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.     

The oxidative stress-sensitive yap1 null strain of Saccharomyces cerevisiae becomes resistant due to increased carotenoid levels upon the introduction of the Chlamydomonas reinhardtii cDNA, coding for the 60S ribosomal protein L10a

The Saccharomyces cerevisiae yap1 null strain was transformed with a Chlamydomonas reinhardtii cDNA expression library. A 688-bp cDNA fragment, coding for the 60S ribosomal protein L10a (RPL10a), restored the capacity of the S. cerevisiae yap1 null strain to resist oxidative stress. The rpl10a gene is a single-copy gene in C. reinhardtii and encodes a constitutively produced 1.35-kb mRNA. The deduced 214-residue amino acid sequence was highly related with RPL10a proteins from eukarya (between 46.1 and 63.7% identity) and archaea (between 24.5 and 29.2% identity). Resistant transformants were pink, due to increased carotenoid levels, with the same chemical structure as torularhodin, the main carotenoid of the pink yeast Rhodotorula mucilaginosa. The pink transformants showed high resistance levels against H(2)O(2), paraquat, menadione, and UV light. Partial inhibition of the carotenoid synthesis by diphenylamine reduced the resistance levels, demonstrating the role of excess carotenoid synthesis in the resistance mechanism.     

Effects of Cr(VI) long-term and low-dose action on mammalian antioxidant enzymes (an in vitro study)

In order to investigate the low-dose long-term Cr(VI) action on antioxidant enzymes in cultured mammalian cells we estimated the activity of glutathione dependent antioxidant enzymes, catalase and superoxide dismutase (SOD) under various chromium concentrations in human epithelial-like L-41 cells. The long-term action of 20 microM causes the toxicity that results in losing of the cell viability by activating the apoptotic process, as identified by morphological analysis, the activation of caspase-3, and DNA fragmentation. The toxic chromium concentration totally destroys glutathione antioxidant system, and diminishes the activity of catalase and cytosolic Cu, ZnSOD. The non-toxic concentration (2 microM) causes the activation of the antioxidant defense systems, and they neutralize the oxidative impact.     

Cloned prokaryotic iron superoxide dismutase protects yeast cells against oxidative stress depending on mitochondrial location

Superoxide dismutase (SOD) is considered to be the first line of defense against oxygen toxicity. It exists as a family of three metalloproteins with copper,zinc (Cu,ZnSOD), manganese (MnSOD), and iron (FeSOD) forms. In this work, we have targeted Escherichia coli FeSOD to the mitochondrial intermembrane space (IMS) of yeast cells deficient in mitochondrial MnSOD. Our results show that FeSOD in the IMS increases the growth rate of the cells growing in minimal medium in air but does not protect the MnSOD-deficient yeast cells when exposed to induced oxidative stress. Cloned FeSOD must be targeted to the mitochondrial matrix to protect the cells from both physiological and induced oxidative stress. This confirms that the superoxide radical is mainly generated on the matrix side of the inner mitochondrial membrane of yeast cells, without excluding its potential appearance in the mitochondrial IMS where its elimination by SOD is beneficial to the cells.     

Relationship among carotenoid production,copper bioremediation and oxidative stress in Rhodotorula mucilaginosa RCL-11

Rhodotorula mucilaginosa RCL-11, a pigmented yeast isolated from a filter plant of a copper mine in the province of Tucumán, Argentina, supports high concentrations of the heavy metal Cu(II). Copper overload augmented carotenoid biosynthesis in this yeast, modifying at the same time the relative proportion of the pigments produced. Inhibition of the synthesis pathway with diphenylamine suggests an inverse relationship between carotenoid and copper biosorption by R. mucilaginosa RCL-11. The increased activity of superoxide dismutase and catalase measured under inhibition of carotenoid biosynthesis could explain these observations. Exposure to H₂O₂, a second oxidative stress agent, alone or in combination with Cu(II) also modified the carotenoid content, both qualitatively and quantitatively. The change in the relative proportion of the carotenoids torularhodin, torulene and beta-carotena, as well as the detection of gamma-carotene in the presence of H₂O₂ and Cu(II) allows to hypothesize that the carotenoids produced by R. mucilaginosa RCL-11 plays different roles in the oxidative stress response of this yeast.     

