditions. Recently, dietary antioxidants have gained significant attention as potential preventive and therapeutic agents against ROS-generated aging and pathological conditions. We previously demonstrated that food-derived antioxidants prevented intracellular oxidative stress under proteasome inhibition conditions, which was attributed to mitochondrial dysfunction and ROS generation, followed by cell death. Here, we further screened dietary antioxidants for their activity as redox modulators by visualization of the redox state using Redoxfluor, a fluorescent protein redox probe. Direct alleviation of ROS by antioxidants, but not induction of antioxidative enzymes, prevented mitochondria-mediated intracellular oxidation. The effective antioxidants scavenged mitochondrial ROS and suppressed cell death. Our study indicates that redox visualization under mitochondria-mediated oxidative stress is useful for screening potential antioxidants to counteract mitochondrial dysfunction, which has been implicated in aging and the pathogenesis of aging-related diseases.     

Zeaxanthin in combination with ascorbic acid or alpha-tocopherol protects ARPE-19 cells against photosensitized peroxidation of lipids

The antioxidant action of carotenoids is believed to involve quenching of singlet oxygen and scavenging of reactive oxygen radicals. However, the exact mechanism by which carotenoids protect cells against oxidative damage, particularly in the presence of other antioxidants, remains to be elucidated. This study was carried out to examine the ability of exogenous zeaxanthin alone and in combination with vitamin E or C, to protect cultured human retinal pigment epithelium cells against oxidative stress. The survival of ARPE-19 cells, subjected to merocyanine 540-mediated photodynamic action, was determined by the MTT test and the content of lipid hydroperoxides in photosensitized cells was analyzed by HPLC with electrochemical detection. We found that zeaxanthin-supplemented cells, in the presence of either alpha-tocopherol or ascorbic acid, were significantly more resistant to photoinduced oxidative stress. Cells with added antioxidants exhibited increased viability and accumulated less lipid hydroperoxides than cells without the antioxidant supplementation. Such a synergistic action of zeaxanthin and vitamin E or C indicates the importance of the antioxidant interaction in efficient protection of cell membranes against oxidative damage induced by photosensitized reactions.     

The role of thiol antioxidants in restoring mitochondrial functions modified by microbial metabolites

The effects of phenolic acids of microbial origin on mitochondrial functions and the possibility of removing their effects by thiol antioxidants dithiotreitol and N-acetylcysteine were studied. The action of some phenolic acids on the redox state of NADH, the membrane potential and calcium capacity of mitochondria is due to their interaction with thiol groups. Partial restoration of mitochondrial functions occurred in the presence of dithiotreitol and N-acetylcysteine, full recovery (short-term duration) was promoted by the combined action of dithiotreitol and menadione (vitamin K₃). It was found that the protective effect of thiol antioxidants became prooxidant if the medium contained free iron and compounds with quinone structure, capable of entering into a redox cycle with thiols. It is shown that the interaction of thiols with iron and menadione is accompanied by absorption of oxygen to form superoxide anion. Prooxidant effect of thiol antioxidants may explain the absence of the protective effect at the later stages of sepsis and systemic inflammatory syndrome.     

Orally active multi-functional antioxidants are neuroprotective in a rat model of light-induced retinal damage

Background

Progression of age-related macular degeneration has been linked to iron dysregulation and oxidative stress that induce apoptosis of neural retinal cells. Since both antioxidants and chelating agents have been reported to reduce the progression of retinal lesions associated with AMD in experimental animals, the present study evaluates the ability of multi-functional antioxidants containing functional groups that can independently chelate redox metals and quench free radicals to protect the retina against light-induced retinal degeneration, a rat model of dry atrophic AMD.

