Prooxidant effects of NGF withdrawal and MEK inhibition in sympathetic neurons

An increase of mitochondrial-derived reactive oxygen species (ROS) occurs in nerve growth factor (NGF)-deprived sympathetic neurons undergoing apoptotic death. It has been reported that NGF suppresses increased ROS production by the mitochondria in these cells through a mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein (MAP) kinase pathway because NGF withdrawal inactivates this pathway and the MEK inhibitor, PD98059, increases ROS in the presence of NGF. We show here that treating rat sympathetic neurons in cell culture with PD98059 greatly decreased cellular concentrations of reduced glutathione (GSH), a major cellular antioxidant. Therefore, it is likely that this inhibitor induces a cellular prooxidant state in NGF-maintained sympathetic neurons primarily by decreasing GSH concentration rather than by causing increased mitochondrial ROS production. These data suggest that the MEK/MAP kinase signaling pathway regulates cellular GSH concentration.     

ROS formation and glutathione levels in human oral fibroblasts exposed to TEGDMA and camphorquinone

Glutathione (GSH) is important for the self-protection of cells against oxidative stress and toxic xenobiotics, whereas reactive oxygen species (ROS) at elevated concentrations may cause detrimental alterations of cell membranes, DNA, and other cellular structures. The present investigation addressed the effects of triethylene-glycoldimethacrylate (TEGDMA) and camphorquinone (CQ) on glutathione metabolism and the formation of ROS in oral cells. Primary human pulp fibroblasts were exposed to various concentrations of TEGDMA and CQ (0.1-5 mM). Subsequently, GSH concentration and ROS formation were analyzed with the use of the monobromobimane assay (GSH) and 2',7'-dichlorofluorescein diacetate (DCFH-DA) (ROS). The endogenous ROS hydrogen peroxide (H2O2) was used as a positive control (0.02-2 mM). TEGDMA significantly decreased GSH at concentrations between 0.5 and 5 mM (p<0.05), but did not elevate ROS levels. Contrary, CQ increased ROS formation at concentrations>or=1 mM, but had only a moderate effect on GSH at the highest test concentration. Hydrogen peroxide increased ROS and simultaneously decreased GSH at concentrations of >or=0.2 mM. These data show that the investigated substances may cause cell damage due to various mechanisms, GSH decrease and/or ROS increase. As a consequence, TEGDMA and CQ released into an aqueous environment from resinous materials might interact, thus generating significant cytotoxic effects even at low concentrations.     

Alteration of gene expressions by the overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (mtPHGPx)

To determine the effect on gene expression of trace levels of reactive oxygen species from mitochondria, we used the mRNA differential display technique to compare gene expression in two cell lines: M15, which overexpresses mitochondrial phospholipid hydroperoxide glutathione peroxidase (mtPHGPx), in rat basophilic leukemia RBL-2H3 cells, and a control cell line, S1. We isolated 27 differentially expressed genes, including 10 previously unreported sequences. These genes included cytoskeletal proteins (beta-tubulin, nonmuscle myosin alkali light chain, and vimentin), growth or proliferation regulators [growth differentiation factor 1 (Gdf-1), Rap1a, and inhibitor of growth 3 (Ing3)], and others. Although the expression of most of the isolated genes did not respond to ROS (hydrogen peroxide) or antioxidant (pyrolidine dithiocarbamate) treatment, the expression of Gdf-1 was downregulated by hydrogen peroxide treatment. Thus, low levels of ROS produced in mitochondria during normal cellular metabolism can modulate gene expression.     

Attenuation of hyperoxia-induced growth inhibition in H441 cells by gene transfer of mitochondrially targeted glutathione reductase

