The influence of extracorporeal circulation on the susceptibility of erythrocytes to oxidative stress

Extracorporeal circulation (ECC), a necessary and integral part of cardiac surgery, can itself induce deleterious effects in patients. The pathogenesis of diffuse damage of several tissues is multifactorial. It is believed that circulation of blood extracorporeally through plastic tubes causes a whole body inflammatory response and a severe shear stress to blood cells. The aim of this study was to evaluate the level of oxidative stress and its deleterious effect on red blood cell (RBC) before (pre-ECC), immediately after (per-ECC) and 24 h after an ECC (24 h post-ECC). Several indicators of extracellular oxidative status were evaluated. The ascorbyl free radical (AFR) was directly measured in plasma using electron spin resonance (ESR) spectroscopy and expressed with respect to vitamin C levels in order to obtain a direct index of oxidative stress. Allophycocyanin assay was also used to investigate the plasma antioxidant status (PAS). Indirect parameters of antioxidant capacities of plasma such as vitamin E, thiol and uric acid levels were also quantified. RBC alterations were evaluated through potassium efflux and carbonyl levels after action of AAPH, a compound generating carbon centered free radicals. No changes in plasma uric acid and thiols levels were observed after ECC. However, vitamin E levels and PAS were decreased in per-ECC and 24 h post-ECC samples. Vitamin C levels were significantly lower in 24 h post-ECC and the AFR/ vitamin C ratio was increased. Differences in results had been noted when measurements took account of hemodilution. Increases of uric acid and thiols levels were observed after ECC. Vitamin E levels were not modified. However after hemodilution correction a significant decrease of vitamin C level was noted in 24 h post-ECC samples as compared to per-ECC sample. Whatever the way of measurement, vitamin C levels decreased suggesting the occurrence of ECC induced-oxidative stress. Concerning RBC, in the absence of AAPH, extracellular potassium remained unchanged between pre-, per- and 24 h post-ECC. AAPH induced a significant increase in extracellular potassium and carbonyls levels of RBC membranes, which was not modified by ECC. These results suggest the absence of alterations of RBC membrane during ECC despite the occurrence of disturbances in PAS. Such protection is of particular importance in a cell engaged in the transport of oxygen and suggests that RBC are equipped with mechanisms affording a protection against free radicals.     

Functional deletion of Txndc2 and Txndc3 increases the susceptibility of spermatozoa to age-related oxidative stress

Oxidative stress in the male germ line is known to be a key factor in both the etiology of male infertility and the high levels of DNA damage encountered in human spermatozoa. Because the latter has been associated with a variety of adverse clinical outcomes, including miscarriage and developmental abnormalities in the offspring, the mechanisms that spermatozoa use to defend themselves against oxidative stress are of great interest. In this context, the male germ line expresses three unique forms of thioredoxin, known as thioredoxin domain-containing proteins (Txndc2, Txndc3, and Txndc8). Two of these proteins, Txndc2 and Txndc3, retain association with the spermatozoa after spermiation and potentially play an important role in regulating the redox status of the mature gamete. To address this area, we have functionally deleted the sperm-specific thioredoxins from the male germ line of mice by either exon deletion (Txndc2) or mutation of the bioactive cysteines (Txndc3). The combined inactivation of these Txndc isoforms did not have an overall impact on spermatogenesis, epididymal sperm maturation, or fertility. However, Txndc deficiency in spermatozoa did lead to age-dependent changes in these cells as reflected by accelerated motility loss, high rates of DNA damage, increases in reactive oxygen species generation, enhanced formation of lipid aldehyde–protein adducts, and impaired protamination of the sperm chromatin. These results suggest that although there is considerable redundancy in the systems employed by spermatozoa to defend themselves against oxidative stress, the sperm-specific thioredoxins, Txndc2 and Txndc3, are critically important in protecting these cells against the increases in oxidative stress associated with paternal age.     