Methods/Results

Proof of concept studies were conducted to evaluate the ability of 4-(5-hydroxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 4) and 4-(5-hydroxy-4,6-dimethoxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 8) to reduce retinal damage in 2-week dark adapted Wistar rats exposed to 1000 lx of light for 3 hours. Assessment of the oxidative stress markers 4- hydroxynonenal and nitrotyrosine modified proteins and Thioredoxin by ELISA and Western blots indicated that these compounds reduced the oxidative insult caused by light exposure. The beneficial antioxidant effects of these compounds in providing significant functional and structural protection were confirmed by electroretinography and quantitative histology of the retina.

Conclusions/Significance

The present study suggests that multi-functional compounds may be effective candidates for preventive therapy of AMD.     

Anticarcinogenic actions of melatonin which involve antioxidative processes: comparison with other antioxidants

The complex processes of carcinogenesis often involve oxidative stress. Numerous indicators of oxidative damage are enhanced as the result of the action of carcinogens. Several antioxidants, with different efficacies, protect against oxidative abuse caused by carcinogens. Recently, melatonin (N-acetyl-5-methoxytryptamine) and related indoleamines have attracted attention because of their high antioxidant and anticarcinogenic activity. Some antioxidants, e.g. ascorbic acid, play an ambivalent role in antioxidative defense, since, under specific conditions, they are strongly prooxidant. Among known antioxidants, melatonin has been an often investigated experimental agent in reducing cancer initiation and inhibiting the growth of established tumors. The indoleamine has been shown to protect macromolecules from oxidative mutilation induced by carcinogens. In these studies, a variety of in vitro and in vivo models were used and numerous indices of oxidative damage were evaluated. The protective effects of melatonin and several other indoleamine antioxidants against cellular damage caused by carcinogens make them potential supplements in the treatment or co-treatment at several stages of cancer.     

Resveratrol Couples Apoptosis with Autophagy in UVB-Irradiated HaCaT Cells

UVB radiation causes about 90% of non-melanoma skin cancers by damaging DNA either directly or indirectly by increasing levels of reactive oxygen species (ROS). Skin, chronically exposed to both endogenous and environmental pro-oxidant agents, contains a well-organised system of chemical and enzymatic antioxidants. However, increased or prolonged free radical action can overwhelm ROS defence mechanisms, contributing to the development of cutaneous diseases. Thus, new strategies for skin protection comprise the use of food antioxidants to counteract oxidative stress. Resveratrol, a phytoalexin from grape, has gained a great interest for its ability to influence several biological mechanisms like redox balance, cell proliferation, signal transduction pathways, immune and inflammatory response. Therefore, the potential of resveratrol to modify skin cell response to UVB exposure could turn out to be a useful option to protect skin from sunlight-induced degenerative diseases. To investigate into this matter, HaCaT cells, a largely used model for human skin keratinocytes, were treated with 25 or 100 µM resveratrol for 2 and 24 hours prior to UVB irradiation (10 to 100 mJ/cm²). Cell viability and molecular markers of proliferation, oxidative stress, apoptosis, and autophagy were analyzed. In HaCaT cells resveratrol pretreatment: reduces UVB-induced ROS formation, enhances the detrimental effect of UVB on HaCaT cell vitality, increases UVB-induced caspase 8, PARP cleavage, and induces autophagy. These findings suggest that resveratrol could exert photochemopreventive effects by enhancing UVB-induced apoptosis and by inducing autophagy, thus reducing the odds that damaged cells could escape programmed cell death and initiate malignant transformation.     

Drought-induced changes in the redox state of alpha-tocopherol,ascorbate, and the diterpene carnosic acid in chloroplasts of Labiatae species differing in carnosic acid contents