Reactive oxygen species (ROS) are implicated as agents of cellular damage in pulmonary oxygen toxicity. Glutathione (GSH) and GSH-dependent antioxidant enzymes protect against damage by ROS, and recycling of glutathione disulfide (GSSG) to GSH by glutathione reductase (GR) is essential for the optimum functioning of this system. Exposure to hyperoxia inhibits lung development in newborn animals and humans, and attenuates cell growth in proliferating cell cultures. Considerable evidence supports a role for ROS as growth-altering molecules. Previously, we have observed that gene transfer of GR to mitochondria in H441 cells, using a vector containing a mitochondrial leader sequence (LGR), protected these cells against t-BuOOH-induced cytotoxicity. The present studies tested the hypothesis that gene transfer of LGR would attenuate the cytostatic effects of hyperoxia exposure in H441 cells. H441 cells (0.9 x 10(6) cells/plate) transfected with adenovirus containing LGR or the complementary DNA (cDNA) for manganese superoxide dismutase in reverse orientation (DOS) as a control construct, and untransfected cells (CON) were maintained in 21% oxygen (normoxia) or 95% oxygen (hyperoxia) for 48 h, and cell growth was assessed by cell counts and by reduction of the tetrazolium dye 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan. Cells maintained in normoxia achieved normal growth (CON, 1.98; DOS, 1.91; LGR, 2.0 x 10(6) cells/plate). Hyperoxia inhibited cell growth and the reduction of MTT; however, cells transfected with LGR had greater mitochondrial GR activities (CON, 16+/-2; DOS, 19+/-3; LGR, 322+/-18 mU/mg of protein), sustained more normal growth patterns (CON, 1.25+/-0.12; DOS, 1.24 +/-0.21, LGR, 1.8+/-0.25 x 10(6) cells/plate), and had less inhibition of MTT reduction (CON, 29; DOS, 27; LGR, 16% inhibition, P<0.01) after exposure to hyperoxia for 48 h than was observed in cells transfected with DOS or in control cells not infected with virus. In addition, resistant cells had higher mitochondrial GSH levels and maintained mitochondrial GSH/GSSG ratios in hyperoxia, suggesting that maintaining mitochondrial GSH homeostasis determined critical aspects of cell division in these studies. The mechanisms for sustaining cell growth during hyperoxia in H441 cells with enhanced mitochondrial GR activities are unknown, but similar effects in infants exposed to supplemental oxygen could be highly beneficial.     

Depletion of intracellular glutathione mediates zinc-induced cell death in rat primary astrocytes

In the present study, we investigated the possible mechanisms of cellular injury induced by zinc in rat primary astrocytes and C6 glioma cells. Reactive oxygen species (ROS) production, cellular glutathione (GSH) level and mitochondrial transmembrane potential were examined. Exposure to 200-300 microM Zn2+ for 24 h resulted in significant lactate dehydrogenase (LDH) release in rat primary astrocytes and C6 glioma cells. An exposure of 200 microM Zn2+ resulted in profound morphological changes, for example, shrunken and fragmented nuclei. Pretreatment of a protein synthesis inhibitor, cycloheximide, did not attenuate cellular toxicity induced by Zn2+. Zn2+ exposure increased intracellular ROS levels by about 250%, and depleted cellular GSH within 2 h, which preceded observable LDH release from the cell. Addition of GSH, N-acetylcysteine (NAC) and ascorbic acid substantially attenuated cellular death induced by Zn+ in a concentration dependent manner. ROS production and morphological changes induced by zinc were also inhibited by co-treatment of GSH or NAC with Zn2+. Zn2+ significantly depolarized mitochondrial transmembrane potential, which was reversed by co-treatment of GSH or NAC with zinc. In summary, ROS generation, GSH depletion and mitochondrial dysfunction may be key factors in Zn2+-induced glial toxicity.     

Protective effects of the citrus flavanones to PC12 cells against cytotoxicity induced by hydrogen peroxide

Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities. One of the plausible ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of endogenous antioxidant defense capacity. In this study, we investigated the protective actions of citrus flavanones naringin and nobiletin against the cytotoxicity induced by exposure to hydrogen peroxide (H₂O₂) (150 μM, 3 h) in PC12 cells. The results showed that naringin and nobiletin inhibited the decrease of cell viability (MTT reduction), prevented membrane damage (LDH release), scavenged ROS formation, reduced caspase-3 activity, and attenuated the decrease of mitochondrial membrane potential (MMP), respectively, in H₂O₂-induced PC12 cells. Meanwhile, naringin and nobiletin increased superoxide dismutase (SOD) and glutathione (GSH) activity, while decreased malondialdehyde (MDA), the production of lipid peroxidation, in H₂O₂-induced PC12 cells. In addition, the percentage of cells undergoing H₂O₂-induced apoptosis was decreased in the presence of naringin and nobiletin. These results first demonstrate that naringin and nobiletin, even at physiological concentrations, have neuroprotective effects against H₂O₂-induced cytotoxicity in PC12 cells. All the above results suggest that these dietary antioxidants are potential candidates for use in the intervention for neurodegenerative diseases.     

Parkinson's disease: A disorder due to nigral glutathione deficiency?