Use of cyanide and diethyldithiocarbamate in the assay of superoxide dismutases.

Eucaryotes have two major forms of superoxide dismutase (SOD), Cu,ZnSOD and MnSOD; in most tissues Cu,ZnSOD is present in higher amounts than MnSOD. To assay MnSOD, Cu,ZnSOD can be inhibited selectively by millimolar concentrations of cyanide ion. However, calculation of MnSOD activity from the differential cyanide inhibition assay is complex and small experimental errors can cause large errors in the calculated MnSOD activity. We have assessed how interaction of cyanide and hydrogen peroxide with cytochrome c can lead to further errors in the xanthine oxidase-cytochrome c assay for SOD. Alternatively, Cu,ZnSOD can be completely inactivated by 50 mM diethyldithiocarbamate (DDC) at 30 degrees C for 1 h without affecting the activity of MnSOD. Since DDC reduces cytochrome c, the treated samples must be thoroughly dialyzed or desalted before assay. In the case of lung homogenates, dialysis is not an extra step since fresh, untreated samples must also be dialyzed or desalted before assaying by the cytochrome c method. Cu,ZnSOD activity is equal to the activity in the untreated sample minus the activity in the DDC-treated portion of the sample. Another copper chelator, triethylenetetramine, did not inactivate Cu,ZnSOD and could not be used instead of DDC. For accurate measurement of both enzymes in samples where MnSOD contributes only a small fraction of the total SOD activity, the DDC method has the advantage that it provides a direct measure of the MnSOD activity without interference by Cu,ZnSOD.     

Regulation of Cu,Zn-superoxide dismutase in bovine pulmonary artery endothelial cells.

To evaluate the regulation of endothelial cell Cu,Zn-SOD, we have exposed bovine pulmonary artery endothelial cells in culture to hyperoxia and hypoxia, second messengers or related agonists, hormones, free radical generating systems, endotoxin, and cytokines and have measured Cu,Zn-SOD protein of these cells by an ELISA developed in our laboratory. Control preconfluent and confluent cells in room air contained 196 +/- 18 ng Cu,Zn-SOD/10(6) cells. A23187 (0.33 microM), forskolin (10 microM), isobutylmethylxanthine (0.1 mM), dexamethasone (1 microM), triiodothyronine (1 microM) and retinoic acid (1 microM) failed to alter this level of Cu,Zn-SOD. Exposure to anoxia and hyperoxia both elevated the level approximately 1.5-2.0-fold over 20% oxygen-exposed controls at 48-72 hr. Similarly, exposures to glucose oxidase (0.0075 units/ml), menadione (12.5 microM), xanthine-xanthine oxidase (10 microM, 0.03 units/ml) and H2O2 (0.0005%) increased the level up to two-threefold over controls at 24-48 hr. Lipopolysaccharide, TGF beta 1, TNF alpha, and Il-1 also increased levels of cellular Cu,Zn-SOD, but only in proliferating cells. Il-2, Il-4, interferon-gamma, and GM-CSF had no effect on Cu,Zn-SOD. All treatments that elevated SOD resulted in inhibition of cellular growth, but decreased growth of cells at confluence alone was not associated with increased Cu,Zn-SOD. We propose from these studies that Cu,Zn-SOD of endothelial cells is not under conventional second messenger or hormonal regulation, but that up-regulation of the enzyme is associated with (and perhaps stimulated by) free-radical or oxidant production that also may be influenced by availability of certain cytokines under replicating conditions.     

