Iron-mediated oxidative stress in erythrocytes.

Erythrocytes subjected extracellularly to iron-mediated oxidant stress undergo haemoglobin oxidation and membrane damage, which can be modulated by maintaining the energy requirements of the cells. The results presented here suggest that a balance exists between the oxidation state of the haemoglobin and the oxidative deterioration of the membrane lipids, which is dependent on the metabolic state of the erythrocytes. These findings have important implications for thalassaemic erythrocytes that may be exposed to excess plasma iron levels, in which excessive membrane-bound iron in the form of haemichromes is a characteristic feature and in which cellular ATP levels are lowered.     

Enhanced sensitivity to oxidative stress in an Arabidopsis nitric oxide synthase mutant

The possible involvement of nitric oxide (NO) in oxidative stress tolerance was studied using Arabidopsis thaliana wild type (WT) and Atnos1 mutant plants, in which endogenous NO production is greatly diminished because 80% of nitric oxide synthase (NOS) activity is eliminated due to T-DNA insertion in the first exon of the NOS1 gene. Compared with WT, Atnos1 mutant plants showed increased hypersensitivity to salt stress and methyl viologen (MV) treatment. The maximal photochemical efficiency of photosystem II (F(v)/F(m)) and membrane integrity decreased in WT and Atnos1 mutant plants under stresses, but the extent was higher in the mutant. Treatment with sodium nitroprusside (SNP) (a NO donor) to Atnos1 mutant plants alleviated the damage. Instead, inhibition of nitric oxide accumulation in the WT plants produced opposite effects. Hydrogen peroxide and lipid peroxidation increased and the extent was higher in Atnos1 mutant plants than that in WT plants under MV stress. These results indicated that nitric oxide could protect the damage against NaCl and MV treatments.     

Copper-specific damage in human erythrocytes exposed to oxidative stress

Ascorbate and complexes of Cu(II) and Fe(III) are capable of generating significant levels of oxygen free radicals. Exposure of erythrocytes to such oxidative stress leads to increased levels of methemoglobin and extensive changes in cell morphology. Cu(II) per mole is much more effective than Fe(III). However, isolated hemoglobin is oxidized more rapidly and completely by Fe(III)- than by Cu(II)-complexes. Both Fe(III) and Cu(II) are capable of inhibiting a number of the key enzymes of erythrocyte metabolism. The mechanism for the enhanced activity of Cu(II) has not been previously established. Using intact erythrocytes and hemolysates we demonstrate that Cu(II)-, but not Fe(III)-complexes in the presence of ascorbate block NADH-methemoglobin reductase. Complexes of Cu(II) alone are not inhibitory. The relative inability of Fe(III)-complexes and ascorbate to cause methemoglobin accumulation is not owing to Fe(III) association with the membrane, or its failure to enter the erythrocytes. The toxicity of Cu(II) and ascorbate appears to be a result of site-specific oxidative damage of erythrocyte NADH-methemoglobin reductase and the enzyme's subsequent inability to reduce the oxidized hemoglobin.     

The balance between Cu,Zn-superoxide dismutase and catalase affects the sensitivity of mouse epidermal cells to oxidative stress.

Oxidants are toxic, but at low doses they can stimulate rather than inhibit the growth of mammalian cells and play a role in the etiology of cancer and fibrosis. The effect of oxidants on cells is modulated by multiple interacting antioxidant defense systems. We have studied the individual roles and the interaction of Cu,Zn-superoxide dismutase (SOD) and catalase (CAT) in transfectants with human cDNAs of mouse epidermal cells JB6 clone 41. Since only moderate increases in these enzymes are physiologically meaningful, we chose the following five clones for in-depth characterization: CAT 4 and CAT 12 with 2.6-fold and 4.2-fold increased catalase activities, respectively, SOD 15 and SOD 3 with 2.3-fold and 3.6-fold increased Cu,Zn-SOD activities, respectively, and SOCAT 3 with a 3-fold higher catalase activity and 1.7-fold higher Cu,Zn-SOD activity than the parent JB6 clone 41. While the increases in enzyme activities were moderate, the human cDNAs were highly expressed in the transfectants. As demonstrated for the clone SOD 15, this discordance between message concentrations and enzyme activities may be due to the low stability of the human Cu,Zn-SOD mRNA in the mouse recipient cells. According to immunoblots the content of Mn-SOD was unaltered in the transfectants. While the activities of glutathione peroxidase were comparable in all strains, the concentrations of reduced glutathione (GSH) were significantly lower in SOD 3 and SOD 15. This decrease in GSH may reflect a chronic prooxidant state in these Cu,Zn-SOD overproducers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced glucose consumption in erythrocytes under mechanical stress