Amino acid analysis of autopsied human brain showed reduced glutathione (GSH) content significantly lower in the substantia nigra than in other brain regions. GSH was virtually absent in the nigra of patients with Parkinson's disease. Oxidative degradation of l-DOPA and dopamine in vivo may generate reactive oxygen species (hydrogen peroxide, superoxide, hydroxyl radical, or singlet oxygen) which can damage membranes and other cellular components. Since GSH is an important natural antioxidant, a deficiency of GSH in the substantia nigra could make this region vulnerable to oxidative injury. If confirmed, the hypothesis that loss of nigrostriatal dopaminergic neurons results from a regional GSH deficiency could have important therapeutic implications for the management and prevention of Parkinson's disease.     

A postulated role of garlic organosulfur compounds in prevention of valproic acid hepatotoxicity

Valproic acid (2-propyl-pentanoic acid, VPA) is well established as a first-line and widely used antiepileptic agent. VPA is well tolerated in most patients and has an impressive safety profile. VPA induced hepatotoxicity is rare, but often there is fatal complication of this drug and it is more frequent in children under 2 years of age and in those taking multiple drugs. Several findings showed that oxidative stress induced by reactive oxygen species (ROS) over production and/or compromised antioxidant capacity play an important role in the development of hepatotoxicity in VPA treated patients. Reduced glutathione (GSH) and its related enzymes are important cellular defense against oxidative stress in which VPA induced oxidative stress impairs their function in hepatocytes. Consequently any mechanism which removes ROS or prevents hepatic GSH depletion or induce activation and production of GSH dependent enzymes may provide protection for hepatotoxicity in VPA-treated patient. As garlic organosulfur compounds enhance cellular antioxidant activity by free radical scavenging and augmentation of endogenous antioxidants via prevention of GSH depletion and alteration of GSH dependent enzymes activity and/or their gene expression, we propose the hypothesis that garlic organosulfur compounds can prevent valproic acid hepatotoxicity.     

Glutathione peroxidase-1 in health and disease: from molecular mechanisms to therapeutic opportunities

Reactive oxygen species, such as superoxide and hydrogen peroxide, are generated in all cells by mitochondrial and enzymatic sources. Left unchecked, these reactive species can cause oxidative damage to DNA, proteins, and membrane lipids. Glutathione peroxidase-1 (GPx-1) is an intracellular antioxidant enzyme that enzymatically reduces hydrogen peroxide to water to limit its harmful effects. Certain reactive oxygen species, such as hydrogen peroxide, are also essential for growth factor-mediated signal transduction, mitochondrial function, and maintenance of normal thiol redox-balance. Thus, by limiting hydrogen peroxide accumulation, GPx-1 also modulates these processes. This review explores the molecular mechanisms involved in regulating the expression and function of GPx-1, with an emphasis on the role of GPx-1 in modulating cellular oxidant stress and redox-mediated responses. As a selenocysteine-containing enzyme, GPx-1 expression is subject to unique forms of regulation involving the trace mineral selenium and selenocysteine incorporation during translation. In addition, GPx-1 has been implicated in the development and prevention of many common and complex diseases, including cancer and cardiovascular disease. This review discusses the role of GPx-1 in these diseases and speculates on potential future therapies to harness the beneficial effects of this ubiquitous antioxidant enzyme.     

Evaluation of epirubicin-induced acute oxidative stress toxicity in rat liver cells and mitochondria,and the prevention of toxicity through quercetin administration

Anticancer therapy with epirubicin (EPI) results in acute hepatotoxicity, likely due to the generation of free radicals. However, the oxidative status of rat liver cells and mitochondria after EPI toxicity has not been investigated. In the present study, we first investigated the pro-oxidant effect of EPI on both hepatic cells and mitochondrial function. Injection of EPI into rats at a dose of 9 mg/kg (cumulative dose in human chemotherapy), induced hepatic dysfunction, as revealed by a significant increase in serum glutamate oxaloacetate transaminases (SGOT) and glutamate pyruvate transaminases (SGPT). Oxidative stress in liver cells and mitochondria was provoked by EPI because a statistically significant reduction of catalase (CAT), superoxide dismutase (SOD) and cytosolic glutathione (GSH) levels, and a significant increase in malonedialdehyde (MDA) levels – an indicator of lipid peroxidation that can perforate biological membranes – were observed. Second, the protective effect of quercetin (QE) (0.33 mg/kg) against EPI-induced oxidative stress was also investigated. Indeed, the pretreatment of rats with QE protected liver cells and mitochondria from oxidative stress. This treatment prevented hepatic dysfunction by maintaining normal levels of serum transaminases following the inhibition of their hepatic leakage by preventing lipid peroxidation. Thus, QE works through the prevention of cellular membrane perforation and the antioxidant defense system of mitochondria from liver cells, which represent compartments for the permanent production of reactive oxygen species (ROS) through the respiratory chain.     