<Emphasis Type="Italic">Rhodotorula mucilaginosa</Emphasis>, a carotenoid producing yeast strain from a patagonian high-altitude lake

The red yeast Rhodotorula mucilaginosa strain CRUB 0138 (previously identified as R. lactosa) was isolated from a high-altitude Patagonian Lake Toncek (1700 m a.s.l.), and assigned with mucilaginosa species. Its biochemical, physiological and molecular features were assessed and compared to R. mucilaginosa PYCC 5166 type strain using a polyphasic approach; in addition, biomass and carotenoid pigment production at different C/N ratios were determined in an incubator shaker. Phenetic characterization by means of 70 current physiological tests including assimilation of aldaric acids and aromatic compounds, and also the ability to grow with amino acids as sole carbon sources, was carried out. According to numerical taxonomy calculations, similarity indexes between R. mucilaginosa CRUB 0138 and PYCC 5166 type strain were 0.86 and 0.77, corresponding to a complete set of physiological tests and MSP-PCR (Mini/Micro Satellite Primed PCR; (GTG)5, M13 and (GAC)5 primers were employed) fingerprinting. Killer activity against 2 native strains, Rhodosporidium kratochvilovae and R. mucilaginosa was detected. Maximum biomass-glucose conversion efficiency (87%) and maximum carotenoid yield (2.32 mg/L) were obtained at C/N = 5 in culture medium containing 10 and 40 g/L glucose, respectively. Different C/N ratios did not influence carotenoid pigment production but low C/N enhanced biomass yield.     

Binding of polyaminocarboxylate chelators to the active-site copper inhibits the GSNO-reductase activity but not the superoxide dismutase activity of Cu,Zn-superoxide dismutase

In addition to its superoxide dismutase (SOD) activity, Cu,Zn-superoxide dismutase (CuZnSOD) catalyzes the reductive decomposition of S-nitroso-L-glutathione (GSNO) in the presence of thiols such as L-glutathione (GSH). The GSNO-reductase activity but not the superoxide dismutase (SOD) activity of CuZnSOD is inhibited by the commonly used polyaminocarboxylate metal ion chelators, EDTA and DTPA. The basis for this selective inhibition is systematically investigated here. Incubation with EDTA or DTPA caused a time-dependent decrease in the 680 nm d-d absorption of Cu(II)ZnSOD but no loss in SOD activity or in the level of metal loading of the enzyme as determined by ICP-MS. The chelators also protected the SOD activity against inhibition by the arginine-specific reagent, phenylglyoxal. Measurements of both the time course of SNO absorption decay at 333 nm and oxymyoglobin scavenging of the NO that is released confirmed that the chelators inhibit CuZnSOD catalysis of GSNO reductive decomposition by GSH. The decreased GSNO-reductase activity is correlated with decreased rates of Cu(II)ZnSOD reduction by GSH in the presence of the chelators as monitored spectrophotometrically at 680 nm. The aggregate data suggest binding of the chelators to CuZnSOD, which was detected by isothermal titration calorimetry (ITC). Dissociation constants of 0.08 +/- 0.02 and 8.3 +/- 0.2 microM were calculated from the ITC thermograms for the binding of a single EDTA and DTPA, respectively, to the CuZnSOD homodimer. No association was detected under the same conditions with the metal-free enzyme (EESOD). Thus, EDTA and DTPA must bind to the solvent-exposed active-site copper of one subunit without removing the metal. This induces a conformational change at the second active site that inhibits the GSNO-reductase but not the SOD activity of the enzyme.     

