Wood smoke extract induces oxidative stress-mediated caspase-independent apoptosis in human lung endothelial cells: role of AIF and EndoG

Although a link between toxic smoke and oxidant lung vascular injury has been indicated, the cellular mechanisms of smoke-induced injury to lung endothelial cells are unknown. We investigated oxidative stress and apoptosis induced by wood smoke extract (SE) in human pulmonary artery endothelial cells (HPAECs) and delineated their relationship. We found that SE increased intracellular reactive oxygen species (ROS), depleted intracellular glutathione, and upregulated Cu/Zn superoxide dismutase and heme oxygenase-1 (2 antioxidant enzymes), but it failed to alter the expression of catalase and glutathione peroxidase. In addition, SE promoted apoptosis as indicated by the external exposure of membrane phosphatidylserine, the loss of mitochondrial membrane potential, an increase in the level of Bax (a proapoptotic protein), and enhanced DNA fragmentation. This apoptosis was associated with mitochondrial-to-nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) (2 apoptogenic proteins) but was independent of caspase cascade activation. Whereas N-acetylcysteine (an antioxidant) effectively reversed the SE-induced increase in ROS and depletion of glutathione, it also suppressed SE-induced nuclear translocation of either AIF or EndoG and prevented the enhanced DNA fragmentation that would have resulted from this. We conclude that 1) although SE upregulates Cu/Zn superoxide dismutase and heme oxygenase-1, it nevertheless increases intracellular oxidative stress in HPAECs, and 2) SE promotes oxidative stress-mediated caspase-independent HPAEC apoptosis that involves mitochondrial-to-nuclear translocation of AIF and EndoG. Thus modulations of the expression of antioxidant enzymes and the caspase-independent apoptotic pathway are possible target choices for potential therapeutic regimes to treat smoke-induced lung injury.     

Augmentation of antioxidant enzymes in vascular endothelium

The endothelium is a key site of injury from reactive oxygen species that can potentially be protected by the antioxidant enzymes superoxide dismutase and catalase. Large proteins, such as superoxide dismutase and catalase, do not readily penetrate cell membranes, which limits their efficacy in protecting cells from cellular reactions involving both intracellularly and extracellularly generated reactive oxygen species. Two methods are described that promote enzyme delivery to cultured endothelial cells and confer increased resistance to oxidative stress. The first method is to entrap the antioxidant enzymes within liposomes, which then become incorporated by endothelial cells and can increase enzyme specific activities by as much as 44-fold within 2 h. The second method involves covalent conjugation of polyethylene glycol (PEG) to superoxide dismutase and catalase, a technique that increases circulatory half-life and reduces protein immunogenicity. Conjugation of PEG to superoxide dismutase and catalase increased cellular-specific activities of these enzymes in cultured endothelial cells (but at a slower rate than for liposome entrapped enzymes) and rendered these cells more resistant to oxidative stress. Both liposome-mediated delivery and PEG conjugation offer an additional benefit over native superoxide dismutase and catalase because they can increase cellular antioxidant activities in a manner that can provide protection from both intracellular and extracellular superoxide and hydrogen peroxide.     

Functional relevance of Gedunin as a bona fide ligand of NADPH oxidase 5 and ROS scavenger: An in silico and in vitro assessment in a hyperglycemic RBC model

Clinical evidence suggests that type 2 diabetes therapy can greatly benefit from the suppression of reactive oxygen species generation and the activation or restoration of cellular antioxidant mechanisms. In human, NADPH oxidase (NOX) is the main producer of reactive oxygen species (ROS) that supress the activity of endogenous antioxidant enzymes. In the present study, the antioxidant potential of Gedunin was studied. In silico findings reveal its strong binding affinity with NOX5 C terminal HSP90 binding site that disrupts NOX5 stability and its ability to generate ROS, leading to restoration antioxidant enzymes activities. It was found that Gedunin suppressed hyperglycaemia induced oxidative stress in an in vitro RBC model and markedly reversed glucose induced changes including haemoglobin glycosylation and lipid peroxidation. A significant restoration of activities of cellular antioxidant enzymes; superoxide dismutase, catalase and glutathione peroxidase in the presence of Gedunin revealed its ability to reduce oxidative stress. These results substantiated Gedunin as a bona fide inhibitor of human NOX5 and a ROS scavenging antioxidant with promising therapeutic attributes including its natural origin and inhibition of multiple diabetic targets.     

Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance

Covalent conjugation of superoxide dismutase and catalase with polyethylene glycol (PEG) increases the circulatory half-lives of these enzymes from less than 10 min to 40 h, reduces immunogenicity, and decreases sensitivity to proteolysis. Because PEG has surface active properties and can induce cell fusion, we hypothesized that PEG conjugation could enhance cell binding and association of normally membrane-impermeable enzymes. Incubation of cultured porcine aortic endothelial cells with 125I-PEG-catalase or 125I-PEG-superoxide dismutase produced a linear, concentration-dependent increase in cellular enzyme activity and radioactivity. Fluorescently labeled PEG-superoxide dismutase incubated with endothelial cells showed a vesicular localization. Mechanical injury to cell monolayers, which is known to stimulate endocytosis, further increased the uptake of fluorescent PEG-superoxide dismutase. Endothelial cell cultures incubated with PEG-superoxide dismutase and PEG-catalase for 24 h and then extensively washed were protected from the damaging effects of reactive oxygen species derived from exogenous xanthine oxidase as judged by two criteria: decreased release of intracellular 51Cr-labeled proteins and free radical-induced changes in membrane fluidity, measured by electron paramagnetic resonance spectroscopy of endothelial membrane proteins covalently labeled with 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. Addition of PEG and PEG-conjugated enzymes perturbed the spin-label binding environment, indicative of producing an increase in plasma membrane fluidity. Thus, PEG conjugation to superoxide dismutase and catalase enhances cell association of these enzymes in a manner which increases cellular enzyme activities and provides prolonged protection from partially reduced oxygen species.     

Increased oxygen radical-dependent inactivation of metabolic enzymes by liver microsomes after chronic ethanol consumption

Enzymatic and nonenzymatic mixed-function oxidase systems have been shown to generate an oxidant that catalyzes the inactivation of glutamine synthetase and other metabolic enzymes. Recent studies have shown that microsomes isolated from rats chronically fed ethanol generate reactive oxygen intermediates at elevated rates compared with controls. Microsomes from rats fed ethanol were found to be more effective than control microsomes in catalyzing the inactivation of enzymes added to the incubation system. The enzymes studied were alcohol dehydrogenase, lactic dehydrogenase, and pyruvate kinase. The inactivation process by both types of microsomal preparations was sensitive to catalase and glutathione plus glutathione peroxidase, but was not affected by superoxide dismutase or hydroxyl radical scavengers. Iron was required for the inactivation of the added enzymes; microsomes from the rats fed ethanol remained more effective than control microsomes in catalyzing the inactivation of enzymes in the absence or presence of several ferric complexes. The inactivation of enzymes was enhanced by the addition of menadione or paraquat to the microsomes, and rates of inactivation were higher with the microsomes from the ethanol-fed rats. The enhanced generation of reactive oxygen intermediates and increased inactivation of enzymes by microsomes may contribute toward the hepatotoxic effects associated with ethanol consumption.     

Implications of Reactive Oxygen Species in Heat Shock Induced Germination and Early Growth Impairment in Amaranthus lividus L.

An effort has been made to assess the role of reactive oxygen species in germination and subsequent growth of Amaranthus lividus under elevated temperature. Transfer of A. lividus seeds from 25 to 45 °C for 4, 8 and 12 h, during early imbibitional period reduced percentage of germination, relative germination performance, relative growth index and seedling length. Heat shock during early germination decreased also the activities of free radical scavenging enzymes like catalase, peroxidase and superoxide dismutase, increased the accumulation of superoxide, hydrogen peroxide and induced lipoxygenase mediated membrane lipid peroxidation. Membrane injury index and relative leakage ratio revealed a rise with concomitant reduction in membrane protein thiol content in heat shock raised seedlings. The results indicate that heat shock in A. lividus seeds induced an excessive generation of ROS and led to an oxidative membrane damage, causing early growth impairment.     

Overexpression of Manganese Superoxide Dismutase Promotes the Survival of Prostate Cancer Cells Exposed to Hyperthermia

It has been hypothesized that exposure of cells to hyperthermia results in an increased flux of reactive oxygen species (ROS), primarily superoxide anion radicals, and that increasing antioxidant enzyme levels will result in protection of cells from the toxicity of these ROS. In this study, the prostate cancer cell line, PC-3, and its manganese superoxide dismutase (MnSOD)-overexpressing clones were subjected to hyperthermia (43°C, 1?h). Increased expression of MnSOD increased the mitochondrial membrane potential (MMP). Hyperthermic exposure of PC-3 cells resulted in increased ROS production, as determined by aconitase inactivation, lipid peroxidation, and H₂O₂ formation with a reduction in cell survival. In contrast, PC-3 cells overexpressing MnSOD had less ROS production, less lipid peroxidation, and greater cell survival compared to PC-3 Wt cells. Since MnSOD removes superoxide, these results suggest that superoxide free radical or its reaction products are responsible for part of the cytotoxicity associated with hyperthermia and that MnSOD can reduce cellular injury and thereby enhance heat tolerance.     