Effect of <Emphasis Type="Italic">Gymnema montanum</Emphasis> leaves on red blood cell resistance to oxidative stress in experimental diabetes

The aim of the present study was to evaluate the protective effect of Gymnema montanum on red blood cell (RBC) membrane in diabetic rats during lipid peroxidation. Ethanol extract of G. montanum leaves (GLEt) was administered orally to alloxan-induced diabetic rats for 3 weeks, and the effects on blood glucose, insulin, lipid peroxidation markers, thiobarbituric acid reactive substances, hydroperoxides in plasma and antioxidant enzymes including superoxide dismutase, catalase and glutathione peroxidase activities in erythrocytes were studied. Administration of GLEt to diabetic animals at doses of 50, 100, and 200 mg/kg body weight lowered elevated blood glucose levels by 24, 35, and 66%, respectively, relative to untreated diabetic rats. In comparison, treatment with the known antidiabetic drug, glibenclamide (600 μg/kg body weight) decreased blood glucose concentrations by 51%. Plasma insulin concentrations were increased in the diabetic rat by 73% with GLEt (200 mg/kg body weight) and 45% with glibenclamide (600 μg/kg body weight). Although a significant decrease in the lipid peroxidation markers was observed in plasma on treatment with GLEt and glibenclamide, the RBC antioxidant levels were increased significantly in diabetic rats. Furthermore, erythrocytes from the GLEt-treated animals were found to be more resistant to H₂O₂-induced peroxidation than that of untreated diabetic animals. The chemical characterization of the polyphenolics of the extract showed the presence of gallic acid (5.29% w/w), resveratrol (2.2% w/w), and quercetin (16.6% w/w). The results of this study suggest that G. montanum may be useful for the control, management, and prevention of oxidative stress associated with diabetes.     

Repetitive static muscle contractions in humans —a trigger of metabolic and oxidative stress?

Repetitive static exercise (RSE) is a repetitive condition of partial ischaemia/reperfusion and may therefore be connected to the formation of oxygen-derived free radicals and tissue damage. Seven subjects performed two-legged intermittent knee extension exercise repeating at 10 s on and 10 s off at a target force corresponding to about 30% of the maximal voluntary contraction force. The RSE was continued for 80 min (n = 4) or to fatigue (n = 3). Four of the subjects also performed submaximal dynamic exercise (DE) at an intensity of about 60% maximal oxygen uptake (VO2max) for the same period. Whole body oxygen uptake (VO2) increased gradually with time during RSE (P less than 0.05), indicating a decreased mechanical efficiency. This was further supported by a slow increase in leg blood flow (P less than 0.05) and leg oxygen utilization (n.s.) during RSE. In contrast, prolonged RSE had no effect on VO2 during submaximal cycling. Maximal force (measured in six additional subjects) declined gradually during RSE and was not completely restored after 60 min of recovery. After 20 and 80 min (or at fatigue) RSE phosphocreatine (PC) dropped to 74% and 60% of the initial value, respectively. A similar decrease in PC occurred during DE. Muscle and arterial lactate concentrations remained low during both RSE and DE. The three subjects who were unable to continue RSE for 80 min showed no signs of a more severe energy imbalance than the other subjects. A continuous release of K+ occurred during both RSE and DE.(ABSTRACT TRUNCATED AT 250 WORDS)     

A study of oxidative stress in cervical cancer- an institutional study

Cervical cancer is the second most common cause of cancer-related death among women worldwide, especially in developing countries. Oxidative stress has been associated with cervical cancer. Many studies demonstrated that the low level of antioxidants induces the production of free radicals that cause lipid peroxidation, DNA, and protein damage leading to mutations that favors malignant transformation. This is a case-control institutional study conducted to evaluate the level of oxidative stress in cervical cancer patients and the age-matched healthy controls. We measured level of TBARS expressed as MDA, activity of SOD and GSH level by the spectrophotometric method, and level of 8-OHdG was estimated using a competitive sandwich ELISA assay. Our results showed a significant increase in the level of lipid peroxidation in group IV when compared to the control, group II and group III (p < 0.001). The activity of SOD was also significantly higher in group IV when compared to the control group (p < 0.001), group II (p < 0.001), and group III (p < 0.001). The level of GSH was also significantly lower in group IV when compared to the control group (p < 0.01), group II (p < 0.01), and group III (p < 0.01). The level of 8-OHdG was significantly higher in group IV than in the other groups (p < 0.01). The results suggest that oxidative stress is involved in the pathogenesis of cervical cancer, which is demonstrated by an increased level of lipid peroxidation and higher levels of 8-OHdG and an altered antioxidant defense system.     