Superoxide dismutase activity in callus from the C3-CAM intermediate plant Mesembryanthemum crystallinum

In light-grown callus obtained from M. crystallinum hypocotyls, three classes of superoxide dismutase (SOD): Mn-, Fe- and Cu/ZnSOD were identified. Callus cultured on a medium containing 0.4 M NaCl showed an increase in FeSOD activity on day 4 of the experiment. In contrast, Cu/ZnSOD activity was higher over 16 days of the experiment. Salinity stress induces oxidative stress mainly for the cytosolic SOD form (Cu/ZnSOD). After 16 days of callus culture on salt-containing medium, diurnal malate oscillations, and an increase in NADP-malic enzyme activity were noticed. These results strongly suggest that C₃-CAM transition can also be expressed at the cellular level. Therefore, callus tissue could be a useful model, similar to a whole plant, for investigation of mechanisms of stress responses in M. crystallinum.     

Superoxide dismutase in Cryptococcus neoformans varieties gattii, grubi, and neoformans

Some clear dissimilarities occur among the varieties of Cryptococcus neoformans but there are few studies about the differences among individual yeast antioxidant enzymes. The total superoxide dismutase (SOD) activities and the copper, zinc-depend SOD (Cu,ZnSOD) and manganese-dependent SOD (MnSOD) isoenzymes of five reference C. neoformans strains belonged to A, B, C, AD and D serotypes (Table I) and other nine C. neoformans isolates (Table II) were determined. There were significant differences (p < 0.01 and p < 0.05) in total SOD activity among the varietie gattii (serotype C) and the other varieties. Cu,ZnSOD showed difference (p < 0.05) between A and D serotypes. These results point out a variety and serotype-independent SOD activity in C. neoformans reference strains and the other isolates that were evaluated.     

Increased Tolerance to Mn Deficiency in Transgenic Tobacco Overproducing Superoxide Dismutase

The effect of Mn deficiency on plant growth and activities ofsuperoxide dismutase (SOD) was studied in hydroponically-grownseedlings of transgenic tobacco (Nicotiana tabacum L.) engineeredto overexpress FeSOD in chloroplasts or MnSOD in chloroplastsor mitochondria. In comparison to the non-transgenic parentalline, the activity of MnSOD in the lines overproducing MnSODwas 1.6-fold greater, and the activity of FeSOD in the FeSOD-overproducinglines was 3.2-fold greater, regardless of the Mn treatment (deficientor sufficient). The MnSOD activities decreased due to Mn deficiency,while activities of FeSOD and Cu/ZnSOD remained unaffected 25d after transplanting (DAT). With an increased duration of theMn deficiency stress (45 DAT), FeSOD activity decreased, andthat of MnSOD continued to decrease, while Cu/ZnSOD activitysimultaneously increased. Under Mn sufficiency, non-transgenicparental plants had greater shoot biomass than the transgenics;however, when subjected to Mn deficiency stress, non-transgenicparents suffered a proportionally greater growth reduction thantransgenic lines. Thus, overproduction of MnSOD in chloroplastsmay provide protection from oxidative stress caused by Mn deficiency.Copyright 1999 Annals of Botany Company Manganese deficiency, Nicotiana tabacum, superoxide dismutase (SOD), transgenic tobacco.     

Comparative effects of pitavastatin and probucol on oxidative stress, Cu/Zn superoxide dismutase, PPAR-gamma, and aortic stiffness in hypercholesterolemia