In this paper we study the question of whether the unique features of erythrocyte (biconcave shape, extremely high deformability etc.) result only from the specific structural characteristics of the membrane or whether the maintenance of these features is conditioned by the supply of chemical energy. It is shown that glucose, the main source of cell energy, is consumed at a markedly increased rate when there is appreciable mechanical stress of the cell. This observation supports the hypothesis that there is utilization of chemical energy for the mechanical needs of the membrane.     

Differential effects of short-term lindane administration on parameters related to oxidative stress in rat liver and erythrocytes

Parameters related to oxidative stress in rat liver and erythrocytes were studied after short-term administration (60 and 90 days) of 1000 ppm of lindane in the diet. Lindane induced an oxidative stress condition in the liver, which is related to an enhancement in microsomal NADPH-cyto-chrome c reductase and NADPH oxidase activities, superoxide radical formation and cytochrome P450 content, produced independently of the time of treatment. Also, decreased activities of glutathione peroxidase and catalase were concomitantly observed. Although these changes were paralleled by an increase in lipid peroxidation indices, such as production of thiobarbituric acid reactants and spontaneous chemiluminescence, no evidence of liver injury was obtained. Lindane treatment did not exert quantitatively important changes in the pro-oxidant/anti-oxidant status of the erythrocyte, with reduction in the red blood cell mass possibly reflecting actions of the insecticide on the eryth-ropoietic process.     

Acetaminophen protects human erythrocytes against oxidative stress

Acetaminophen protects human erythrocytes against various modes of oxidative stress. Protection against ozone-induced damage can be explained by a direct scavenging reaction between the drug and ozone. With t-butylhydroperoxide acetaminophen appeared to be an effective scavenger of radicals, generated in secondary reactions. The protection by acetaminophen against t-butylhydroperoxide- and hydrogen peroxide-induced lipid peroxidation and K+-leakage can be explained along these lines. In all cases the protective effect of acetaminophen was attended with covalent binding of acetaminophen to membrane proteins.     

Protective effect of vitamin E in dimethoate and malathion induced oxidative stress in rat erythrocytes

Organophosphate (OP) pesticides such as dimethoate and malathion intoxication has been shown to produce oxidative stress due to the generation of free radicals and alter the antioxidant defense system in erythrocytes. It is possible that vitamin E being present at the cell membrane site may prevent OP-induced oxidative damage. In the present study, rats were pretreated orally with vitamin E (250 mg/kg body wt, twice a week for 6 weeks) prior to oral administration of a single low dose of dimethoate and/or malathion (0.01% LD(50)). The result showed that treatment with OP increased lipid peroxidation (LPO) in erythrocytes, however, vitamin E pretreated rats administered OP's showed decreased LPO in erythrocytes. The increase in the activities of superoxide dismutase (SOD) and catalase (CAT) and total-SH content in erythrocytes from dimethoate and/or malathion treated rats as compared to control appears to be a response towards increased oxidative stress. Vitamin E pretreated animals administered OP's showed a lowering in these parameters as compared to OP treated rats which indicates that vitamin E provide protection against OP-induced oxidative stress. The glutathione-S-transferase (GST) activity in erythrocytes was inhibited in OP intoxicated rats which partially recovered in vitamin E pretreated animals administered OP's. Inhibition in erythrocyte and serum acetylcholinesterase (AChE) activity was not relieved in vitamin E pretreated rats administered OP's probably due to the competitive nature of enzyme inhibition by OP's. The results show that vitamin E may amelierate OP-induced oxidative stress by decreasing LPO and altering antioxidant defense system in erthrocytes.     

