N-acetylcysteine amide (AD4) attenuates oxidative stress in beta-thalassemia blood cells

Many aspects of the pathology in beta-hemoglobinopathies (beta-thalassemia and sickle cell anemia) are mediated by oxidative stress. In the present study we tested a novel thiol compound, N-acetylcysteine amide (AD4), the amide form of N-acetyl cysteine (NAC) for its antioxidant effects. Using flow-cytometry, we showed that in vitro treatment of blood cells from beta-thalassemic patients with AD4 elevated the reduced glutathione (GSH) content of red blood cells (RBC), platelets and polymorphonuclear (PMN) leukocytes, and reduced their ROS. These effects resulted in a significant reduced sensitivity of thalassemic RBC to hemolysis and phagocytosis by macrophages. Intra-peritoneal injection of AD4 to beta-thalassemic mice (150 mg/kg) reduced the parameters of oxidative stress (p<0.001). Our results show the superiority of AD4, compared to NAC, in reducing oxidative stress markers in thalassemic cells both in vitro and in vivo.     

Vitamin E protects against acetone-induced oxidative stress in rat red blood cells

Acetone may induce oxidative stress leading to disturbance of the biochemical and physiological functions of red blood cells (RBCs) thereby affecting membrane integrity. Vitamin E (vit E) is believed to function as an antioxidant in vivo protecting membranes from lipid peroxidation. The aim of the present study was the evaluation of possible protective effects of vit E treatment against acetone-induced oxidative stress in rat RBCs. Thirty healthy male Wistar albino rats, weighing 200–230 g and averaging 12 weeks old were randomly allotted into one of three experimental groups: Control (A), acetone-treated (B) and acetone + vit E-treated groups (C), each containing ten animals. Group A received only drinking water. Acetone, 5% (v/v), was given with drinking water to B and C groups. In addition, C group received vit E dose of 200 mg/kg/day i.m. The experiment continued for 10 days. At the end of the 10th day, the blood samples were obtained for biochemical and morphological investigation. Acetone treatment resulted in RBC membrane destruction and hemolysis, increased thiobarbituric acid reactive substance (TBARS) levels in plasma and RBC, and decreased RBC vit E levels. Vit E treatment decreased elevated TBARS levels in plasma and RBC and also increased reduced RBC vit E levels, and prevented RBC membrane destruction and hemolysis. In conclusion, vit E treatment appears to be beneficial in preventing acetone-induced oxidative RBC damage, and therefore, it can improve RBC rheology.     

Desferrioxamine protects human red blood cells from hemin-induced hemolysis

Hemin binding to red cell membranes, its effect on red cell hemolysis, and it interaction with desferrioxamine (DFO) in these processes were investigated. DFO interacted with hemin via the iron moiety. Blockage of the binding groups in DFO prevented interaction of DFO with hemin, implying the importance of the hydroxamic acid groups in DFO-hemin interactions. Since hemolysis is a result of hemin association with the membrane components, its binding in the presence and absence of DFO was studied. DFO strongly inhibited hemin-induced lysis in a concentration-dependent manner. With 50 microM hemin, 1 mM DFO completely inhibited lysis. Preincubation of ghost membranes with DFO (1 mM) inhibited binding of hemin (50 microM) to membranes by 42%. After ghost membranes were preincubated with hemin (50 microM), the addition of DFO (1 mM) removed 20% of the membrane-bound hemin. It is suggested that DFO may have an important role in alleviating the hemin-induced deleterious effects on the red cell membrane, especially in hemolytic anemias associated with unstable, autoxidized hemoglobins.     

N-acetylcysteine amide,a thiol antioxidant,prevents bleomycin-induced toxicity in human alveolar basal epithelial cells (A549)

Abstract

Bleomycin (BLM), a glycopeptide antibiotic from Streptomyces verticillus, is an effective antineoplastic drug. However, its clinical use is restricted due to the wide range of associated toxicities, especially pulmonary toxicity. Oxidative stress has been implicated as an important factor in the development of BLM-induced pulmonary toxicity. Previous studies have indicated disruption of thiol-redox status in lungs (lung epithelial cells) upon BLM treatment. Therefore, this study focused on (1) investigating the oxidative effects of BLM on lung epithelial cells (A549) and (2) elucidating whether a well-known thiol antioxidant, N-acetylcysteine amide (NACA), provides any protection against BLM-induced toxicity. Oxidative stress parameters, such as glutathione (GSH), malondialdehyde (MDA), and antioxidant enzyme activities were altered upon BLM treatment. Loss of mitochondrial membrane potential (ΔΨm), as assessed by fluorescence microscopy, indicated that cytotoxicity is possibly mediated through mitochondrial dysfunction. Pretreatment with NACA reversed the oxidative effects of BLM. NACA decreased the reactive oxygen species (ROS) and MDA levels and restored the intracellular GSH levels. Our data showed that BLM induced A549 cell death by a mechanism involving oxidative stress and mitochondrial dysfunction. NACA had a protective role against BLM-induced toxicity by inhibiting lipid peroxidation, scavenging ROS, and preserving intracellular GSH and ΔΨm. NACA can potentially be developed into a promising adjunctive therapeutic option for patients undergoing chemotherapy with BLM.     