Reactive oxygen species-scavenging enzyme Cu/Zn superoxide dismutase (SOD) regulated by peroxisome proliferator-activated receptors (PPARs) plays an important role in vascular responsiveness. However, it remains unknown whether statin restores vascular dysfunction through the activation of reactive oxygen species-scavenging enzymes in vivo. We hypothesized that pitavastatin restores vascular function by modulating oxidative stress through the activation of Cu/ZnSOD and PPAR-gamma in hypercholesterolemia. New Zealand White male rabbits were fed either normal chow or a 1% cholesterol (CHO) diet for 14 wk. After the first 7 wk, the CHO-fed rabbits were further divided into three groups: those fed with CHO feed only (HC), those additionally given pitavastatin, and those additionally given an antioxidant, probucol. The extent of atherosclerosis was assessed by examining aortic stiffness. When compared with the HC group, both the pitavastatin and probucol groups showed improved aortic stiffness by reducing aortic levels of reactive oxidative stress, nitrotyrosine, and collagen, without affecting serum cholesterol or blood pressure levels. Pitavastatin restored both Cu/ZnSOD activity (P < 0.005) and PPAR-gamma expression and activity (P < 0.01) and inhibited NAD(P)H oxidase activity (P < 0.0001) in the aorta, whereas probucol inhibited NAD(P)H oxidase activity more than did pitavastatin (P < 0.0005) without affecting Cu/ZnSOD activity or PPAR-gamma expression and activity. Importantly, Cu/ZnSOD activity was positively correlated with the PPAR-gamma activity in the aorta (P < 0.005), both of which were negatively correlated with aortic stiffness (P < 0.05). Vascular Cu/ZnSOD and PPAR-gamma may play a crucial role in the antiatherogenic effects of pitavastatin in hypercholesterolemia in vivo.     

Effects of beta-carotene on antioxidant enzyme activity, intracellular reactive oxygen and membrane integrity within post confluent Caco-2 intestinal cells

As encountered with a plethora of other natural products, the antioxidant activity of beta-carotene has been proposed as one of the mechanisms by which diets rich in this pro-vitamin A active carotenoid apparently afford chemoprevention. Here, we report the ability of beta-carotene to alter endogenous reactive oxygen levels and antioxidant defences within non-stressed 'differentiated' monolayers of an intestinal epithelial cell line (Caco-2) and to subsequently effect resistance to general oxidative insult. The differentiated monolayers efficiently absorbed beta-carotene. Between 3 and 8 days post confluence, cultures exhibited a progressive increase in antioxidant enzyme activity and a corresponding reduction to intracellular ROS levels. The profile for antioxidant enzyme activity was unaffected by sustained daily supplementation with beta-carotene. However, after two daily treatments with 50 microM beta-carotene intracellular ROS levels were significantly reduced and there was a trend towards reduced intracellular ROS within monolayers subject to five daily treatments with 0.5 and 5 microM beta-carotene. Prolonged supplementation with 0.1 and 0.5 microM beta-carotene or short supplementation periods with 5 and 50 microM beta-carotene did not alter susceptibility to H(2)O(2). However, cultures treated daily between 3 and 8 days post confluence with 5 or 50 microM beta-carotene exhibited enhanced LDH release, increased non-adherence and enhanced Trypan blue staining when challenged with 10 mM H(2)O(2). In the absence of H(2)O(2), the beta-carotene treatments were not overtly toxic to the monolayers. These results indicate that beta-carotene does not enhance antioxidant defences within Caco-2 monolayers. The enhancement of H(2)O(2) toxicity by persistent, high doses of beta-carotene may contribute to the failure of this carotenoid to protect high risk individuals from certain degenerative conditions.     

Effect of iron chelators on the cytotoxic and genotoxic action of hyperoxia in Chinese hamster ovary cells.

The iron chelators o-phenanthroline and desferrioxamine were tested for their ability to protect Chinese hamster ovary cells against the cytotoxic and genotoxic effects of normobaric hyperoxia. Desferrioxamine added at sub-toxic concentrations (up to 2.5 microM) over a period of several days had no protective effect on hyperoxia-induced clonogenic cell killing and growth inhibition. The clastogenic effect of hyperoxia was strongly potentiated by desferrioxamine, while the induction of sister-chromatid exchanges (SCEs) by hyperoxia was unaffected. Similarly, o-phenanthroline (up to 0.25 microM) had no protective effect on hyperoxia-induced cell killing, growth inhibition, and SCE induction, while also this compound potentiated the clastogenic effect of hyperoxia. These results do not support a critical role for cellular iron in the mechanism of toxicity by normobaric hyperoxia in CHO cells. However, the results may still be consistent with a critical involvement of particular iron fraction(s) not susceptible to the chelators used. Furthermore, our results show that concentrations of iron chelators known to protect against short-term (up to 1 h) toxic exposure to oxidative stress become toxic themselves when applied chronically, i.e., in the order of days.     