Antioxidant activity of 7,8-dihydroxyflavone provides neuroprotection against glutamate-induced toxicity

Glutamate, an excitatory neurotransmitter in the central nervous system, plays an important role in neurological disorders. Previous studies have shown that excess glutamate can cause oxidative stress in a hippocampal HT-22 cell line. 7,8-Dihydroxyflavone (7,8-DHF), a member of the flavonoid family, is a selective tyrosine kinase receptor B (TrkB) agonist that has neurotrophic effects in various neurological diseases such as stroke and Parkinson's disease. In this study, we found that there is no TrkB receptor in HT-22 cells. Despite this, our data demonstrate that 7,8-DHF still protects against glutamate-induced toxicity in HT-22 cells in a concentration-dependent manner, indicating that 7,8-DHF prevents cell death through other mechanisms rather than TrkB receptors in this cell model. We further show that 7,8-DHF increases cellular glutathione levels and reduces reactive oxygen species (ROS) production caused by glutamate in HT-22 cells. Finally, our data demonstrate that 7,8-DHF protects against hydrogen peroxide and menadione-induced cell death, suggesting that 7,8-DHF has an antioxidant effect. In summary, although 7,8-DHF is considered as a selective TrkB agonist, our results demonstrate that 7,8-DHF can still confer neuroprotection against glutamate-induced toxicity in HT-22 cells via its antioxidant activity.     

Mitochondrial oxidative stress in the lungs of cystic fibrosis transmembrane conductance regulator protein mutant mice

Cystic fibrosis is a fatal genetic disorder involving dysfunction of the cystic fibrosis transmembrane regulator protein (CFTR) resulting in progressive respiratory failure. Previous studies indicate that CFTR regulates cellular glutathione (GSH) transport and that dysfunctional CFTR is associated with chronic pulmonary oxidative stress. The cause and the source of this oxidative stress remain unknown. The current study examines the role of the mitochondria in CFTR-mediated pulmonary oxidative stress. Mitochondrial GSH levels and markers of DNA and protein oxidation were assessed in the lung mitochondria from CFTR-knockout mice. In addition, in vitro models using human CFTR-sufficient and -deficient lung epithelial cells were also employed. Mitochondrial GSH levels were found to be decreased up to 85% in CFTR-knockout mice, and 43% in human lung epithelial cells deficient in CFTR. A concomitant 29% increase in the oxidation of mitochondrial DNA, and a 30% loss of aconitase activity confirmed the existence of a mitochondrial oxidative stress. Flow cytometry revealed significantly elevated levels of cellular reactive oxygen species (ROS) in CFTR-deficient human lung cells. These studies suggest that dysfunctional CFTR leads to an increase in the level of ROS and mitochondrial oxidative stress. This oxidative stress, however, appears to be a consequence of lower mitochondrial GSH levels and not increased oxidation of GSH. Further studies are needed to determine how CFTR deficiency contributes to mitochondrial oxidative stress and the role this plays in CFTR-mediated lung pathophysiology.     

The mitochondrial protein frataxin prevents nuclear damage

The mitochondrial protein frataxin helps maintain appropriate iron levels in the mitochondria of yeast and humans. A deficiency of this protein in humans causes Friedreich's ataxia, while its complete absence in yeast (Delta yfh1 mutant) results in loss of mitochondrial DNA, apparently due to radicals generated by excess iron. We found that the absence of frataxin in yeast also leads to nuclear damage, as evidenced by inducibility of a nuclear DNA damage reporter, increased chromosomal instability including recombination and mutation, and greater sensitivity to DNA-damaging agents, as well as slow growth. Addition of a human frataxin mutant did not prevent nuclear damage, although it partially complemented the Delta yfh1 mutant in preventing mitochondrial DNA loss. The effects in Delta yfh1 mutants result from reactive oxygen species (ROS), since (i) Delta yfh1 cells produce more hydrogen peroxide, (ii) the effects are alleviated by a radical scavenger and (iii) the glutathione peroxidase gene prevents an increase in mutation rates. Thus, the frataxin protein is concluded to have a protective role for the nucleus as well as the mitochondria.     