Proteasome inhibition induces glutathione synthesis and protects cells from oxidative stress: relevance to Parkinson disease

The cause of selective dopaminergic neuronal degeneration in Parkinson disease has still not been resolved, but it has been hypothesized that oxidative stress and the ubiquitin-proteasome system are important in the pathogenesis. In this report, we investigated the effect of proteasome inhibition on oxidative stress-induced cytotoxicity in PC12 cells, an in vitro model of Parkinson disease. Treatment with proteasome inhibitors provided significant protection against toxicity by 6-hydroxydopamine and H(2)O(2) in a concentration-dependent manner. The measurement of intracellular reactive oxygen species using 2',7'-dichlorofluorescein diacetate demonstrated that lactacystin, a proteasome inhibitor, significantly reduced 6-hydroxydopamineand H(2)O(2)-induced reactive oxygen species production. Proteasome inhibitors elevated the amount of glutathione and phosphorylated p38 mitogen-activated protein kinase (MAPK) prior to glutathione elevation. The treatment with lactacystin induced the nuclear translocation of NF-E2-related factor 2 (Nrf2) and increased the level of mRNA for gamma-glutamylcysteine synthetase, a rate-limiting enzyme in glutathione synthesis. Furthermore, SB203580, an inhibitor of p38 MAPK, abolished glutathione elevation and cytoprotection by lactacystin. These data suggest that proteasome inhibition afforded cytoprotection against oxidative stress by the elevation of glutathione content, and its elevation was mediated by p38 MAPK phosphorylation.     

N-acetylcysteine counteracts oxidative stress and protects alveolar epithelial cells from lung contusion-induced apoptosis in rats with blunt chest trauma

The aim of this study was to investigate the protective effects of N-acetylcysteine (NAC) on peroxidative and apoptotic changes in the contused lungs of rats following blunt chest trauma. The rats were randomly divided into three groups: control, contusion, and contusion + NAC. All the rats, apart from those in the control group, performed moderate lung contusion. A daily intramuscular NAC injection (150 mg/kg) was given immediately following the blunt chest trauma and was continued for two additional days following cessation of the trauma. Samples of lung tissue were taken in order to evaluate the tissue malondialdehyde (MDA) level, histopathology, and epithelial cell apoptosis using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and active caspase-3 immunostaining. In addition, we immunohistochemically evaluated the expression of surfactant protein D (SP-D) in the lung tissue. The blunt chest trauma-induced lung contusion resulted in severe histopathological injury, as well as an increase in the MDA level and in the number of cells identified on TUNEL assay together with active caspase-3 positive epithelial cells, but a decrease in the number of SP-D positive alveolar type 2 (AT-2) cells. NAC treatment effectively attenuated histopathologic, peroxidative, and apoptotic changes, as well as reducing alterations in SP-D expression in the lung tissue. These findings indicate that the beneficial effects of NAC administrated following blunt chest trauma is related to the regulation of oxidative stress and apoptosis.     

A novel neuroprotectant PAN-811 protects neurons from oxidative stress

Hydrogen peroxide (H₂O₂), a major non-radical reactive oxygen species (ROS) could elicit intracellular oxidative damage and/or cause extracellular free calcium influx by activating the NMDA receptor or through calcium channels. In the present study, NMDA receptor antagonist MK-801 fully blocked H₂O₂-induced neuronal cell death, whereas green tea (GT) extract containing-antioxidants only partially suppressed the neurotoxicity of H₂O₂. These suggest that majority of ROS overproduction is downstream of H₂O₂-induced calcium influx. A novel neuroprotectant PAN-811 was previously demonstrated to efficiently attenuate ischemic neurotoxicity. PAN-811 hereby fully blocks H₂O₂-elicited neuronal cell death with a more advanced neuroprotective profile than that of GT extract. PAN-811 was also shown to protect against CaCl₂-elicited neurotoxicity. Efficient protection against oxidative stress-induced neurotoxicity by PAN-811 indicates its potential application in treatment of ROS-mediated neurodegenerative diseases. W.P. and C.M.D. had equal contributions to this project     

