Human polynucleotide phosphorylase reduces oxidative RNA damage and protects HeLa cell against oxidative stress

We examined HeLa cell viability and RNA oxidative damage in response to hydrogen peroxide (H₂O₂) treatment. The level of damaged RNA, measured by the content of 8-hydroxyguanosine (7,8-dihydro-8-oxoguanosine, 8-oxoG), increases depending on H₂O₂ dosage and is inversely correlated with cell viability. The elevated level of 8-oxoG in RNA decreases after removal of oxidative challenge, suggesting the existence of surveillance mechanism(s) for cleaning up oxidized RNA. Human polynucleotide phosphorylase (hPNPase), an exoribonuclease primarily located in mitochondria, has been previously shown to bind 8-oxoG-RNA with high affinity. The role of hPNPase in HeLa cell under oxidative stress conditions is examined here. Overexpression of hPNPase reduces RNA oxidation and increases cell viability against H₂O₂ insult. Conversely, hPNPase knockdown decreases viability and increases 8-oxoG level in HeLa cell exposed to H₂O₂. Our results suggest that hPNPase plays an important role in protecting cells and limiting damaged RNA under oxidative stress.     

Resveratrol ameliorates oxidative DNA damage and protects against acrylamide-induced oxidative stress in rats

Acrylamide (ACR), used in many fields from industrial manufacturing to laboratory personnel work is also formed during the heating process through interactions of amino acids. Therefore ACR poses a significant risk to human health. This study aimed to elucidate whether resveratrol (RVT) treatment could modulate ACR-induced oxidative DNA damage and oxidative changes in rat brain, lung, liver, kidney and testes tissues. Rats were divided into four groups as control (C); RVT (30 mg/kg i.p. dissolved in 0.9% NaCl), ACR (40 mg/kg i.p.) and RVT + ACR groups. After 10 days rats were decapitated and tissues were excised. 8-hydroxydeoxyguanosine (8-OHdG) is a biomarker of oxidative DNA damage. 8-OHdG content in the extracted DNA solution was determined by enzyme-linked immunosorbent assay method. Malondialdehyde (MDA), glutathione (GSH) levels and myeloperoxidase activity (MPO) were determined in tissues, while oxidant-induced tissue fibrosis was determined by collagen contents. Serum enzyme activities, cytokine levels, leukocyte apoptosis were assayed in plasma. As an indicator of oxidative DNA damage, 8-OHdG levels significantly increased in ACR group and this was reversed significantly by RVT treatment. In ACR group, GSH levels decreased significantly while the MDA levels, MPO activity and collagen content increased in the tissues suggesting oxidative organ damage. In RVT-treated ACR group, oxidant responses reversed significantly. Serum enzyme activities, cytokine levels and leukocyte late apoptosis which increased following ACR administration, decreased with RVT treatment. Therefore supplementing with RVT can be useful in individuals at risk of ACR toxicity.     

Intraperitoneal gardiquimod protects against hepatotoxicity through inhibition of oxidative stress and inflammation in mice with sepsis

Many reports recapitulate the contribution of reactive oxygen species (ROS) over‐accumulation to the organ damage; it is of significance to strictly target ROS production. In this study, we evaluated the potential role of TLR7 agonist gardiquimod (GDQ) in oxidative stress (OS) in liver injury induced by sepsis. Here, we observed that intraperitoneal pretreatment with GDQ dramatically elevated the septic survival rate and effectively attenuated the septic liver injury. Interestingly, the increased ROS and inflammatory factor IL‐6 levels were reversed after GDQ intervention. Subsequently, Western blot was employed to determine the definite mechanism. As expected, it was showed that the upregulation of c‐Jun N‐terminal kinase (JNK)/c‐Jun pathway in liver of septic animals was considerably suppressed by GDQ pre‐exposure. Our current result highlight that pre‐administration of GDQ ameliorated sepsis induced hepatotoxicity and reduced the generation of IL‐6 and OS responses, which was associated with downregulation of JNK/c‐Jun pathway. Our strategies might be ultimately beneficial in mitigating liver injury symptom.     