Leishmania donovani iron superoxide dismutase A is targeted to the mitochondria by its N-terminal positively charged amino acids

Many reports have shown that Leishmania species are susceptible to reactive oxygen species (ROS) and reactive nitrogen species (RNS)-mediated killing. The superoxide dismutase (SOD) is one of the antioxidant defense enzymes important for parasite survival through its detoxification of superoxide into hydrogen peroxide and oxygen. The mitochondria produce numerous superoxide radicals as a by-product of cellular respiration and hence targeting of SODs to the mitochondria is critical in maintaining healthy mitochondria. This study examines the characteristic determinants for mitochondrial localization of Leishmania donovani FeSODA. We show that FeSODA is localized to the mitochondria and that the N-terminal 31 amino acid extension is important for its localization. Interestingly, further dissection of the 31 amino acid extension revealed that the first 8 amino acids of the FeSODA protein are sufficient for targeting to the mitochondria. In addition, we found that the four basic amino acid residues contained within the N-terminal extension are also important for targeting. These studies highlight important features of mitochondrial targeting sequences in kinetoplastids.     

病毒硫氧还蛋白基因在Neuro2A细胞中的重组与表达

背景：目前对抗氧化基因硫氧还蛋白的研究逐步受到重视,但从基因治疗的角度对其研究较少。 目的：采用硫氧还蛋白转染Neuro-2A细胞,观察细胞内表达相应蛋白因子对细胞的具体保护效果,并分析其发挥保护作用的可能机制。 方法：以质粒PIRES2-EGFP-TRX转染Neuro-2A细胞,经RT-PCR鉴定,建立能够稳定表达硫氧还蛋白的细胞株。利用不同浓度的H₂O₂处理正常细胞及转染细胞,建立氧化应激模型。观察受到氧化损伤后两组细胞的形态学、存活率、还原型谷胱甘肽的浓度及细胞内DNA链断裂情况。 结果与结论：经H₂O₂作用后,两组细胞形态均出现损伤,但转染组细胞比正常组细胞损伤减轻、细胞存活率升高、细胞内还原型谷胱甘肽水平增高、DNA链断裂程度减轻。说明人类硫氧还蛋白基因可以在Neuro-2A细胞中被重组并顺利表达,对细胞具有一定的保护作用;这一作用可能是通过清除氧自由基,维持细胞内还原型谷胱甘肽水平,从而保护细胞DNA免受氧化性损伤来实现的。     

(R)-alpha-lipoic acid reverses the age-associated increase in susceptibility of hepatocytes to tert-butylhydroperoxide both in vitro and in vivo

Hepatocytes were isolated from young (3-5 months) and old (24-28 months) rats and incubated with various concentrations of tert-butylhydroperoxide (t-BuOOH). The t-BuOOH concentration that killed 50% of cells (LC50) in 2 hr declined nearly two-fold from 721 +/- 32 microM in cells from young rats to 391 +/- 31 microM in cells from old rats. This increased sensitivity of hepatocytes from old rats may be due, in part, to changes in glutathione (GSH) levels, because total cellular and mitochondrial GSH were 37.7% and 58.3% lower, respectively, compared to cells from young rats. Cells from old animals were incubated with either (R)- or (S)-lipoic acid (100 microM) for 30 min prior to the addition of 300 microM t-BuOOH. The physiologically relevant (R)-form, a coenzyme in mitochondria, as opposed to the (S)-form significantly protected hepatocytes against t-BuOOH toxicity. Dietary supplementation of (R)-lipoic acid [0.5% (wt/wt)] for 2 weeks also completely reversed the age-related decline in hepatocellular GSH levels and the increased vulnerability to t-BuOOH as well. An identical supplemental diet fed to young rats did not enhance the resistance to t-BuOOH, indicating that antioxidant protection was already optimal in young rats. Thus, this study shows that cells from old animals are more susceptible to oxidant insult and (R)-lipoic acid, after reduction to an antioxidant in the mitochondria, effectively reverses this age-related increase in oxidant vulnerability.     