Nanoparticle-mediated catalase delivery protects human neurons from oxidative stress

Several neurodegenerative diseases and brain injury involve reactive oxygen species and implicate oxidative stress in disease mechanisms. Hydrogen peroxide (H₂O₂) formation due to mitochondrial superoxide leakage perpetuates oxidative stress in neuronal injury. Catalase, an H₂O₂-degrading enzyme, thus remains an important antioxidant therapy target. However, catalase therapy is restricted by its labile nature and inadequate delivery. Here, a nanotechnology approach was evaluated using catalase-loaded, poly(lactic co-glycolic acid) nanoparticles (NPs) in human neuronal protection against oxidative damage. This study showed highly efficient catalase encapsulation capable of retaining∼99% enzymatic activity. NPs released catalase rapidly, and antioxidant activity was sustained for over a month. NP uptake in human neurons was rapid and nontoxic. Although human neurons were highly sensitive to H₂O₂, NP-mediated catalase delivery successfully protected cultured neurons from H₂O₂-induced oxidative stress. Catalase-loaded NPs significantly reduced H₂O₂-induced protein oxidation, DNA damage, mitochondrial membrane transition pore opening and loss of cell membrane integrity and restored neuronal morphology, neurite network and microtubule-associated protein-2 levels. Further, catalase-loaded NPs improved neuronal recovery from H₂O₂ pre-exposure better than free catalase, suggesting possible applications in ameliorating stroke-relevant oxidative stress. Brain targeting of catalase-loaded NPs may find wide therapeutic applications for oxidative stress-associated acute and chronic neurodegenerative disorders.     

Activation of stimulatory heterotrimeric G proteins increases glutathione and protects neuronal cells against oxidative stress

Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for cell death by oxidative stress, where an excess of extracellular glutamate inhibits import of cystine, a building block of the antioxidant glutathione. The subsequent decrease in glutathione then leads to the accumulation of reactive oxygen species (ROS) and programmed cell death. We used pharmacological compounds known to interact with heterotrimeric G-protein signalling and studied their effects on cell survival, morphology, and intracellular events that ultimately lead to cell death. Cholera toxin and phorbol esters were most effective and prevented cell death through independent pathways. Treating HT22 cells with cholera toxin attenuated the glutamate-induced accumulation of ROS and calcium influx. This was, at least in part, caused by an increase in glutathione due to improved uptake of cystine mediated by the induction of the glutamate/cystine-antiporter subunit xCT or, additionally, by the up-regulation of the antiapoptotic protein Bcl-2. Gs activation also protected HT22 cells from hydrogen peroxide or inhibition of glutathione synthesis by buthionine sulfoximine, and immature cortical neurones from oxidative glutamate toxicity. Thus, this pathway might be more generally implicated in protection from neuronal death by oxidative stress.     

Quercetin protects human granulosa cells against oxidative stress via thioredoxin system

Granulosa Cells (GCs) are sensitive to excessive production of reactive oxygen species (ROS). Quercetin (QUR) is a free radical scavenger which can alleviate oxidative stress through nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/antioxidant response element (ARE) pathway and thioredoxin (Trx) system. We aimed to explore the probable protective role of QUR on cultured human GCs treated with hydrogen peroxide (H₂O₂) as an inducer of oxidative stress. MTT assay was applied for evaluating the cell cytotoxicity of QUR and H₂O₂. The rate of apoptotic cells and intracellular ROS generation were determined by Annexin V-FITC/PI staining and 2′-7′-dichlorodihydro?uorescein diacetate ?uorescent probes (DCFH-DA), respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and western blot analysis were used to evaluate the gene and protein expression of Nrf2 and kelch-like ech-associated protein 1 (Keap1)1. The Nrf2 and Trx activities were measured by Enzyme-linked Immunosorbent Assay (ELISA). The results indicated that QUR pretreatment can decrease ROS production and apoptosis induced by H₂O₂. In addition, QUR increased Nrf2 gene and protein expression, as well as its nuclear translocation. Moreover, in QUR-treated group, a lower level of Keap1 protein was observed, which was not reported as significant. The results also indicated a significant correlation between the expression of Nrf2 and Keap1 in QUR-treated group. Further, QUR protected GCs from oxidative stress by increasing Trx gene expression and activity. This study suggests that QUR as a supplementary factor may protect GCs from oxidative stress in diseases related to this condition.     