PUMA inactivation protects against oxidative stress through p21/Bcl-XL inhibition of bax death

The tumor suppressor protein p53 activates growth arrest and proapoptotic genes in response to DNA damage. It is known that negative feedback by p21(Cip1/Waf1/Sdi1) represses p53-dependent transactivation of PUMA. The current study investigates PUMA feedback on p53 during oxidative stress from hyperoxia and the subsequent effects on cell survival mediated through p21 and Bcl-X(L). Deletion of PUMA in HCT116 colon carcinoma cells increased levels of p53 and p21, resulting in a larger G(1) population during hyperoxia. P21-dependent increase in Bcl-X(L) levels protected PUMA-deficient cells against hyperoxic cell death. Bax and Bak were both able to promote hyperoxic cell death. Bcl-X(L) protection against hyperoxic death was lost in cells lacking Bax, not PUMA, suggesting that Bcl-X(L) acts to inhibit Bax-dependent death. These results indicate that PUMA exerts a negative feedback on p53 and p21, leading to p21-dependent growth suppressive and survival changes. Enhanced survival was associated with increased Bcl-X(L) to block Bax activated cell death during oxidative stress.     

ARC protects rat cardiomyocytes against oxidative stress through inhibition of caspase-2 mediated mitochondrial pathway

Apoptosis repressor with a CARD domain (ARC) has been demonstrated to protect heart cells against ischemia/reperfusion (I/R) injury. In this study, we investigated the mechanism by which ARC protects heart cells against oxidative stress. We monitored the extent of apoptosis and activity of multiple components of the intrinsic apoptotic pathway in rat cardiac myoblast cell line H9c2 with either reduced or increased expression of ARC during oxidative stress. Overexpression of ARC-inhibited oxidative stress-induced caspase-2/3 activation, cytochrome c release, and translocation of Bax to mitochondria. Furthermore, phosphorylation of ARC at threonine 149 was found to be critical to its function. ARC containing a T149A mutation failed to translocate to mitochondria, did not inhibit caspase-2 activation, and had a dominant negative effect against the protective effect of endogenous ARC during oxidative stress. In addition, wild-type ARC but not the T149A mutant inhibited cell death induced by overexpression of caspase-2. Using a yeast two-hybrid (YTH) screening approach and co-immunoprecipitation (Co-IP), we found that protein phosphatase 2C (PP2C) interacted with ARC and that PP2C mediated-dephosphorylation of ARC inhibited its anti-apoptotic activity. Eliminating either the N-terminal CARD domain or the C-terminal P/E domain also abolished the anti-apoptotic function of ARC, suggesting that full-length ARC is required for its apoptotic inhibition. These results indicate that ARC plays an important role in protection of H9c2 cells against oxidative stress-induced apoptosis by phosphorylation-dependent suppression of the mitochondria-mediated intrinsic pathway, partially initiated through the activation of caspase-2.     

Green tea metabolite EGCG protects membranes against oxidative damage in vitro

Green tea polyphenols like epigallocatechin gallate (EGCG) have been proposed as a cancer chemopreventative. Several studies have shown that EGCG can act as an antioxidant by trapping proxyl radicals and inhibiting lipid peroxidation. The main propose of this study is to investigate the antioxidant capacity of EGCG using erythrocyte membrane-bound ATPases as a model. The effects of EGCG on t-butylhydroperoxide-induced lipid peroxidation and the activity of membrane-bound ATPases in human erythrocyte membranes were studied. The extent of oxidative damage in membranes was assessed by measuring lipid peroxidation, (TBARS, thiobarbituric acid reactive substances formation) and the activity of ATPases (Na(+)/K(+), Ca(2+), and CaM-activated Ca(2+) pump ATPases). EGCG blocked t-BHP induced lipid peroxidation in erythrocyte membranes, significantly (0.45 +/- 0.02 vs 0.20 +/- 0.01; t-BHP vs t-BHP + EGCG respectively, microm/L TBARS) (p < 0.05). EGCG also protected ATPases against t-BHP induced damage; for Na/K ATPase (2.4 +/- 0.2 vs 1.6 +/- 0.1 vs 2.44 +/- 0.2, nmol Pi/min/mg protein, control vs t-BHP vs t-BHP and EGCG respectively), for Ca ATPase (5.8 +/- 0.4 vs 3.9 +/- 0.3 vs 5.6 +/- 0.34, nmol Pi/min/mg protein, control vs t-BHP vs t-BHP and EGCG respectively) and for CaM-Ca ATPase (14.7 +/- 0.7 vs 7.3 +/- 0.4 vs 11.6 +/- 0.55, nmol Pi/min/mg protein, control vs t-BHP vs t-BHP and EGCG respectively) (p < 0.05). In conclusion our results indicate that EGCG is a powerful antioxidant that is capable protecting erythrocyte membrane-bound ATPases against oxidative stress.     