Antioxidant activity of Terminalia arjuna bark extract on N-nitrosodiethylamine induced hepatocellular carcinoma in rats

Role of oxidative stress and Na⁺,K⁺-ATPase in the cytotoxicity of hexachlorocyclohexane (HCH) on Ehrlich Ascites tumor (EAT) cells has been studied. HCH caused dose dependent cell death as measured by trypan blue exclusion and lactate dehydrogenase (LDH) leakage from the cells. HCH induced oxidative stress in EAT cells which was characterized by glutathione depletion, lipid peroxidation (LPO), reactive oxygen species (ROS) production and inhibition of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT). Protective effect of antioxidants on HCH induced oxidative stress was assessed, among the antioxidants used only quercetin inhibited HCH-induced LPO and ROS production as well as cell death whereas α -tocopherol, ascorbic acid and BHA inhibited LPO but not cell death. Inhibition of membrane bound Na⁺,K⁺-ATPase was a characteristic feature of HCH cytotoxicity in EAT cells. Experimental evidence indicates that HCH-induced cell death involves oxidative stress due to ROS production and membrane perturbation in EAT cells.     

Stabilization of superoxide dismutase by acetyl-l-carnitine in human brain endothelium during alcohol exposure: novel protective approach

Oxidative damage of the endothelium disrupts the integrity of the blood-brain barrier (BBB). We have shown before that alcohol exposure increases the levels of reactive oxygen species (ROS; superoxide and hydroxyl radical) and nitric oxide (NO) in brain endothelial cells by activating NADPH oxidase and inducible nitric oxide synthase. We hypothesize that impairment of antioxidant systems, such as a reduction in catalase and superoxide dismutase (SOD) activity, by ethanol exposure may elevate the levels of ROS/NO in endothelium, resulting in BBB damage. This study examines whether stabilization of antioxidant enzyme activity results in suppression of ROS levels by anti-inflammatory agents. To address this idea, we determined the effects of ethanol on the kinetic profile of SOD and catalase activity and ROS/NO generation in primary human brain endothelial cells (hBECs). We observed an enhanced production of ROS and NO levels due to the metabolism of ethanol in hBECs. Similar increases were found after exposure of hBECs to acetaldehyde, the major metabolite of ethanol. Ethanol simultaneously augmented ROS generation and the activity of antioxidative enzymes. SOD activity was increased for a much longer period of time than catalase activity. A decline in SOD activity and protein levels preceded elevation of oxidant levels. SOD stabilization by the antioxidant and mitochondria-protecting agent acetyl-L-carnitine (ALC) and the anti-inflammatory agent rosiglitazone suppressed ROS levels, with a marginal increase in NO levels. Mitochondrial membrane protein damage and decreased membrane potential after ethanol exposure indicated mitochondrial injury. These changes were prevented by ALC. Our findings suggest the counteracting mechanisms of oxidants and antioxidants during alcohol-induced oxidative stress at the BBB. The presence of enzymatic stabilizers favors the ROS-neutralizing antioxidant redox of the BBB, suggesting an underlying protective mechanism of NO for brain vascular tone and vasodilation.     

beta-sitosterol decreases irradiation-induced thymocyte early damage by regulation of the intracellular redox balance and maintenance of mitochondrial membrane stability

Both radiation injury and oxidation toxicity occur when cells are exposed to ion irradiation (IR), ultimately leading to apoptosis. This study was designed to determine the effect of beta-sitosterol (BSS) on early cellular damage in irradiated thymocytes and a possible mechanism of effect on irradiation-mediated activation of the apoptotic pathways. Thymocytes were irradiated (6 Gy) with or without BSS. Cell apoptosis and apoptosis-related proteins were evaluated. BSS decreased irradiation-induced cell death and nuclear DNA strand breaks while attenuating intracellular reactive oxygen species (ROS) and increasing the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). BSS decreased the release of cytochrome c from mitochondria to the cytosol and the mitochondrio-nuclear translocation of apoptosis-inducing factor (AIF). Furthermore, BSS partially inhibited the radiation-induced increase of cleaved caspase 3 and cleaved PARP, and attenuated the activation of JNK and AP-1. In addition, evidence suggests that ROS generated by irradiation are involved in this course of cell damage. The results indicate that BSS confers a radioprotective effect on thymocytes by regulation of the intracellular redox balance which is carried out via the scavenging of ROS and maintenance of mitochondrial membrane stability.     