Quercetin protects HCT116 cells from Dichlorvos-induced oxidative stress and apoptosis

The present study was designed to assess the possible protective effects of Quercetin (QUER), a flavonoid with well-known pharmacological effects, against Dichlorvos (DDVP)-induced toxicity in vitro using HCT116 cells. The cytotoxicity was monitored by cell viability, reactive oxygen species (ROS) generation, anti-oxidant enzyme activities, malondialdehyde (MDA) production, and DNA fragmentation. The apoptosis was assessed through the measurement of the mitochondrial transmembrane potential (ΔΨm) and caspase activation. The results indicated that pretreatment of HCT116 cells with QUER, 2 h prior to DDVP exposure, significantly decreased the DDVP-induced cell death, inhibited the ROS generation, modulated the activities of catalase (CAT) and superoxide dismutase (SOD), and reduced the MDA level. The reductions in mitochondrial membrane potential, DNA fragmentation, and caspase activation were also attenuated by QUER. These findings suggest that dietary QUER can protect HCT116 cells against DDVP-induced oxidative stress and apoptosis.     

Endosulfan-induced apoptosis and glutathione depletion in human peripheral blood mononuclear cells: Attenuation by N-acetylcysteine

Present study investigated whether endosulfan, an organochlorine pesticide is able to deplete glutathione (GSH) and induce apoptosis in human peripheral blood mononuclear cells (PBMC) in vitro. The role of oxidative stress in the induction of apoptosis was also evaluated by the measurement of the GSH level in cell lysate. The protective role of N-acetylcysteine (NAC) on endosulfan-induced apoptosis was also studied. Isolated human PBMC were exposed to increasing concentrations (0-100 microM) of endosulfan (alpha/beta at 70:30 mixture) alone and in combination with NAC (20 microM) up to 24 h. Apoptotic cell death was determined by Annexin-V Cy3.18 binding and DNA fragmentation assays. Cellular GSH level was measured using dithionitrobenzene. Endosulfan at low concentrations, i.e., 5 and 10 microM, did not cause significant death during 6 h/12 h incubation, whereas a concentration-dependent cell death was observed at 24 h. DNA fragmentation analysis revealed no appreciable difference between control cells and 5 microM/10 microM endosulfan treated cells, where only high molecular weight DNA band was observed. Significant ladder formation was observed at higher concentration, which is indicative of apoptotic cell death. Intracellular GSH levels decreased significantly in endosulfan-treated cells in a dose-dependent manner, showing a close correlation between oxidative stress and degree of apoptosis of PBMC. Cotreatment with NAC attenuated GSH depletion as well as apoptosis. Our results provide experimental evidence of involvement of oxidative stress in endosulfan-mediated apoptosis in human PBMC in vitro.     

Effects of N-acetylcysteine amide (NACA), a thiol antioxidant on radiation-induced cytotoxicity in Chinese hamster ovary cells

Ionizing radiation is known to cause tissue damage in biological systems, mainly due to its ability to produce reactive oxygen species (ROS) in cells. Many thiol antioxidants have been used previously as radioprotectors, but their application has been limited by their toxicity. In this investigation, we have explored the possible radioprotective effects of a newly synthesized thiol antioxidant, N-acetylcysteine amide (NACA), in comparison with N-acetylcysteine (NAC), a commonly used antioxidant. Protective effects of NACA and NAC were assessed using Chinese hamster ovary (CHO) cells, irradiated with 6 gray (Gy) radiation. Oxidative stress parameters, including levels of reduced glutathione (GSH), cysteine, malondialdehyde (MDA), and activities of antioxidant enzymes like glutathione peroxidase, glutathione reductase, and catalase, were measured. Results indicate that NACA was capable of restoring GSH levels in irradiated cells in a dose dependent manner. In addition, NACA prevented radiation-induced loss in cell viability. NACA further restored levels of malondialdehyde, caspase-3 activity, and antioxidant enzyme activities to control levels. Although NAC affected cells in a similar manner to NACA, its effects were not as significant. Further, NAC was also found to be cytotoxic to cells at higher concentrations, whereas NACA was non-toxic at similar concentrations. These results suggest that NACA may be able to attenuate radiation-induced cytotoxicity, possibly by its ability to provide thiols to cells.