Photochemiluminescent detection of antiradical activity. V. Application in combination with the hydrogen peroxide-initiated chemiluminescence of blood plasma proteins to evaluate antioxidant homeostasis in humans

Abstract

Disturbance of the steady state between pro- and antioxidants in tissues is an important aetiopathogenetic factor. Two methods - (i) photosensitized chemiluminescence for detection of antiradical activity and (ii) hydrogen peroxide-initiated chemiluminescence of plasma proteins (CLP) and erythrocytes (CLE) - were tested in 136 healthy donors and 82 patients with untreated breast tumours for their applicability to detecting disturbances in antioxidant homeostasis in humans. The total antiradical capacity of water-soluble substances (ACW) and its urate-independent proportion (ACU) were lower (P <0.05) and CLP higher (P <0.001) in smokers in comparison to non-smokers. A significant negative correlation was found between the content of ascorbate in plasma and the intensity of CLP: r = 0.39, P <0.001. A significant reduction in ACU and increased values of CLP and CLE were seen according to the stage of disease in breast cancer patients. On the basis of these observations and model experiments we suggest that hydrogen peroxide-initiated chemiluminescence can serve as a parameter of oxidative modification of blood components and, in combination with the antioxidant parameters, can be used to describe the antioxidant homeostasis in humans and possibly to have value as a predictor of disease states.     

7-Ketocholesterol enhances the expression of adhesion molecules on human aortic endothelial cells by increasing the production of reactive oxygen species

Abstract

The aim of the present study was to assess the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), monocytic adhesion of human aortic endothelial cells (HAECs), and the production of intracellular reactive oxygen species (ROS), when HAECs were stimulated by 7-ketocholesterol. 7-ketocholesterol enhances surface expression of ICAM-1 and VCAM-1 as determined by EIA, induces their mRNA expression by RT-PCR, and stimulates adhesiveness of HAECs to U937 monocytic cells. We confirmed up-regulation of ROS production of HAECs treated with 7-ketocholesterol. Although the surface expression of ICAM-1 and VCAM-1 on HAECs treated with 7-ketocholesterol increased in a time-dependent manner, α-tocopherol inhibited this increase of the surface expression of ICAM-1 and VCAM-1. In the monocytic adhesion assay, adhesion of U937 to HAECs treated with 7-ketocholesterol was enhanced, but monoclonal anti-ICAM-1 and VCAM-1 antibodies reduced the endothelial adhesiveness. In conclusion, this study suggests that the endothelial adhesiveness to monocytic cells that was increased by 7-ketocholesterol was associated with enhanced expression of ICAM-1 and VCAM-1 mediated by ROS production.     

Hemin toxicity: a preventable source of brain damage following hemorrhagic stroke

Abstract

Hemorrhagic stroke is a common cause of permanent brain damage, with a significant amount of the damage occurring in the weeks following a stroke. This secondary damage is partly due to the toxic effects of hemin, a breakdown product of hemoglobin. The serum proteins hemopexin and albumin can bind hemin, but these natural defenses are insufficient to cope with the extremely high amounts of hemin (10 mM) that can potentially be liberated from hemoglobin in a hematoma. The present review discusses how hemin gets into brain cells, and examines the multiple routes through which hemin can be toxic. These include the release of redox-active iron, the depletion of cellular stores of NADPH and glutathione, the production of superoxide and hydroxyl radicals, and the peroxidation of membrane lipids. Important gaps are revealed in contemporary knowledge about the metabolism of hemin by brain cells, particularly regarding how hemin interacts with hydrogen peroxide. Strategies currently being developed for the reduction of hemin toxicity after hemorrhagic stroke include chelation therapy, antioxidant therapy and the modulation of heme oxygenase activity. Future strategies may be directed at preventing the uptake of hemin into brain cells to limit the opportunity for toxic interactions.     

Sickle hemoglobin instability: a mechanism for malarial protection

Abstract

Heterozygosity for the mutant sickle hemoglobin confers protection from severe Plasmodium falciparum malaria. It is here proposed that this protection derives from the instability of sickle hemoglobin, which clusters red cell membrane protein band 3 and triggers accelerated removal by phagocytic cells. This explanation requires that sickle trait cells manifest greater hemoglobin instability than normal red cells, something that could derive from their content of sickle hemoglobin. The mechanism also implicates splenic function as a determinant of the protective effect.     