Age-related sensitivity to lung oxidative stress during ozone exposure

As immature and aged rats could be more sensitive to ozone (O(3))-linked lung oxidative stress we have attempted to shed more light on age-related susceptibility to O(3) with focusing our interest on lung mitochondrial respiration, reactive oxygen species (ROS) production and lung pro/antioxidant status. For this purpose, we exposed to fresh air or O(3) (500 ppb 12 h per day, for 7 days) 3 week- (immature), 6 month- (adult) and 20 month-old rats (aged). We determined, in lung, H(2)O(2) release by mitochondria, activities of major antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)], heat shock protein (HSP(72)) content and 8-oxodG and dG-HNE nDNA contents, as DNA oxidative damage markers. In adult rats we did not observe alteration of pro/antioxidant status. In contrast to adults, immature rats exposed to O(3) higher nDNA 8-oxodG content and HSP(72) and without antioxidant enzymes modification. Aged rats displayed mild uncoupled lung mitochondria, increased SOD and GPx activities, and higher 8-oxodG content after O(3) exposure. Thus, in contrast to adults, immature and aged rats displayed lung oxidative stress after O(3) exposure. Higher sensitivity of immature to O(3) was partly related to ventilatory parameters and to the absence of antioxidant enzyme response. In aged rats, the increase in cytosolic SOD and GPx activities during O(3) exposure was not sufficient to prevent the impairment in mitochondrial function and accumulation in lung 8- oxodG. Finally, we showed that mitochondria seem not to be a major source of ROS under O(3) exposure.     

Intermittent hypobaric hypoxia-induced oxidative stress in rat erythrocytes: protective effects of vitamin E, vitamin C, and carnitine

This study was aimed at determining the effect of vitamin E, vitamin C, and carnitine on intermittent hypobaric-hypoxia-induced oxidative stress (OS) in erythrocytes. For this purpose, male Wistar rats of 4 months of age were orally supplemented with one of the antioxidants prior to exposure to altitudes of 5700 m or 6300 m. Hemoglobin (Hb) and OS indices such as osmotic fragility and hemolysis were measured together with lipid peroxidation (LPO) and protein oxidation. The increase in Hb was accompanied by increase in activities of antioxidant enzymes, superoxide dismutase (SOD), and catalase (CAT) during exposure to both the altitudes without any further elevation by supplements. The extent of reduction in osmotic fragility and hemolysis by vitamin E and carnitine was greater at 6300 m than at 5700 m. Increase in LPO products, for example, malondialdehyde (MDA) and lipofuscin-like autofluorescent substances (AFS) was noticeable at both the altitudes, and vitamin E and carnitine were effective in reducing LPO. While protein oxidation products such as carbonyl content (PrC) and advanced oxidation protein products (AOPP) increased at 6300 m, protein sulphydryl (P-SH) content decreased. P-SH levels were restored on supplementation of antioxidants. Hence, our results indicate that vitamin E, vitamin C, and carnitine may be beneficial in overcoming OS and hemolysis under situations such as intermittent hypobaric hypoxia (IHH) and hypobarotherapy wherein hypoxia is used to correct many pathological situations in humans. Further, this study suggests that supplementation of vitamin E, vitamin C, and L-carnitine alone and not in combination can be beneficial in attenuating the OS associated with IHH compared to the unsupplemented rats exposed to two different altitudes.     

Mitochondrial membrane potential in density-separated trout erythrocytes exposed to oxidative stress in vitro

Previous literature reports have demonstrated that nucleated trout erythrocytes in condition of oxidative stress are subjected to DNA and membrane damage, and inactivation of glutathione peroxidase. The present study was undertaken to investigate if mitochondrial membrane potential in stressed conditions was also influenced. Density-separated trout erythrocyte fractions, obtained using a discontinuous Percoll gradient, were submitted to stress conditions and the mitochondrial membrane potential was determined by means of cytofluorimetric analysis after incubation of each subfraction with JC-1, a mitochondrial specific fluorescent probe. The results clearly show that the mitochondrial membrane potential decreased significantly in all erythrocyte fractions, also if the oxidative effect on mitochondria is more severe with increased density (age) of the cell. Ebselen was very effective in preventing mitochondrial depolarization in young as well as in old erythrocytes.     