Mitochondrial dysfunction and oxidative stress: cause and consequence of epileptic seizures

Mitochondrial dysfunction has been implicated as a contributing factor in diverse acute and chronic neurological disorders. However, its role in the epilepsies has only recently emerged. Animal studies show that epileptic seizures result in free radical production and oxidative damage to cellular proteins, lipids, and DNA. Mitochondria contribute to the majority of seizure-induced free radical production. Seizure-induced mitochondrial superoxide production, consequent inactivation of susceptible iron–sulfur enzymes, e.g., aconitase, and resultant iron-mediated toxicity may mediate seizure-induced neuronal death. Epileptic seizures are a common feature of mitochondrial dysfunction associated with mitochondrial encephalopathies. Recent work suggests that chronic mitochondrial oxidative stress and resultant dysfunction can render the brain more susceptible to epileptic seizures. This review focuses on the emerging role of oxidative stress and mitochondrial dysfunction both as a consequence and as a cause of epileptic seizures.     

Inborn defects in the antioxidant systems of human red blood cells

Red blood cells (RBCs) contain large amounts of iron and operate in highly oxygenated tissues. As a result, these cells encounter a continuous oxidative stress. Protective mechanisms against oxidation include prevention of formation of reactive oxygen species (ROS), scavenging of various forms of ROS, and repair of oxidized cellular contents. In general, a partial defect in any of these systems can harm RBCs and promote senescence, but is without chronic hemolytic complaints. In this review we summarize the often rare inborn defects that interfere with the various protective mechanisms present in RBCs. NADPH is the main source of reduction equivalents in RBCs, used by most of the protective systems. When NADPH becomes limiting, red cells are prone to being damaged. In many of the severe RBC enzyme deficiencies, a lack of protective enzyme activity is frustrating erythropoiesis or is not restricted to RBCs. Common hereditary RBC disorders, such as thalassemia, sickle-cell trait, and unstable hemoglobins, give rise to increased oxidative stress caused by free heme and iron generated from hemoglobin. The beneficial effect of thalassemia minor, sickle-cell trait, and glucose-6-phosphate dehydrogenase deficiency on survival of malaria infection may well be due to the shared feature of enhanced oxidative stress. This may inhibit parasite growth, enhance uptake of infected RBCs by spleen macrophages, and/or cause less cytoadherence of the infected cells to capillary endothelium.     

Oxidative stress in sickle cell disease: An overview of erythrocyte redox metabolism and current antioxidant therapeutic strategies

Erythrocytes have an environment of continuous pro-oxidant generation due to the presence of hemoglobin (Hb), which represents an additional and quantitatively significant source of superoxide (O₂⁻) generation in biological systems. To counteract oxidative stress, erythrocytes have a self-sustaining antioxidant defense system. Thus, red blood cells uniquely function to protect Hb via a selective barrier allowing gaseous and other ligand transport as well as providing antioxidant protection not only to themselves but also to other tissues and organs in the body. Sickle hemoglobin molecules suffer repeated polymerization/depolymerization generating greater amounts of reactive oxygen species, which can lead to a cyclic cascade characterized by blood cell adhesion, hemolysis, vaso-occlusion, and ischemia–reperfusion injury. In other words, sickle cell disease is intimately linked to a pathophysiologic condition of multiple sources of pro-oxidant processes with consequent chronic and systemic oxidative stress. For this reason, newer therapeutic agents that can target oxidative stress may constitute a valuable means for preventing or delaying the development of organ complications.     

Hyperglycemia-induced oxidative stress in diabetic complications

Reactive oxygen species are increased by hyperglycemia. Hyperglycemia, which occurs during diabetes (both type 1 and type 2) and, to a lesser extent, during insulin resistance, causes oxidative stress. Free fatty acids, which may be elevated during inadequate glycemic control, may also be contributory. In this review, we will discuss the role of oxidative stress in diabetic complications. Oxidative stress may be important in diabetes, not just because of its role in the development of complications, but because persistent hyperglycemia, secondary to insulin resistance, may induce oxidative stress and contribute to beta cell destruction in type 2 diabetes. The focus of this review will be on the role of oxidative stress in the etiology of diabetic complications.     

Chitin oligosaccharides inhibit oxidative stress in live cells

The aim of this research is to identify the cellular antioxidant effects of chitin oligosaccharides (NA-COS; M_w 229.21–593.12 Da) produced by acidic hydrolysis of crab chitin. The inhibitory effect of NA-COS on myeloperoxidase (MPO) activity in human myeloid cells (HL-60) and oxidation of DNA and protein in mouse macrophages (Raw 264.7) were identified. Furthermore, their direct radical scavenging effect by 2′,7′-dichlorofluorescein (DCF) intensity and intracellular glutathione (GSH) level were significantly increased in a time dependent manner, respectively. These results suggest that NA-COS act as a potent antioxidant in live cells.     