Glucose-6-phosphate dehydrogenase – from oxidative stress to cellular functions and degenerative diseases

Abstract

Glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme of the pentose phosphate pathway, is indispensable to maintenance of the cytosolic pool of NADPH and thus the cellular redox balance. The role of G6PD as an antioxidant enzyme has been recognized in erythrocytes for a long time, as its deficiency is associated with neonatal jaundice, drug- or infection-mediated hemolytic crisis, favism and, less commonly, chronic non-spherocytic hemolytic anemia. To a large extent, advances in the field were made on the pathophysiology of G6PD-deficient erythrocytes, and the molecular characterization of different G6PD variants. Not until recently did numerous studies cast light on the importance of G6PD in other aspects of the physiology of both cells and organisms. Deficiency in G6PD activity, and hence a disturbance in redox homeostasis, can lead to dysregulation of cell growth and signaling, anomalous embryonic development, altered susceptibility to viral infection as well as increased susceptibility to degenerative diseases. The present review covers recent developments in this field. Additionally, molecular characterization of G6PD variants, especially those frequently found in Taiwan and Southern China, is also addressed.     

Evidence of reactive oxygen species generation in synovial fluid from patients with temporomandibular disease by electron spin resonance spectroscopy

Abstract

Reactive oxygen species (ROS) have been implicated in the pathogenesis of temporomandibular disorders. In the present study, we provide the first evidence of ROS generation in the synovial fluid from human temporomandibular disorder patients, as shown by electron spin resonance (ESR) and spin trapping. Three distinct ESR spectra of DMPO spin adducts were observed in the synovial fluid. They corresponded to three free radical species: hydroxyl radical (HO^?), hydrogen radical (H^?), and carbon-center radical (R^?). Among them, the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OH spectrum was the most prominent, suggesting that HO^? was dominantly generated in the synovial fluid from temporomandibular disorder patients. Desferrioxamine (DFO), an iron chelator, strongly depressed the DMPO-OH signal intensity in the synovial fluid from patients with temporomandibular disorders. We successfully demonstrated ROS-induced oxidative stress in the synovial fluid from temporomandibular disorder patients. ROS generation in the temporomandibular joint could lead to exacerbation of inflammation and activation of cartilage matrix degrading enzymes that proceed to degenerative change of the temporomandibular joint. Thus, iron-dependent generation of HO ^? might have a crucial role in the pathogenesis of temporomandibular disorders.     

Critical role of exposure time to endogenous oxidative stress in hepatocyte apoptosis

Abstract

We have previously shown that inhibition of catalase and glutathione peroxidase activities in rat primary hepatocytes by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS) results in sustained oxidative stress, followed by apoptosis. To examine the effects of duration of oxidative stress, ATZ and MS were removed from culture medium at 3, 6 and 9 h after treatment with both inhibitors. Oxidative stress was induced for periods of time by ATZ and MS exposures in primary hepatocytes. Treatment with ATZ and MS reduced catalase (CAT) and glutathione peroxidase (GPx) activities, and decreased CAT and GPx activities recovered to normal values upon withdrawal. Although oxidative stress of up to 6 h duration did not cause cell death, sustained oxidative stress (over 9 h) induced apoptosis. The increase in the glutathione disulfide/reduced glutathione ratio under oxidative stress up to 6 h was transient and reversible, while that due to sustained oxidative stress was irreversible. These results suggest that irreversible redox shifts resulting from sustained oxidative stress play a critical role in the induction of hepatocyte apoptosis in this experimental system.     

N-Acetyl cysteine restores viability and function of rat odontoblast-like cells impaired by polymethylmethacrylate dental resin extract

Abstract

There is concern that dental-resin materials directly loaded on a prepared tooth adversely affect dental pulp tissue by releasing the resin chemicals through dentinal tubes. This study determined whether self-curing polymethyl methacrylate (PMMA)-based dental resin extract adversely affected the viability and function of odontoblast-like cells and whether the cytotoxicity of this resin, if any, could be eliminated by N-acetyl cysteine, an antioxidant amino acid derivative. Odontoblast-like cells isolated from rat maxillary incisor dental pulp tissue were exposed to a PMMA resin extract with or without N-acetyl cysteine for 1 h and then cultured in osteoblastic media. The percentage of viable cells 24 h after seeding was 20% in cells exposed to the resin extract without N-acetyl cysteine, whereas 45% of cells were viable after exposure to the N-acetyl cysteine-supplemented extract. The cells that had been exposed to the extract showed a strong tendency for apoptosis associated with the increased reactive oxygen species production and decreased intracellular glutathione level, which was improved by the addition of N-acetyl cysteine. N-Acetyl cysteine supplementation almost completely restored the significantly reduced alkaline phosphatase activity and matrix mineralization by the resin extract. These results conclusively demonstrated that exposure of odontoblast-like cells to the resin extract impaired the cell viability and function and, more intriguingly, N-acetyl cysteine supplementation to the extract significantly prevented these toxic effects.