Store-operated calcium entry in human oocytes and sensitivity to oxidative stress

Calcium signaling is a cellular event that plays a key role at many steps of fertilization and early development. However, little is known regarding the contribution of extracellular Ca(2+) influx into the cell to this signaling in gametes and early embryos. To better know the significance of calcium entry on oocyte physiology, we have evaluated the mechanism of store-operated calcium entry (SOCE) in human metaphase II (MII) oocytes and its sensitivity to oxidative stress, one of the major factors implicated in the outcome of in vitro fertilization (IVF) techniques. We show that depletion of intracellular Ca(2+) stores through inhibition of sarco(endo)plasmic Ca(2+)-ATPase with thapsigargin triggers Ca(2+) entry in resting human oocytes. Ba(2+) and Mn(2+) influx was also stimulated following inhibition, and Ca(2+) entry was sensitive to pharmacological inhibition because the SOCE blocker 2-aminoethoxydiphenylborate (2-APB) reduced calcium and barium entry. These results support the conclusion that there is a plasma membrane mechanism responsible for the capacitative divalent cation entry in human oocytes. Moreover, the Ca(2+) entry mechanism described in MII oocytes was found to be highly sensitive to oxidative stress. Hydrogen peroxide, at micromolar concentrations that could mimic culture conditions in IVF, elicited an increase of [Ca(2+)](i) that was dependent on the presence of extracellular Ca(2+). This rise was preventable by 2-APB, indicating that it was mainly due to the enhanced influx through store-operated calcium channels. In sum, our results demonstrate the occurrence of SOCE in human MII oocytes and the modification of this pathway due to oxidative stress, with possible consequences in IVF.     

Genome-wide association for sensitivity to chronic oxidative stress in Drosophila melanogaster

Reactive oxygen species (ROS) are a common byproduct of mitochondrial energy metabolism, and can also be induced by exogenous sources, including UV light, radiation, and environmental toxins. ROS generation is essential for maintaining homeostasis by triggering cellular signaling pathways and host defense mechanisms. However, an imbalance of ROS induces oxidative stress and cellular death and is associated with human disease, including age-related locomotor impairment. To identify genes affecting sensitivity and resistance to ROS-induced locomotor decline, we assessed locomotion of aged flies of the sequenced, wild-derived lines from the Drosophila melanogaster Genetics Reference Panel on standard medium and following chronic exposure to medium supplemented with 3 mM menadione sodium bisulfite (MSB). We found substantial genetic variation in sensitivity to oxidative stress with respect to locomotor phenotypes. We performed genome-wide association analyses to identify candidate genes associated with variation in sensitivity to ROS-induced decline in locomotor performance, and confirmed the effects for 13 of 16 mutations tested in these candidate genes. Candidate genes associated with variation in sensitivity to MSB-induced oxidative stress form networks of genes involved in neural development, immunity, and signal transduction. Many of these genes have human orthologs, highlighting the utility of genome-wide association in Drosophila for studying complex human disease.     

Exercise-induced oxidative stress affects erythrocytes in sedentary rats but not exercise-trained rats.