To assess antioxidative protection by carnosic acid (CA) in combination with that of other low-molecular weight (M(r)) antioxidants (alpha-tocopherol [alpha-T] and ascorbate [Asc]) in chloroplasts, we measured endogenous concentrations of these antioxidants, their redox states, and other indicators of oxidative stress in chloroplasts of three Labiatae species, differing in their CA contents, exposed to drought stress in the field. Damage to the photosynthetic apparatus was observed neither in CA-containing species (rosemary [Rosmarinus officinalis]) and sage [Salvia officinalis]) nor in CA-free species (lemon balm [Melissa officinalis]) at relative leaf water contents between 86% and 58%, as indicated by constant maximum efficiency of photosystem II photochemistry ratios and malondialdehyde levels in chloroplasts. The three species showed significant increases in alpha-T, a shift of the redox state of alpha-T toward its reduced state, and increased Asc levels in chloroplasts under stress. Lemon balm showed the highest increases in alpha-T and Asc in chloroplasts under stress, which might compensate for the lack of CA. Besides, whereas in rosemary and sage, the redox state of CA was shifted toward its oxidized state and the redox state of Asc was kept constant, lemon balm displayed a shift of the redox state of Asc toward its oxidized state under stress. In vitro experiments showed that both CA and Asc protect alpha-T and photosynthetic membranes against oxidative damage. These results are consistent with the contention that CA, in combination with other low-M(r) antioxidants, helps to prevent oxidative damage in chloroplasts of water-stressed plants, and they show functional interdependence among different low-M(r) antioxidants in chloroplasts.     

Tyrphostins protect neuronal cells from oxidative stress

Tyrphostins are a family of tyrosine kinase inhibitors originally synthesized as potential anticarcinogenic compounds. Because tyrphostins have chemical structures similar to those of the phenolic antioxidants, we decided to test the protective efficacy of tyrphostins against oxidative stress-induced nerve cell death (oxytosis). Many commercially available tyrphostins, at concentrations ranging from 0.5 to 200 microm, protect both HT-22 hippocampal cells and rat primary neurons from oxytosis brought about by treatment with glutamate, as well as by treatment with homocysteic acid and buthionine sulfoximine. The tyrphostins protect nerve cells by three distinct mechanisms. Some tyrphostins, such as A25, act as antioxidants and eliminate the reactive oxygen species that accumulate as a result of glutamate treatment. These tyrphostins also protect cells from hydrogen peroxide and act as antioxidants in an in vitro assay. In contrast, tyrphostins A9 and AG126 act as mitochondrial uncouplers, collapsing the mitochondrial membrane potential and thereby reducing the generation of reactive oxygen species from mitochondria during glutamate toxicity. Finally, the third group of tyrphostins does not appear to be effective as antioxidants but rather protects cells by increasing the basal level of cellular glutathione. Therefore, the effects of tyrphostins on cells are not limited to their ability to inhibit tyrosine kinases.     

Protection of yeast lacking the Ure2 protein against the toxicity of heavy metals and hydroperoxides by antioxidants

The aim of this study was to examine the protection of the yeast lacking the “antioxidant-like” prion precursor protein (Ure2p), by antioxidants and to elucidate how modification of redox homeostasis affects toxicity of agents inducing oxidative stress in the Δure2 cells. We found a diverse ability of a range of antioxidants to ameliorate the hypersensitivity of the Δure2 disruptant to oxidants and heavy metal ions. Glutathione and then ascorbate were the most effective antioxidants; Tempol, Trolox and melatonin were much less effective or even hampered the growth of the Δure2 cells exposed to tested agents. The intracellular level of ROS was augmented in the Δure2 mutant under normal growth conditions (1.7-fold), and after treatment with H₂O₂ (2.3-fold) and Cd(II) (2.8-fold), with respect to its wild-type counterpart. Glutathione was unable to prevent the increase in ROS production caused by CdCl₂. The Δure2 disruptant was also hypersensitive to heat shock, like mutants lacking glutathione S-transferases.     

