Oxidative stress and antioxidant deficiencies in asthma: potential modification by diet

The lungs of asthmatic patients are exposed to oxidative stress due to the generation of reactive oxygen and nitrogen species as a consequence of chronic airway inflammation. Increased concentrations of NO*, H2O2 and 8-isoprostane have been measured in exhaled breath and induced sputum of asthmatic patients. O2*-, NO*, and halides interact to form highly reactive species such as peroxynitrite and HOBr, which in turn cause nitration and bromination of protein tyrosine residues. Oxidative stress may also reduce glutathione levels and cause inactivation of antioxidant enzymes such as superoxide dismutase, with a consequent increase in apoptosis, shedding of airway epithelial cells and airway remodelling. The oxidant/antioxidant equilibrium in asthmatic patients may be further perturbed by low dietary intakes of the antioxidant vitamins C and E, selenium and flavonoids, with a consequent lowering of the concentrations of these and other non-dietary antioxidants such as bilirubin and albumin in plasma and airway epithelial lining fluid. Although supplementation with vitamins C and E appears to offer protection against the adverse effects of ozone, recent randomised, placebo-controlled trials of vitamin C or E supplements for patients with mild asthma have not shown significant benefits over standard therapy. However, genetic variation in glutathione S-transferase may influence the susceptibility of asthmatic individuals to oxidative stress and the extent to which they are likely to benefit from antioxidant supplementation. Long-term prospective trials are required to determine whether modification of dietary intake will benefit asthma patients and reduce the socio-economic burden of asthma in the community.     

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.     

Polyphenols suppress hydrogen peroxide‐induced oxidative stress in human bone‐marrow derived mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are considered a highly promising candidate cell type for cell‐based tissue engineering and regeneration because of their self‐renewal and multi‐lineage differentiation characteristics. Increased levels of reactive oxygen/nitrogen species (ROS/RNS) are associated with tissue injury and inflammation, impact a number of cellular processes, including cell adhesion, migration, and proliferation, and have been linked to cellular senescence in MSCs, potentially compromising their activities. Naturally occurring polyphenolic compounds (polyphenols), epigallocatechin‐3‐gallate (EGCG), and curcumin, block ROS/RNS and are potent inflammation‐modulating agents. However, their potential protective effects against oxidative stress in hMSCs have not been examined. In this study, we carried out a systematic analysis of the effects of polyphenols on hMSCs in their response to oxidative stress in the form of treatment with H₂O₂ and S‐nitroso‐N‐acetylpenicillamine (SNAP), respectively. Parameters measured included colony forming activity, apoptosis, and the levels of antioxidant enzymes and free reactive species. We found that polyphenols reversed H₂O₂‐induced loss of colony forming activity in hMSCs. In a dose‐dependent manner, polyphenols inhibited increased levels of ROS and NO, produced by H₂O₂ or SNAP, respectively, in MSCs. Notably, polyphenols rapidly and almost completely blocked H₂O₂‐induced ROS in the absence of significant direct effect on H₂O₂ itself. Polyphenols also protected the antioxidant enzymes and reduced apoptotic cell death caused by H₂O₂ exposure. Taken together, these findings demonstrate that EGCG and curcumin are capable of suppressing inducible oxidative stress in hMSCs, and suggest a possible new approach to maintain MSC viability and potency for clinical application. J. Cell. Biochem. 114: 1163–1173, 2013. © 2012 Wiley Periodicals, Inc.     

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.     

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.     

Neutrophils and oxygen-induced lung injury: a case of when a few is still too many

The role of neutrophils in acute oxidative lung injury in preterm babies is presently unclear, with some investigators maintaining they contribute to tissue injury while others believe they do not. The aim of the present study was to determine whether neutropenia, induced by a specific neutrophil antibody, influenced the time course or extent of oxygen-induced injury of the immature lung. Preterm guinea pigs, delivered by caesarean section at 65 days' gestation (term=68 days), were injected intraperitoneally with either control serum (CS) or neutrophil antiserum (NAS; 200 μl/100 g body weight) once daily for 5 days. Pups were exposed to 95% oxygen for the first 72 h, and then allowed to recover in 21% oxygen for the subsequent 48 h. Groups of treated animals were also maintained in 21% oxygen for 5 days. Lungs were examined by bronchoalveolar lavage (BAL) at 72 h or 120 h. In CS-treated pups, exposure to 95% oxygen increased both the number of circulating neutrophils and those recovered by BAL at both 72 h and 120 h. Protein concentration in BAL fluid, an index of lung microvascular permeability, and BAL elastase and β-glucuronidase activities, indices of neutrophil activation, were significantly increased in pups exposed to 95% oxygen. Pups exposed to 95% oxygen and treated with NAS showed a decrease in numbers of circulating neutrophils (72 h, 9.53 vs 0.66 x 10⁵/ml, P<0.0005; 120 h, 4.9 vs 0.08 x 10⁵/ml, P<0.0005) and BAL fluid neutrophils (72 h, 3.1 vs 0.7 x 10⁵/ml, P<0.05; 120 h, 12.4 vs 3.8 x 10⁵/ml, P<0.05). BAL protein concentration, neutrophil elastase and β-glucuronidase activities in hyperoxia-exposed pups were similar following treatment with either CS or NAS. Although the number of circulating neutrophils were markedly depleted and expansion of the alveolar neutrophil pool was restricted in NAS-treated pups, the neutrophils recruited to the lung were activated and could have contributed to the increase in microvascular permeability in hyperoxia-exposed pups.     