Glutaredoxin 2 knockout increases sensitivity to oxidative stress in mouse lens epithelial cells

Glutaredoxin belongs to the oxidoreductase family, with cytosolic glutaredoxin 1 (Grx1) and mitochondrial glutaredoxin 2 (Grx2) isoforms. Of the two isozymes, the function of Grx2 is not well understood. This paper describes the effects of Grx2 deletion on cellular function using primary lens epithelial cell cultures isolated from Grx2 gene knockout (KO) and wild-type (WT) mice. We found that both cell types showed similar growth patterns and morphology and comparable mitochondrial glutathione pool and complex I activity. Cells with deleted Grx2 did not show affected Grx1 or thioredoxin expression but exhibited high sensitivity to oxidative stress. Under treatment with H(2)O(2), the KO cells showed less viability, higher membrane leakage, enhanced ATP loss and complex I inactivation, and weakened ability to detoxify H(2)O(2) in comparison with the WT cells. The KO cells had higher glutathionylation in the mitochondrial proteins, particularly the 75-kDa subunit of complex I. Recombinant Grx2 deglutathionylated complex I and restored most of its activity. We conclude that Grx2 has a function that protects cells against H(2)O(2)-induced injury via its peroxidase and dethiolase activities; particularly, Grx2 prevents complex I inactivation and preserves mitochondrial function.     

Pharmacological consequences of oxidative stress in ocular tissues

The eye is a unique organ because of its constant exposure to radiation, atmospheric oxygen, environmental chemicals and physical abrasion. That oxidative stress mechanisms in ocular tissues have been hypothesized to play a role in diseases such as glaucoma, cataract, uveitis, retrolental fibroplasias, age-related macular degeneration and various forms of retinopathy provides an opportunity for new approaches to their prevention and treatment, In the anterior uvea, both H₂O₂ and synthetic peroxides exert pharmacological/toxicological actions tissues of the anterior uvea especially on the sympathetic nerves and smooth muscles of the iris–ciliary bodies of several mammalian species. Effects produced by peroxides require the presence of trace amounts of extracellular calcium and the functional integrity of mitochondrial calcium stores. Arachidonic acid metabolites appear to be involved in both the excitatory action of peroxides on sympathetic neurotransmission and their inhibitory effect on contractility of the iris smooth muscle to muscarinic receptor activation. In addition to the peroxides, isoprostanes (products of free radical catalyzed peroxidation of arachidonic acid independent of the cyclo-oxygenase enzyme) can also alter sympathetic neurotransmission in anterior uveal tissues. In the retina, both H₂O₂ and synthetic peroxides produced an inhibitory action on potassium depolarization induced release of [³H] d-aspartate, in vitro and on the endogenous glutamate and glycine concentrations in vivo. Effects caused by peroxides in the retina are mediated, at least in part, by second messengers such as nitric oxide, prostaglandins and isoprostanes. The ability of H₂O₂ to alter the integrity of neurotransmitter pools from sympathetic nerves in the anterior uvea and glutaminergic nerves in the retina could underlie its role in the etiology of glaucoma.     

Ubiquitin-proteasome pathway and cellular responses to oxidative stress

The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Substrate proteins of the canonical UPP are first tagged by multiple ubiquitin molecules and then degraded by the 26S proteasome. However, in noncanonical UPP, proteins can be degraded by the 26S or the 20S proteasome without being ubiquitinated. It is clear that a proteasome is responsible for selective degradation of oxidized proteins, but the extent to which ubiquitination is involved in this process remains a subject of debate. Whereas many publications suggest that the 20S proteasome degrades oxidized proteins independent of ubiquitin, there is also solid evidence indicating that ubiquitin and ubiquitination are involved in degradation of some forms of oxidized proteins. A fully functional UPP is required for cells to cope with oxidative stress and the activity of the UPP is also modulated by cellular redox status. Mild or transient oxidative stress up-regulates the ubiquitination system and proteasome activity in cells and tissues and transiently enhances intracellular proteolysis. Severe or sustained oxidative stress impairs the function of the UPP and decreases intracellular proteolysis. Both the ubiquitin-conjugating enzymes and the proteasome can be inactivated by sustained oxidative stress, especially the 26S proteasome. Differential susceptibilities of the ubiquitin-conjugating enzymes and the 26S proteasome to oxidative damage lead to an accumulation of ubiquitin conjugates in cells in response to mild oxidative stress. Thus, increased levels of ubiquitin conjugates in cells seem to be an indicator of mild oxidative stress.     