Overexpression of manganese superoxide dismutase promotes the survival of prostate cancer cells exposed to hyperthermia

It has been hypothesized that exposure of cells to hyperthermia results in an increased flux of reactive oxygen species (ROS), primarily superoxide anion radicals, and that increasing antioxidant enzyme levels will result in protection of cells from the toxicity of these ROS. In this study, the prostate cancer cell line, PC-3, and its manganese superoxide dismutase (MnSOD)-overexpressing clones were subjected to hyperthermia (43°C, 1 h). Increased expression of MnSOD increased the mitochondrial membrane potential (MMP). Hyperthermic exposure of PC-3 cells resulted in increased ROS production, as determined by aconitase inactivation, lipid peroxidation, and H₂O₂ formation with a reduction in cell survival. In contrast, PC-3 cells overexpressing MnSOD had less ROS production, less lipid peroxidation, and greater cell survival compared to PC-3 Wt cells. Since MnSOD removes superoxide, these results suggest that superoxide free radical or its reaction products are responsible for part of the cytotoxicity associated with hyperthermia and that MnSOD can reduce cellular injury and thereby enhance heat tolerance.     

Opposite effects of endotoxin on mitochondrial and endoplasmic reticulum functions

In this study, we determined functional integrity and reactive oxygen species generation in mitochondria and endoplasmic reticulum in liver of rats subjected to endotoxic shock to clarify whether intracellular reactive oxygen species (ROS) destabilize cellular integrity causing necrosis in rats challenged with lipopolysaccharide (LPS). LPS caused drastically increased plasma levels of alanine aminotransferase, suggesting damage to plasma membranes of liver cells. Liver necrosis was confirmed by histological examination. LPS induced a significant increase in ROS production in rat liver mitochondria (RLM), but did not impair mitochondrial function. In contrast to mitochondria, enzymatic activity and ROS production of cytochrome P450 were lower in microsomal fraction obtained from LPS-treated animals, suggesting the dysfunction of endoplasmic reticulum. Protein patterns obtained from RLM by two-dimensional electrophoresis showed significant upregulation of mitochondrial superoxide dismutase by LPS. We hypothesize that upregulation of this enzyme protects mitochondria against mitochondrial ROS, but does not protect other cellular compartments such as endoplasmic reticulum and plasma membrane causing necrosis.     

Effect of stress on the antioxidant enzymes and gastric ulceration

The effect of cold-restraint stress on the antioxidant enzymes of the rat gastric mucosa was studied with a view to finding out their role in stress induced gastric ulceration. Histological examination revealed stress induced extensive damage of the surface epithelial cell with lesions extending upto submucosa in some cases. Stress causes time-dependent increase in histamine and pepsin content but decrease in acid content of the gastric fluid with the progress of ulceration (ulcer index) for two hours. The tissue lipid peroxidation was significantly increased as evidenced by accumulation of malondialdehyde. Since lipid peroxidation results from the generation of reactive oxygen species, stress effect was studied on some antioxidant enzymes such as superoxide dismutase, peroxidases and prostaglandin synthetase as a function of time. The time dependent increase in stress ulcer correlates well with the concomitant increase in superoxide dismutase activity and decrease in peroxidase and prostaglandin synthetase activity. This creates a favourable condition for accumulation of endogenous H₂O₂ and more reactive hydroxyl radical (OH·). Administration of antioxidants such as reduced glutathione or sodium benzoate prior to stress causes significant decrease in ulcer index and lipid peroxidation and protection of gastric peroxidase activity suggesting the involvement of reactive oxygen species in stress induced gastric ulceration. This is supported by thein vitro observation that OH· can also inactivate peroxidase and induce lipid peroxidation. As prostaglandin is known to offer cytoprotection, stress-induced loss of prostaglandin synthetase activity appears to aggravate the oxidative damage caused by reactive oxygen species.Abbreviations ROS reactive oxygen species - GPO gastric peroxidase - SOD superoxide dismutase - MDA malondialdehyde - GSH reduced glutathione - TCA trichloroacetic acid     