Quercetin protects mouse oocytes against chromium-induced damage in vitro and in vivo

BackgroundChromium (Cr) is a naturally-occurring element that is used in various fields of industry. Humans may be exposed to hexavalent chromium [Cr(VI)], which is one of the stable valence states of the chromium through contaminated soil, air, and water. Exposure to Cr(VI) through contaminated drinking water, soil and air causes various cancers and also fertility problems in animals and humans. Quercetin (QCT), a common flavonoid compound, has numerous biological effects as an antioxidant and free radical scavenger, but its function and mechanisms in reproductive processes in various species remain unclear. This study aims to determine the chromium effects on mice oocyte quality and the ameliorative effect of QCT in both in vitro and in vivo experimental models.MethodsFor the in vitro experiment, oocytes were collected and divided into the control, sham, QCT-treated, Cr(VI) (potassium dichromate), and treatment [Cr(VI)+QCT] groups. Collected oocytes were cultured in maturation medium with or without 10 µM quercetin and 10 µM Cr(VI) for 14 h based on the defined experimental design. For the in vivo experiment, the mice were randomly divided into the control, sham, QCT-treated, Cr(VI), and Cr(VI) + QCT groups. Control and sham mice received regular drinking water and diet. Cr(VI) group received Cr(VI) (50 ppm in drinking water) and Cr(VI) + QCT group received 50 ppm Cr(VI) with QCT (20 mg/kg body wt, through i.p) for a period of 21 days and then oocytes were collected and cultured for 14 h for in vitro maturation. For both experiments, at the end of the culture period, we examined the ameliorative effect of QCT on oocyte maturation, spindle formation, ROS production, mitochondrial function, and apoptosis.ResultsOur in vitro and in vivo results showed that Cr(VI) disrupt the oocyte maturation and spindle formation (P < 0.001). Furthermore, we found that exposure to Cr(VI) significantly increased ROS levels and decreased mitochondrial membrane potential (P < 0.001). In addition, exposure to Cr(VI) induced early apoptosis and downregulated the Bcl-2 mRNA expression and upregulated the Caspase-3 and Bax mRNAs expression (P < 0.01). Finally, quercetin significantly restored the detrimental effects of Cr(VI).ConclusionThe results indicated that quercetin protects the oocytes against Cr(VI) toxicity through the suppression of oxidative stress and apoptosis. The conclusions drawn from our study's findings suggest that quercetin might be useful agent for oocyte maturation in case of possible exposure to toxic substances such as chromium.     

Caspase inhibition protects against reovirus-induced myocardial injury in vitro and in vivo