Effect of curcumin extract against oxidative stress on both structure and deformation capability of red blood cell

The normal deformability of erythrocytes plays an important role in ensuring blood mobility, erythrocyte longevity, and microcirculation, which is the ability of erythrocytes to change shapes in response to external forces. However, the effects of curcumin extracts on the deformability of erythrocytes have not yet been evaluated. Accordingly, in this study, we explored the effects of pre-treatment with curcumin extract on erythrocyte deformation and erythrocyte band 3 (SLC4A1; EB3) expression. We also evaluated the associations between EB3 expression and erythrocyte deformability induced by hydrogen peroxide. Blood samples were divided into the control group, pre-treatment group (treated with curcumin extract or vitamin C), and negative control group, and oxidant stress parameters, antioxidant status, erythrocyte deformability and elasticity, and EB3 modifications were evaluated using immunoblotting and immunofluorescence staining. Hydrogen peroxide significantly increased oxidative stress parameters, modulus elasticity values and clustered EB3 levels and induced conjugation of membrane proteins to form high-molecular-weight complexes (p < 0.05). Erythrocyte deformability and elasticity were significantly decreased in the treated groups compared with those in the control group. Overall, our findings suggested that pre-treatment with curcumin extracts increased antioxidant status, reduced EB3 cross-linking, and improved erythrocyte deformability, to an even better extent than vitamin C. These results provide important insights into the effects of treatment with curcumin extracts on erythrocyte damage and suggest that curcumin may have applications in antioxidant therapy.     

Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells

Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25–250 μg/ml) to determine whether ROI, specifically superoxide (O₂^•−), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1–24 h increased O₂^•− production 3–6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O₂^•− production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O₂^•− generation. In conclusion, COM increased mitochondrial O₂^•− production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi.     

Mitochondrial oxidative stress elicits chromosomal instability after exposure to isocyanates in human kidney epithelial cells

The role of oxidative stress is often attributed in environmental renal diseases. Isocyanates, a ubiquitous chemical group with diverse industrial applications, are known to undergo bio-transformation reactions upon accidental and occupational exposure. This study delineates the role of isocyanate-mediated mitochondrial oxidative stress in eliciting chromosomal instability in cultured human kidney epithelial cells. Cells treated with 0.005 µM concentration of methyl isocyanate displayed morphological transformation and stress-induced senescence. Along the time course, an increase in DCF fluorescence indicative of oxidative stress, depletion of superoxide dismutase (SOD) and glutathione reductase (GR) and consistent accumulation of 8-oxo-dG were noticed. Thus, endogenous oxidative stress resulted in aberrant expression of p53, p21, cyclin E and CDK2 proteins, suggestive of deregulated cell cycle, chromosomal aberrations, centromeric amplification, aneuploidy and genomic instability.     

Collateral sensitivity: ABCG2-overexpressing cells are more vulnerable to oxidative stress

Multidrug resistance (MDR), which is the main obstacle to cancer chemotherapy, is mainly due to overexpression of ATP-binding cassette (ABC) transporters, especially ABCB1 (P-glycoprotein), ABCC1 (MRP1), and ABCG2 (BCRP). A novel idea to overcome MDR is that of collateral sensitivity, i.e., finding a treatment to which cells overexpressing ABC transporters are more sensitive than cells that do not overexpress them. In this study we demonstrate for the first time that MDCKII-BCRP cells, overexpressing ABCG2, are more vulnerable to exogenous oxidative stress induced by several oxidants, viz. paraquat, menadione, hydrogen peroxide, tert-butylperoxide, and 2,2-azobis(2-methylpropionamidine) dihydrochloride. MDCKII-BCRP cells have significantly decreased glutathione level and decreased activities of glutathione S-transferase and glutathione reductase, which may underlie their augmented vulnerability to oxidative stress. These results suggest the possibility of using agents that induce oxidative stress to selectively kill cells overexpressing BCRP.