The ERalpha/ERbeta ratio determines oxidative stress in breast cancer cell lines in response to 17Beta-estradiol

The effects of 17beta‐estradiol (E2) are mediated through activation of estrogen receptors (ER): ERalpha and ERbeta. It is known that ERalpha/ERbeta ratio is higher in breast tumors than in normal tissue. Since antioxidant enzymes and uncoupling proteins (UCPs) are reactive oxygen species (ROS) production and mitochondrial biogenesis regulators, our aim was to study the E2‐effect on oxidative stress, antioxidant enzyme expression, and UCPs in breast cancer cell lines with different ERalpha/ERbeta ratios. The lower ERalpha/ERbeta ratio T47D cell line showed low ROS production and high UCP5 levels. However, the higher ERalpha/ERbeta ratio MCF‐7 cell line showed an up‐regulation of antioxidant enzymes and UCPs, yet exhibited high oxidative stress. As a result, a decrease in antioxidant enzyme activities and UCP2 protein levels, coupled with an increase in oxidative damage was found. On the whole, these results show different E2‐effects on oxidative stress regulation, modulating UCPs, and antioxidant enzymes, which were ERalpha/ERbeta ratio dependent in breast cancer cell lines. J. Cell. Biochem. 113: 3178–3185, 2012. © 2012 Wiley Periodicals, Inc.     

Differential cytotoxic responses to low- and high-dose photodynamic therapy in human gastric and bladder cancer cells

Here, we present differential cytotoxic responses to two different doses of photodynamic therapies (PDTs; low-dose PDT [LDP] and high-dose PDT [HDP]) using a chlorin-based photosensitizer, DH-II-24, in human gastric and bladder cancer cells. Fluorescence-activated cell sorting analysis using Annexin V and propidium iodide (PI) showed that LDP induced apoptotic cell death, whereas HDP predominantly caused necrotic cell death. The differential cytotoxic responses to the two PDTs were further confirmed by a DiOC(6) and PI double-staining assay via confocal microscopy. LDP, but not HDP, activated caspase-3, which was inhibited by Z-VAD, Trolox, and BAPTA-AM. LDP and HDP demonstrated opposite effects on intracellular reactive oxygen species (ROS)/Ca(2+) signals; LDP stimulated intracellular ROS production, contributing to a transient increase of intracellular Ca(2+) , whereas HDP induced a massive and prolonged elevation of intracellular Ca(2+) responsible for the transient production of intracellular ROS. In addition, the two PDTs also increased in situ transglutaminase 2 (TG2) activity, with a higher stimulation by HDP, and this increase in activity was prevented by Trolox, BAPTA-AM, and TG2-siRNA. LDP-induced apoptotic cell death was strongly inhibited by Trolox and TG2-siRNA and moderately suppressed by BAPTA-AM. However, HDP-mediated necrotic cell death was partially inhibited by BAPTA-AM but not by TG2-siRNA. Thus, these results demonstrate that LDP and HDP induced apoptotic and necrotic cell death by differential signaling mechanisms involving intracellular Ca(2+) , ROS, and TG2.     

Glutathione synthesis by red blood cells in type 2 diabetes mellitus

Abstract

Oxidative stress is implicated in the pathogenesis and complications of type 2 diabetes mellitus (NIDDM). Glycoxidation may damage the enzymes that synthesise glutathione (GSH), an endogenous intracellular antioxidant. Erythrocytes (RBCs) taken from NIDDM subjects, and non-diabetic controls, were GSH-depleted using 1-chloro-2,4-dinitrobenzene, incubated in a solution containing GSH-rebuilding substrates, and sampled for GSH using a 5,5′-γ-dithiobis-(2-nitrobenzoic acid)/enzymatic recycling procedure. NIDDM subjects, on average, had the same GSH concentration and synthesising ability as non-diabetic controls, indicating normal function of the synthesis enzymes. A positive correlation between synthesis and concentration of GSH seen in non-diabetic controls did not exist in NIDDM, due to their putatively larger oxidative load. The results, to the best of our knowledge, provide the first evidence that, despite a higher oxidative load, intact RBCs from NIDDM subjects are able to synthesise GSH normally. It is hypothesised that increased rates of GSH synthesis would maintain a normal steady-state GSH concentration.     

Interplay of reactive oxygen species,intracellular Ca2+ and mitochondrial homeostasis in the apoptosis of prostate cancer cells by deoxypodophyllotoxin

The limited treatment option for recurrent prostate cancer and the eventual resistance to conventional chemotherapy drugs has fueled continued interest in finding new anti‐neoplastic agents of natural product origin. We previously reported anti‐proliferative activity of deoxypodophyllotoxin (DPT) on human prostate cancer cells. Using the PC‐3 cell model of human prostate cancer, the present study reveals that DPT induced apoptosis via a caspase‐3‐dependent pathway that is activated due to dysregulated mitochondrial function. DPT‐treated cells showed accumulation of the reactive oxygen species (ROS), intracellular Ca surge, increased mitochondrial membrane potential (MMP, ΔΨ_m), Bax protein translocation to mitochondria and cytochrome c release to the cytoplasm. This resulted in caspase‐3 activation, which in turn induced apoptosis. The antioxidant N‐acetylcysteine (NAC) reduced ROS accumulation, MMP and Ca surge, on the other hand the Ca²⁺ chelator BAPTA inhibited the Ca overload and MMP without affecting the increase of ROS, indicating that the generation of ROS occurred prior to Ca²⁺ flux. This suggested that both ROS and Ca signaling play roles in the increased MMP via Ca‐dependent and/or ‐independent mechanisms, since ΔΨ_m elevation was reversed by NAC and BAPTA. This study provides the first evidence for the involvement of both ROS‐ and Ca‐activated signals in the disruption of mitochondrial homeostasis and the precedence of ROS production over the failure of Ca²⁺ flux homeostasis. J. Cell. Biochem. 114: 1124–1134, 2013. © 2012 Wiley Periodicals, Inc.