Viral myocarditis is a disease with a high morbidity and mortality. The pathogenesis of this disease remains poorly characterized, with components of both direct virus-mediated and secondary inflammatory and immune responses contributing to disease. Apoptosis has increasingly been viewed as an important mechanism of myocardial injury in noninfectious models of cardiac disease, including ischemia and failure. Using a reovirus murine model of viral myocarditis, we characterized and targeted apoptosis as a key mechanism of virus-associated myocardial injury in vitro and in vivo. We demonstrated caspase-3 activation, in conjunction with terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and annexin binding, in cardiac myocytes after myocarditic viral infection in vitro. We also demonstrated a tight temporal and geographical correlation between caspase-3 activation, histologic injury, and viral load in cardiac tissue after myocarditic viral infection in vivo. Two pharmacologic agents that broadly inhibit caspase activity, Q-VD-OPH and Z-VAD(OMe)-FMK, effectively inhibited virus-induced cellular death in vitro. The inhibition of caspase activity in vivo by the use of pharmacologic agents as well as genetic manipulation reduced virus-induced myocardial injury by 40 to 60% and dramatically improved survival in infected caspase-3-deficient animals. This study indicates that apoptosis plays a critical role in mediating cardiac injury in the setting of viral myocarditis and is the first demonstration that caspase inhibition may serve as a novel therapeutic strategy for this devastating disease.     

Catechin protects against ketoprofen-induced oxidative damage of the gastric mucosa by up-regulating Nrf2 in vitro and in vivo

Nonsteroidal anti-inflammatory drugs (NSAIDs), including ketoprofen, are widely used in clinical medicine. However, these drugs may damage the gastrointestinal mucosa. Some reports have suggested that intestinal diseases, such as ulcers, are associated with lipid peroxidation and oxidative damage in the mucosa. Phytochemicals, such as polyphenols, are common dietary antioxidants that possess many beneficial characteristics, such as antioxidant and anti-inflammatory capabilities. The objective of this study was to investigate the protective effects of polyphenols on ketoprofen-induced oxidative damage in the gastrointestinal mucosa. We evaluated the effects of catechin, theaflavin, malvidin, cyanidin and apigenin on the activity of antioxidant enzymes in human intestinal-407 (Int-407) cells and rat primary gastric cells treated with ketoprofen. The results indicated that catechin significantly (P<.05) decreased the levels of lipid peroxidation (40.5%) and reactive oxygen species (30.0%), and increased the activity of intracellular antioxidant enzymes glutathione peroxidase, glutathione reductase and total sulfhydryl groups. More importantly, the treatment of Sprague–Dawley rats with catechin (35 mg/kg/day) prior to the administration of ketoprofen (50 mg/kg/day) successfully inhibited oxidative damage and reversed the impairment of the antioxidant system in the intestinal mucosa. Western blot analysis revealed that catechin stimulated a time-dependent increase in both the nuclear factor erythroid 2-related factor 2 and total heme oxygenase-1 protein expression in Int-407 cells. These results suggest that catechin may have a protective effect on gastrointestinal ulcers.     

Calmodulin kinase II inhibition protects against myocardial cell apoptosis in vivo

Inhibition of the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) or depletion of sarcoplasmic reticulum (SR) Ca(2+) stores protects against apoptosis from excessive isoproterenol (Iso) stimulation in cultured ventricular myocytes, suggesting that CaMKII inhibition could be a novel approach to reducing cell death in conditions of increased adrenergic tone, such as myocardial infarction (MI), in vivo. We used mice with genetic myocardial CaMKII inhibition due to transgenic expression of a highly specific CaMKII inhibitory peptide (AC3-I) to test whether CaMKII was important for apoptosis in vivo. A second line of mice expressed a scrambled, inactive form of AC3-I (AC3-C). AC3-C and wild-type (WT) littermates were used as controls. AC3-I mice have reduced SR Ca(2+) content and are resistant to Iso- and MI-induced apoptosis compared with AC3-C and WT mice. Phospholamban (PLN) is a target for modulation of SR Ca(2+) content by CaMKII. PLN(-/-) mice have increased susceptibility to Iso-induced apoptosis. Verapamil pretreatment prevented Iso-induced apoptosis in PLN(-/-) mice, indicating the involvement of a Ca(2+)-dependent pathway. AC3-I and AC3-C mice were bred into a PLN(-/-) background. Loss of PLN increased and equalized SR Ca(2+) content in AC3-I, AC3-C, and WT mice and abolished the resistance to apoptosis in AC3-I mice after MI. There was a trend (P = 0.07) for increased Iso-induced apoptosis in AC3-I mice lacking PLN compared with AC3-I mice with PLN. These findings indicate CaMKII is proapoptotic in vivo and suggest that regulation of SR Ca(2+) content by PLN contributes to the antiapoptotic mechanism of CaMKII inhibition.     