Oxidant stress is one of the factors proposed to be responsible for damaged erythrocytes observed during and after exercise. The impact of exertional oxidant stress after acute exhaustive treadmill running on erythrocyte damage was investigated in sedentary (Sed) and exercise-trained (ET) rats treated with or without antioxidant vitamins C and E. Exhaustive exercise led to statistically significant increments in the levels of thiobarbituric acid-reactive substance (TBARS) and H2O2-induced TBARS in Sed rats and resulted in functional and structural alterations in erythrocytes (plasma hemoglobin concentrations, methemoglobin levels, and rise in osmotic fragility of erythrocytes with decrease in erythrocyte deformability). Administration of antioxidant vitamin for 1 mo before exhaustive exercises prevented lipid peroxidation (TBARS, H2O2-induced TBARS) in Sed rats without any functional or structural alterations in erythrocytes. Parameters indicating erythrocyte lipid peroxidation and deterioration after exhaustive exercise in rats trained regularly with treadmill running for 1 mo were not different from those in Sed controls. Erythrocyte lipid peroxidation (TBARS) increased in exhausted-ET rats compared with ET controls; however, the plasma hemoglobin, methemoglobin levels, and erythrocyte osmotic fragility and deformability did not differ. Exhaustive exercise-induced lipid peroxidation in ET rats on antioxidant vitamin treatment was prevented, whereas functional and structural parameters of erythrocytes were not different from those of the ET controls. We conclude that exertional oxidant stress contributed to erythrocyte deterioration due to exercise in Sed but not in ET rats.     

Simulation study of methemoglobin reduction in erythrocytes. Differential contributions of two pathways to tolerance to oxidative stress

Methemoglobin (metHb), an oxidized form of hemoglobin, is unable to bind and carry oxygen. Erythrocytes are continuously subjected to oxidative stress and nitrite exposure, which results in the spontaneous formation of metHb. To avoid the accumulation of metHb, reductive pathways mediated by cytochrome b5 or flavin, coupled with NADH-dependent or NADPH-dependent metHb reductases, respectively, keep the level of metHb in erythrocytes at less than 1% of the total hemoglobin under normal conditions. In this work, a mathematical model has been developed to quantitatively assess the relative contributions of the two major metHb-reducing pathways, taking into consideration the supply of NADH and NADPH from central energy metabolism. The results of the simulation experiments suggest that these pathways have different roles in the reduction of metHb; one has a high response rate to hemoglobin oxidation with a limited reducing flux, and the other has a low response rate with a high capacity flux. On the basis of the results of our model, under normal oxidative conditions, the NADPH-dependent system, the physiological role of which to date has been unclear, is predicted to be responsible for most of the reduction of metHb. In contrast, the cytochrome b5-NADH pathway becomes dominant under conditions of excess metHb accumulation, only after the capacity of the flavin-NADPH pathway has reached its limit. We discuss the potential implications of a system designed with two metHb-reducing pathways in human erythrocytes.     

Cell sensitivity to oxidative stress is influenced by ferritin autophagy

To test the consequences of lysosomal degradation of differently iron-loaded ferritin molecules and to mimic ferritin autophagy under iron-overload and normal conditions, J774 cells were allowed to endocytose heavily iron loaded ferritin, probably with some adventitious iron (Fe-Ft), or iron-free apo-ferritin (apo-Ft). When cells subsequently were exposed to a bolus dose of hydrogen peroxide, apo-Ft prevented lysosomal membrane permeabilization (LMP), whereas Fe-Ft enhanced LMP. A 4-h pulse of Fe-Ft initially increased oxidative stress-mediated LMP that was reversed after another 3h under standard culture conditions, suggesting that lysosomal iron is rapidly exported from lysosomes, with resulting upregulation of apo-ferritin that supposedly is autophagocytosed, thereby preventing LMP by binding intralysosomal redox-active iron. The obtained data suggest that upregulation of the stress protein ferritin is a rapid adaptive mechanism that counteracts LMP and ensuing apoptosis during oxidative stress. In addition, prolonged iron starvation was found to induce apoptotic cell death that, interestingly, was preceded by LMP, suggesting that LMP is a more general phenomenon in apoptosis than so far recognized. The findings provide new insights into aging and neurodegenerative diseases that are associated with enhanced amounts of cellular iron and show that lysosomal iron loading sensitizes to oxidative stress.     