Role of reactive oxygen species in injury-induced insulin resistance

Acute insulin resistance is common after injury, infection, and critical illness. To investigate the role of reactive oxygen species (ROS) in critical illness diabetes, we measured hepatic ROS, which rapidly increased in mouse liver. Overexpression of superoxide dismutase 2, which decreased mitochondrial ROS levels, protected mice from the development of acute hepatic insulin resistance. Insulin-induced intracellular signaling was dramatically decreased, and cellular stress signaling was rapidly increased after injury, resulting in the hyperglycemia of critical illness diabetes. Insulin-induced intracellular signaling, activation of stress (c-Jun N-terminal kinase) signaling, and glucose metabolism were all normalized by superoxide dismutase 2 overexpression or by pretreatment with antioxidants. Thus, ROS play an important role in the development of acute hepatic insulin resistance and activation of stress signaling after injury.     

Zinc inhibition of cellular energy production: implications for mitochondria and neurodegeneration

An increasing body of evidence suggests that high intracellular free zinc promotes neuronal death by inhibiting cellular energy production. A number of targets have been postulated, including complexes of the mitochondrial electron transport chain, components of the tricarboxylic acid cycle, and enzymes of glycolysis. Consequences of cellular zinc overload may include increased cellular reactive oxygen species (ROS) production, loss of mitochondrial membrane potential, and reduced cellular ATP levels. Additionally, zinc toxicity might involve zinc uptake by mitochondria and zinc induction of mitochondrial permeability transition. The present review discusses these processes with special emphasis on their potential involvement in brain injury.     

Vitamin E and selenium administration as a modulator of antioxidant defense system: biochemical assessment and modification

Exposure of cells to ionizing radiation leads to the formation of reactive oxygen species (ROS) that are associated with radiation-induced cytotoxicity. Because of the serious damaging potential of ROS, cells depend on the elaboration of the antioxidant defense system (AODS), both enzymatic and nonenzymatic oxidant defense mechanisms. The deficiency in important components of the endogenous AODS leads to the accumulation of oxidative stress inducing oxidative damage. The antioxidant enzymes superoxide dismutase and glutathione peroxidase are key intracellular antioxidants in the metabolism of ROS. In the current study, we investigated the potential role of these antioxidant enzymes in radioresistance during the evaluation of the compensatory role of some exogenous micronutrients against oxidative stress Animals were categorized into eight groups, receiving vitamin E (α-tocopherol) and/or selenium (Se) with or without whole-body γ-irradiation (6.5 Gy). The results indicate that antioxidant pretreatments before irradiation may have some beneficial effects against irradiation-induced injury. The results also indicate that selenium and vitamin E act alone and in an additive fashion as radioprotecting agents. The results further suggest that selenium confers protection in part by inducing or activating cellular free-radical scavenging systems and by enhancing peroxide breakdown, whereas vitamin E appears to confer its protection by an alternate complementary mechanism.     

Manganese: elemental defence for a life with oxygen

The presence of enzymes such as catalase, peroxidase and superoxide dismutase (SOD) obviates the problems associated with life in an aerobic environment by eliminating the harmful reactive oxygen species (ROS) that arise from respiration. Enzymic detoxification of ROS might not, however, be the only mechanism at work in bacteria. The accumulation of manganese (Mn), an abundant element in many environments, via several, recently identified transporters is thought to form the basis for an alternative, catalytic detoxification of ROS. An increasing body of evidence from work on the genetics and biochemistry of Mn accumulation and its cellular roles reveals that this overlooked defence mechanism is likely to be widespread among bacteria and might also contribute to virulence.     

Hepatocellular protection by nitric oxide or nitrite in ischemia and reperfusion injury

Ischemia and reperfusion (I/R)-induced liver injury occurs in several pathophysiological disorders including hemorrhagic shock and burn as well as resectional and transplantation surgery. One of the earliest events associated with reperfusion of ischemic liver is endothelial dysfunction characterized by the decreased production of endothelial cell-derived nitric oxide (NO). This rapid post-ischemic decrease in NO bioavailability appears to be due to decreased synthesis of NO, enhanced inactivation of NO by the overproduction of superoxide or both. This review presents the most current evidence supporting the concept that decreased bioavailability of NO concomitant with enhanced production of reactive oxygen species initiates hepatocellular injury and that endogenous NO or exogenous NO produced from nitrite play important roles in limiting post-ischemic tissue injury.