Naringenin protects RPE cells from NaIO3-induced oxidative damage in vivo and in vitro through up-regulation of SIRT1

BackgroundDry age-related macular degeneration (dAMD) leads to serious burden of visual impairment and there is no definitive treatment. Previous studies have showed that naringenin (NAR) significantly increased electroretinography (ERG) c-wave in sodium iodate (NaIO₃)-treated rats and viability of NaIO₃-treated ARPE-19 cells. But the underlying mechanism is still unknown.PurposeWe tested the hypothesis that anti-oxidation mediated by Sirtuin 1 (SIRT1) was important to the protective effect of NAR on dAMD.Study design/MethodsNaIO₃-induced mice retinopathy and ARPE-19 cells injury models were established. In vivo, the protective effect of NAR eye drops on retina was evaluated by flash ERG (FERG) recording and histopathological examination. In vitro, viability of ARPE-19 cells, and the levels of lactic dehydrogenase (LDH), reactive oxygen species (ROS) and carbonyl protein were detected. Protein expression of SIRT1 was analyzed by immunochemical staining, immunofluorescence and western blotting.ResultsNAR eye drops improved retinal function and morphology and normalized the protein expression of SIRT1 in mice exposed to NaIO₃. NAR promoted the survival of ARPE-19 cells in a concentration-dependent manner. NAR up-regulated SIRT1 protein expression, and decreased levels of ROS and carbonyl protein. Moreover, EX527, a selective inhibitor of SIRT1, abolished the effects of NAR on the cell viability and ROS. In addition, SRT1720, a selective agonist of SIRT1, improved the viability of cells and suppressed the production of ROS.ConclusionOur findings indicate that SIRT1-mediated anti-oxidation contributes to the protective effect of NAR eye drops on dAMD.     

adapt78 protects cells against stress damage and suppresses cell growth

We have previously identified several genes whose RNA products are induced in HA-1 hamster cells under conditions where a cytoprotective adaptive response is observed. One of these genes, designated adapt78, was found to have a human homolog with some homology to glucose-regulated protein 78 (Grp78). We subsequently determined that adapt78 and grp78 mRNAs are induced by the same stress agents and conclude that adapt78 is a stress-response gene and putative new member of the grp stress gene family. Here we extend these studies to assess the effect of overexpressing adapt78 on stress protection and growth arrest. HA-1 cells stably transfected with adapt78 cDNA were found to exhibit significantly reduced calcium- and hydrogen peroxide-mediated cytotoxicity as compared with control transfectants. In addition, adapt78 stable overexpressors exhibited significantly reduced cell growth. Both cytoprotection and growth arrest accompanied only modest overexpression of adapt78. Flow cytometry revealed that the growth arrest occurred in G(1)-phase. Immunoflourescent analysis revealed that Adapt78 protein exhibits significant perinuclear staining suggestive of endoplasmic reticulum localization in addition to cytoplasmic localization. These data indicate that adapt78 is both cytoprotective and growth suppressive and that these effects may be mediated by Adapt78 protein at the endoplasmic reticulum.     

Metallothionein protection against alcoholic liver injury through inhibition of oxidative stress