Diminution of singlet oxygen-induced DNA damage by curcmin and related antioxidants

Curcumin, the natural antioxidant from turmeric, an Indian spice, and its derivatives have significant abilities to protect plasmid pBR322 against single-strand breaks induced by singlet oxygen (¹O₂), a reactive oxygen species with potential genotoxic/mutagenic properties. ¹O₂ was generated at 37°C in an aqueous buffer system by the thermal dissociation of the endoperoxide of 3,3′-(1,4-naphthylene)dipropionate (NDPO₂). Among the compounds, tested, curcumin was the most effective inhibitor of DNA damage followed by desmethoxycurcumin, bisdesmethoxycurcumin and other derivatives. The observed antioxidant activity was both time-and concentration-dependent. The protectice ability of curcumin was higher than that of the well-known biological antioxidants lipoate, α-tocopherol and β-carotene. However, the highest protective ability with saturating concentrations of curcumin did not exceed 50%. The ability of curcumin and its derivatives to protect DNA against ¹O₂ seems to be related to their structures and may at least partly explain the therapeutic and other beneficial effects of these compounds including anticarcinogenic and antimutagenic properties.     

Antioxidants increase the cryoresistance of GM-CFC

The influence of antioxidative drugs on the cryopreservation of human bone marrow cells was studied. The viability of bone marrow cells was tested after freezing and thawing from--196 degrees C by the growth of GM-CFC in agar culture. The results suggest the ability of antioxidants to protect stem cells against damage caused by freezing and thawing. Addition of tocopherol or penicillin to a cryoprotective medium increases the number of surviving GM-CFC. From the colony-to-cluster ratio can be concluded that antioxidants especially protect more immature haematopoietic (colony forming) cells.     

Astaxanthin protects mitochondrial redox state and functional integrity against oxidative stress

Mitochondria combine the production of energy with an efficient chain of reduction–oxidation (redox) reactions but also with the unavoidable production of reactive oxygen species. Oxidative stress leading to mitochondrial dysfunction is a critical factor in many diseases, such as cancer and neurodegenerative and lifestyle-related diseases. Effective antioxidants thus offer great therapeutic and preventive promise. Investigating the efficacy of antioxidants, we found that a carotenoid, astaxanthin (AX), decreased physiologically occurring oxidative stress and protected cultured cells against strong oxidative stress induced with a respiratory inhibitor. Moreover, AX improved maintenance of a high mitochondrial membrane potential and stimulated respiration. Investigating how AX stimulates and interacts with mitochondria, a redox-sensitive fluorescent protein (roGFP1) was stably expressed in the cytosol and mitochondrial matrix to measure the redox state in the respective compartments. AX at nanomolar concentrations was effective in maintaining mitochondria in a reduced state. Additionally, AX improved the ability of mitochondria to remain in a reduced state under oxidative challenge. Taken together, these results suggest that AX is effective in improving mitochondrial function through retaining mitochondria in the reduced state.     

Mitochondria-targeted antioxidants protect pancreatic β-cells against oxidative stress and improve insulin secretion in glucotoxicity and glucolipotoxicity

Mitochondrial oxidative damage is thought to play a key role in pancreatic β-cell failure in the pathogenesis of type 2 diabetes. Despite this, the potential of mitochondria-targeted antioxidants to protect pancreatic β-cells against oxidative stress has not yet been studied. Therefore, we investigated if mitochondria-targeted antioxidants protect pancreatic β-cells such as RINm5F and HIT-T15 cells against oxidative stress under glucotoxic and glucolipotoxic conditions. When β-cells were incubated under these conditions, the expression levels of mitochondrial electron transport chain complex subunits, mitochondrial antioxidant enzymes (such as MnSOD and Prx3), β-cell apoptosis, lipogenic enzymes (such as ACC, FAS and ABCA1), intracellular lipid accumulation, oxidative stress, ER stress, mitochondrial membrane depolarization, nuclear NF- κB and sterol regulatory element binding protein 1c (SREBP1c) were all increased, in parallel with decreases in intracellular ATP content, citrate synthase enzymatic activity and glucose-stimulated insulin secretion. These changes were consistent with elevated mitochondrial oxidative stress, and incubation with the mitochondria-targeted antioxidants, MitoTempol or Mitoquinone (MitoQ), prevented these effects. In conclusion, mitochondria-targeted antioxidants protect pancreatic β-cells against oxidative stress, promote their survival, and increase insulin secretion in cell models of the glucotoxicity and glucolipotoxicity associated with Type 2 diabetes.