Zygote formation in Blakeslea trispora: morphological peculiarities and relationship with carotenoid synthesis

Changes associated with zygospore formation in the mucorous fungus Blakeslea trispora were studied. Zygospores are dormant cells with thickened cell walls and large central lipid vacuoles containing large amounts of lycopene. We established for the first time that B. trispora gametangia of different sexes differ in their carotenoid content and revealed that zygote formation involves a novel structure that consists of densely intertwined hyphae. Using inhibitory analysis (blocking beta-carotene synthesis with diphenylamine and 2-amino-6-methylpyridine), we showed that suppression of carotene production results in the inhibition of zygote formation. Hence, we established a manifest dependence of zygote formation on beta-carotene synthesis.     

Salt stress induces up-regulation of an efficient chloroplast antioxidant system in the salt-tolerant wild tomato species Lycopersicon pennellii but not in the cultivated species

The response of the chloroplastic antioxidant system of the cultivated tomato Lycopersicon esculentum (Lem) and its wild salt-tolerant related species L. pennellii (Lpa) to NaCl stress was studied. An increase in H₂O₂ level and membrane lipid peroxidation was observed in chloroplasts of salt-stressed Lem. In contrast, a decrease in these indicators of oxidative stress characterized chloroplasts of salt-stressed Lpa plants. This differential response of Lem and Lpa to salinity, correlates with the activities of the antioxidative enzymes in their chloroplasts. Increased activities of total superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), glutathione-S-transferase (GST), phospholipid hydroperoxide glutathione peroxidase (PHGPX) and several isoforms of non-specific peroxidases (POD) were found in chloroplasts of salt-treated Lpa plants. In these chloroplasts, in contrast, activity of lipoxygenase (LOX) decreased while in those of salt-stressed Lem it increased. Although total SOD activity slightly increased in chloroplasts of salt-treated Lem plants, differentiation between SOD types revealed that only stromal Cu/ZnSOD activity increased. In contrast, in chloroplasts of salt-treated Lpa plants FeSOD activity increased while Cu/ZnSOD activity remained unchanged. These data indicate that salt-dependent oxidative stress and damage, suffered by Lem chloroplasts, was effectively alleviated in Lpa chloroplasts by the selective up-regulation of a set of antioxidative enzymes. Further support for the above idea was supplied by leaf discs experiments in which pre-exposure of Lpa plants to salt-treatment conferred cross-tolerance to paraquat-induced oxidative stress while increased oxidative damage by paraquat-treatment was found in salt-stressed Lem plants.     

Superoxide dismutase activity in salt stressed wheat seedlings

We investigated the effect of salt stress on enzymatic activity of superoxide dismutase (SOD) isozymes in shoot and root tissues of salt tolerant and sensitive wheat (Triticum aestivum L. and Triticum durum Defs.) cultivars. Ten day old seedlings were subjected to 0.7 M NaCl stress for 3 and 5 days. Seedlings treated in the same manner without salt stress served as controls. Activity of SOD isozymes in root and shoot extracts was determined by activity staining of native polyacrylamide gels. In both shoot and root extracts of examined cultivars two isozymes of SOD, namely MnSOD and Cu/ZnSOD were identified. Cu/ZnSOD activity comprised 90 % of total SOD activity in both root and shoot tissues. Salt stress caused 1–1.5 fold increase in MnSOD activity of shoots in tolerant cultivars when compared with non-stressed controls. Under stress conditions, compared to controls all cultivars exhibited reduced MnSOD activity in root tissues. Cu/ZnSOD activity, on the other hand, was remarkably enhanced (3–4 fold) in root extracts of the tolerant cultivars, whereas it was reduced in the sensitive ones.