Effect of oltipraz on the susceptibility of adultSchistosoma mansoni to killing by mouse peritoneal exudate cells

Incubation of the adultSchistosoma mansoni with the anti-schistosomal compound oltipraz (OPZ) (40 nM) resulted in a significant decrease in schistosome-reduced glutathione (GSH), a thiol compound which may have a role in protection against oxidant-mediated damage. A significant proportion (20–47%) of worms treated with OPZ became susceptible to in vitro killing by zymosan-stimulated peritoneal exudate cells from mice infected withS. mansoni or inoculated with Bacillus Calmette Guérin (BCG). Killing of the worms was partially inhibited by the addition to the assay system of exogenous glutathione peroxidase with GSH but not by superoxide dismutase. These results suggested that killing of parasites exposed to the drug was partly mediated by cell-generated hydrogen peroxide. They indicate also that depletion of schistosome GSH levels could render the parasites susceptible to killing by oxidative mechanisms, and suggest that there is potential in exploiting schistosome oxidant defense systems and reactive oxygen byproducts in the treatment of schistosomiasis. Inhibition of schistosome oxidant defense systems with drugs may render the parasites susceptible to killing by reactive oxygen byproducts of effector cells.     

Mitochondrial inhibition and oxidative stress: reciprocating players in neurodegeneration

Although the etiology for many neurodegenerative diseases is unknown, the common findings of mitochondrial defects and oxidative damage posit these events as contributing factors. The temporal conundrum of whether mitochondrial defects lead to enhanced reactive oxygen species generation, or conversely, if oxidative stress is the underlying cause of the mitochondrial defects remains enigmatic. This review focuses on evidence to show that either event can lead to the evolution of the other with subsequent neuronal cell loss. Glutathione is a major antioxidant system used by cells and mitochondria for protection and is altered in a number of neurodegenerative and neuropathological conditions. This review also addresses the multiple roles for glutathione during mitochondrial inhibition or oxidative stress. Protein aggregation and inclusions are hallmarks of a number of neurodegenerative diseases. Recent evidence that links protein aggregation to oxidative stress and mitochondrial dysfunction will also be examined. Lastly, current therapies that target mitochondrial dysfunction or oxidative stress are discussed.     

Glutathione peroxidase 1 protects mitochondria against hypoxia/reoxygenation damage in mouse hearts

Glutathione peroxidase 1 (GPx1) plays an important role in preventing cardiac dysfunction following ischemia-reperfusion injury. However, its role in protecting cardiac mitochondria against reoxygenation-induced reactive oxygen species (ROS) generation in vivo is unclear. We examined the role of GPx1 in protecting cardiac mitochondria against hypoxia–reoxygenation (HR) damage by testing for alterations in cardiac mitochondrial function. We used a two-dimensional gel electrophoresis proteomics analysis to examine the effects of reoxygenation on cardiac protein in wild-type (GPx1^+/+) and GPx1 knockout (GPx1^?/?) mouse hearts. We identified 42 protein spots showing differential expression in the two groups. Sixteen of the proteins identified were located in mitochondria and were involved in a number of key metabolic pathways. To verify our proteomics findings functionally, we performed NADH autofluorescence measurements and ATP production assays. The reduced expression of oxidative phosphorylation proteins in GPx1^?/? mice following HR treatment resulted in loss of the mitochondrial membrane potential and decreased mitochondrial respiration. Mitochondrial ROS production and oxidative mtDNA damage were increased markedly during reoxygenation in GPx1^?/? hearts. We also found morphological abnormalities in cardiac mitochondria and myocytes in HR-treated GPx1^?/?. This is the first report of the role of GPx1 in protecting cardiac mitochondria against reoxygenation damage in vivo. These findings will help clarify the mechanisms of HR injury and will aid in the development of antioxidant therapies to prevent cardiac mitochondrial dysfunction associated with reoxygenation.     

Fraxetin prevents rotenone-induced apoptosis by induction of endogenous glutathione in human neuroblastoma cells

Fraxetin belongs to an extensive group of natural phenolic anti-oxidants. In the present study, using a human neuroblastoma SH-SY5Y cells, we have investigated the protective effects of this compound on modifications in endogenous reduced glutathione (GSH), intracellular oxygen species (ROS) and apoptotic death on rotenone-mediated cytoxicity. Incubation of cells with the fraxetin led to a significant elevation dose-dependent of cellular GSH and this was accompanied by a marked protection against rotenone-mediated toxicity, which was also significantly reversed in the cells with buthionine sulfoximine (BSO) co-treatment. Taken together, this study suggested that intracellular GSH appeared to be an important factor in fraxetin-mediated cytoprotection against rotenone-toxicity in SH-SY5Y cells. Fraxetin at 10-100 muM inhibited the formation of ROS, cytochrome c release, activation of caspase-3 and 9, and suppressed the up-regulation of Bax, whereas no significant change occurred in Bcl-2 levels. Our results indicated that the anti-oxidative and anti-apoptotic properties render this natural compound potentially protective against rotenone-induced cytotoxicity.