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.     

Cyclosporine A induced changes to plasma and erythrocyte antioxidant defences

Abstract

Organ transplant recipients develop pronounced cardiovascular disease, and decreased antioxidant capacity in plasma and erythrocytes is associated with the pathogenesis of this disease. These experiments tested the hypothesis that the immunosuppressant cyclosporine A (CsA) alters erythrocyte redox balance and reduces plasma antioxidant capacity. Female Sprague-Dawley rats were randomly assigned to a control or CsA treated group. Treatment animals received 25 mg/kg/day of CsA via intraperitoneal injection for 18 days. Control rats were injected with the same volume of the vehicle. Three hours after the final CsA injection, rats were exsanguinated and plasma analysed for total antioxidant status (TAS), α-tocopherol, malondialdehyde (MDA), and creatinine. Erythrocytes were analysed for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glucose-6-phosphate dehydrogenase (G6PD) activities, α-tocopherol, and MDA. CsA administration resulted in a significant (P < 0.05) decrease in plasma TAS and significant increases (P < 0.05) in plasma creatinine and MDA. Erythrocyte CAT was significantly (P < 0.05) increased in CsA treated rats compared to controls. There were no significant differences (P > 0.05) in erythrocyte SOD, GPX, G6PD, α-tocopherol or MDA between groups. In summary, CsA alters erythrocyte antioxidant defence and decreases plasma total antioxidant capacity.     

The biogenesis of [Fe–S] clusters: the role of the unorthodox ABC ATPase,SufC, and the wider implications for understanding iron metabolism

Abstract

Oxidative stress is the hallmark of various chronic inflammatory lung diseases. Increased concentrations of reactive oxygen species (ROS) in the lungs of such patients are reflected by elevated concentrations of oxidative stress markers in the breath, airways, lung tissue and blood. Traditionally, the measurement of these biomarkers has involved invasive procedures to procure the samples or to examine the affected compartments, to the patient's discomfort. As a consequence, there is a need for less or non-invasive approaches to measure oxidative stress. The collection of exhaled breath condensate (EBC) has recently emerged as a non-invasive sampling method for real-time analysis and evaluation of oxidative stress biomarkers in the lower respiratory tract airways. The biomarkers of oxidative stress such as H₂O₂, F₂-isoprostanes, malondialdehyde, 4-hydroxy-2-nonenal, antioxidants, glutathione and nitrosative stress such as nitrate/nitrite and nitrosated species have been successfully measured in EBC. The reproducibility, sensitivity and specificity of the methodologies used in the measurements of EBC oxidative stress biomarkers are discussed. Oxidative stress biomarkers also have been measured for various antioxidants in disease prognosis. EBC is currently used as a research and diagnostic tool in free radical research, yielding information on redox disturbance and the degree and type of inflammation in the lung. It is expected that EBC can be exploited to detect specific levels of biomarkers and monitor disease severity in response to appropriate prescribed therapy/treatment.     

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.     

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.     

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.     

Antioxidant property of an active component purified from the leaves of paraquat-tolerant Rehmannia glutinosa

Abstract

Acteoside extracted from the leaves of Rehmannia glutinosa was examined to determine the mechanism(s) of its antioxidant properties. The deoxyribose assay system showed that acteoside has a high redox potential as electron donor, which generates hydroxyl radicals in an Fe³⁺-dependent manner similar to ascorbic acid. However, the antioxidant properties of acteoside differ from those of ascorbic acid in that the superoxide anion-mediated reduction of nitroblue tetrazolium was actively inhibited by acteoside but not by ascorbic acid. Acteoside protected cells against glucose oxidase-mediated cytotoxicity and apoptosis in a dose-dependent manner. In addition, acteoside had immune stimulating effects, as shown by the acteoside-mediated increase in the level of DNA synthesis, viability, and cytokine secretion in mouse splenocytes. Moreover, acteoside inhibited the gelatinolytic activity of MMP proteins in a dose-dependent manner. Considering these results and the fact that acteoside is a water-soluble natural product, acteoside might have potential as a preventative treatment for oxidative stress-mediated diseases and have possibilities in the cosmetic industry.     

Hydrogen peroxide is critical for UV-induced apoptosis inhibition

Abstract

Apoptosis is an important cell death system that deletes damaged and mutated cells, preventing the induction of cancer. We previously have reported that UV irradiation inhibited the apoptosis induced by serum starvation and cell detachment. This phenomenon is suitable for clarifying the relationship between cancer and the dysregulation of apoptosis by UV irradiation. Here, we have studied the factors responsible for this inhibition of apoptosis, focusing on reactive oxygen species (ROS) and DNA damage. Treatment with xanthine oxidase in the presence of hypoxanthine, which is known to produce superoxide anion (O₂^??) and hydrogen peroxide (H₂O₂), inhibited the induction of apoptosis. The xanthine oxidase-induced anti-apoptotic effect was suppressed in the presence of an H₂O₂-eliminating enzyme, catalase, but not in the presence of an O₂^??-eliminating enzyme, superoxide dismutase. Treatment with H₂O₂ itself significantly inhibited the induction of apoptosis. Furthermore, the effect of the inhibition of cell death by UVB irradiation and by H₂O₂ treatment decreased in H₂O₂-resistant cells. Although both UVB and H₂O₂ are known to induce DNA damage, other DNA damaging agents, like γ-irradiation and treatment with cisplatin and bleomycin, showed no inhibition of apoptosis. These findings suggested that H₂O₂ was essential to the inhibition of apoptosis, in which DNA damage had no role.     

Differential regulation of intracellular redox state by extracellular matrix proteins in glomerular mesangial cells: potential role in diabetic nephropathy

Abstract

Advanced diabetic nephropathy is characterized by abnormal synthesis of extracellular matrix (ECM) proteins, such as collagen I (COL I). The present experiments were designed to test the hypothesis that the presence of abnormal ECM proteins may be responsible for increased generation of reactive oxygen species (ROS) that are thought to have an important role in the pathogenesis of diabetic nephropathy. SV40 MES 13 murine mesangial cells were plated on COL I or collagen IV (COL IV) for 3 h at 5.5 or 25 mM D-glucose concentration. Increased intracellular ROS generation and reduced intracellular nitric oxide (NO) production was measured in cells attached to COL I compared with cells attached to COL IV. Treatment with N_ω-nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of NO synthase, reduced this difference in ROS generation between cells attached to either COL I or IV. The results using antibodies against integrins also indicated that an α₂ integrin-mediated pathway was involved in the different response in ROS generation caused by ECM proteins. These results suggest that contact between altered ECM proteins that are present in advanced diabetic nephropathy and mesangial cells has the potential to increase intracellular oxidative stress, leading to progressive glomerular damage.     

Kinetic study on the photostability of riboflavin in the presence of barbituric acid

Abstract

The photochemical fate of riboflavin (vitamin B₂) in the presence of barbituric acid was examined employing polarographic detection of dissolved oxygen and steady-state and time-resolved spectroscopy. Under visible light, riboflavin reacts with barbituric acid – the latter being transparent to this type of photo-irradiation – via radicals and reactive oxygen species, such as singlet molecular oxygen [O₂(¹Δ_g)] and superoxide radical anion, which are generated from the excited triplet state of the vitamin. As a result, both the vitamin and barbituric acid are photodegraded. Kinetic and mechanistic studies on the photoreactions of riboflavin in the presence of barbituric acid indicate the excellent quenching ability of the latter towards O₂(¹Δ_g).