Catalase transgenic mice: characterization and sensitivity to oxidative stress

The role of catalase in the antioxidant defense system was studied using transgenic mice [Tg(CAT)] harboring a human genomic clone containing the entire human CAT gene. Catalase activity was 2-fold higher in the tissues of hemizygous [Tg(CAT)(+/o)] mice and 3- to 4-fold higher in the tissues of homozygous [Tg(CAT)(+/+)] mice compared to wild type mice. The human CAT transgene was expressed in a tissue-specific pattern that was similar to the endogenous catalase gene. The levels of other major antioxidant enzymes were not altered in the tissues of the transgenic mice. Hepatocytes and fibroblasts from the Tg(CAT)(+/+) mice were more resistant to hydrogen peroxide-induced cell death but were more sensitive to paraquat and TNFalpha toxicity. Fibroblasts from the Tg(CAT)(+/+) mice showed reduced growth rate in culture without treatment and reduced colony-forming capability after gamma-irradiation compared to fibroblasts from wild type mice. In addition, the Tg(CAT)(+/+) animals were more sensitive to gamma-irradiation.     

The effect of some tannins on trout erythrocytes exposed to oxidative stress

In order to gain more knowledge on the role of tannins as antioxidants, their ability to protect (Salmo irideus) erythrocytes against oxidative stress was investigated. Antioxidant activity of different tannins (tannic, gallic and ellagic acid) was evaluated by chemiluminescence (CL) techniques using lucigenin and luminol as chemiluminogenic probes for the superoxide radical generated by the xanthine/xanthine oxidase system and hydrogen peroxide, respectively. The superoxide-scavenging activity of these tannins was shown for all the compounds; however, it is not clear if this is due to their ability of scavenging the superoxide radical or to their inhibitory activity on xanthine oxidase. Tannic and ellagic acid showed a marked effect on the reduction of H2O2-luminol chemiluminescence. The influence of these tannins on the rate of hemolysis in stressed trout erythrocytes was investigated and the results indicate that tannic acid accelerates the hemolytic event while gallic and ellagic acid have no significant effect. The possible protective action of these compounds against oxidative DNA damage was assessed using the comet assay, a rapid and sensitive single-cell gel electrophoresis technique, used to detect primary DNA damage in individual cells. The results here reported show that tannins under study are capable at low concentrations of protecting DNA breakage, while at high concentrations they can be genotoxic.     

Copper-mediated oxidative stress in rat liver

Copper is an essential trace element with various biological functions. Excess copper, however, is extremely toxic, leading to many pathological conditions that are consistent with oxidative damage to membranes and molecules. Exposure to high levels of copper results in various changes in the tissues. In liver, hypertrophy of hepatocytes, hepatitis, hepatocellular necrosis, and hepatocellular death are the results. Lipid peroxidation causes dysfunction in the cell membrane, decreased fluidity, inactivation of receptors and enzymes, and changes ion permeability. In this study, we aimed to determine the effect of copper on oxidative and antioxidative substances in plasma and liver tissue in a rat model. Sixteen male Sprague—Dawley rats were divided into two groups: Group 1 rats included control rats given tap water. Group 2 rats were given water containing copper in a dose of 100 μg/mL. All rats were sacrificed at 4 wk under ether anesthesia. Plasma and liver superoxide dismutase (SOD) activities, plasma and liver MDA (malondialdehyde) levels, and liver glutathione (GSH) levels were studied. Plasma and liver SOD activities were found to be higher in group 2 than those in group 1. Although plasma MDA levels were higher in group 2, MDA levels in liver tissues were comparable. Liver tissue glutathione levels were lower in group 2. It was concluded that although copper is needed in trace amounts, an excess amount is toxic for the organism. It increases lipid peroxidation and depletes GSH reserves, which makes the organism more vulnerable to other oxidative challenges.     

Studies of oxidative stress in cellular systems. The interaction of monocytes and erythrocytes.

1H spin echo NMR spectroscopy is used to follow the interaction of intact and viable erythrocytes and monocytes obtained from different sources in mixed cultures. After a lag time (270 min) erythrocyte glutathione is observed to become more oxidised. This result is believed to occur as a consequence of monocyte activation generating hydrogen peroxide or hypochlorous acid, which is targeted at the erythrocyte. The red cell in turn employs its sulphydryl system as an anti-oxidant defence.