Antioxidants are likely potential pharmaceutical agents for the treatment of alcoholic liver disease. Metallothionein (MT) is a cysteine-rich protein and functions as an antioxidant. This study was designed to determine whether MT confers resistance to acute alcohol-induced hepatotoxicity and to explore the mechanistic link between oxidative stress and alcoholic liver injury. MT-overexpressing transgenic and wild-type mice were administrated three gastric doses of alcohol at 5 g/kg. Liver injury, oxidative stress, and ethanol metabolism-associated changes were determined. Acute ethanol administration in the wild-type mice caused prominent microvesicular steatosis, along with necrosis and elevation of serum alanine aminotransferase. Ultrastructural changes of the hepatocytes include glycogen and fat accumulation, organelle abnormality, and focal cytoplasmic degeneration. This acute alcohol hepatotoxicity was significantly inhibited in the MT-transgenic mice. Furthermore, ethanol treatment decreased hepatic-reduced glutathione, but increased oxidized glutathione along with lipid peroxidation, protein oxidation, and superoxide generation in the wild-type mice. This hepatic oxidative stress was significantly suppressed in the MT-transgenic mice. However, MT did not affect the ethanol metabolism-associated decrease in NAD(+)/NADH ratio or increase in cytochrome P450 2E1. In conclusion, MT is an effective agent in cytoprotection against alcohol-induced liver injury, and hepatic protection by MT is likely through inhibition of alcohol-induced oxidative stress.     

The stingless bee honey protects against hydrogen peroxide-induced oxidative damage and lipopolysaccharide-induced inflammation in vitro

Oxidative stress, DNA damage, and unresolved inflammation are the predisposing factors of many chronic and degenerative diseases, including cancer. Stingless bee honey (SBH) is recognized to have high medicinal value by traditional medicine practitioners and has been used to treat various illnesses traditionally. This study aimed to determine the antioxidant, anti-inflammatory, and genoprotective effects of SBH by using in vitro cell culture models. The sugar content, total phenolic content, radical scavenging activity, and ferric reducing antioxidant power (FRAP) of SBH were determined in this study. Then, the protective effect of SBH against hydrogen peroxide (H₂O₂)-induced cell death and DNA damage was studied by using WIL2-NS human lymphoblastoid cell line, while the lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophages cell line was used to study the anti-inflammatory effects of SBH. Results from this present study showed that the major sugar contents of SBH were fructose (19.39 + 0.01%) and glucose (14.03 ± 0.03%). Besides, the total phenolic content, the radical scavenging activity, and the FRAP value of SBH were 15.38 ± 0.02 mg GAE/100 g of honey, 34.04 ± 0.21%, and 206.77 + 1.76 μM AAE/100 g honey respectively. Pretreatment with SBH protected WIL2-NS cells from H₂O₂-induced cell death and DNA damage (p < 0.001). Moreover, SBH was also able to attenuate the production of nitric oxide by inhibiting the expression of inducible nitric oxide synthase in LPS-induced RAW 264.7 cells (p < 0.001). In conclusion, SBH is rich in total phenolic content and possesses strong antioxidant, anti-inflammatory, and genoprotective properties. Our current findings suggest that SBH might be useful in the prevention and treatment of many diseases caused by oxidative stress and inflammation assuming the observed effects are also achievable in vivo.     

Scoparia dulcis, a traditional antidiabetic plant, protects against streptozotocin induced oxidative stress and apoptosis in vitro and in vivo

Oxidative stress is implicated in the pathogenesis of diabetic complications. The experiments were performed on normal and experimental male Wistar rats treated with Scoparia dulcis plant extract (SPEt). The effect of SPEt was tested on streptozotocin (STZ) treated Rat insulinoma cell lines (RINm5F cells) and isolated islets in vitro. Administration of an aqueous extract of Scoparia dulcis by intragastric intubation (po) at a dose of 200 mg/kg body weight significantly decreased the blood glucose and lipid peroxidative marker thiobarbituric acid reactive substances (TBARS) with significant increase in the activities of plasma insulin, pancreatic superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) in streptozotocin diabetic rats at the end of 15 days treatment. Streptozotocin at a dose of 10 mug/mL evoked 6-fold stimulation of insulin secretion from isolated islets indicating its insulin secretagogue activity. The extract markedly reduced the STZ-induced lipidperoxidation in RINm5F cells. Further, SPEt protected STZ-mediated cytotoxicity and nitric oxide (NO) production in RINm5F cells. Treatment of RINm5F cells with 5 mM STZ and 10 mug of SPEt completely abrogated apoptosis induced by STZ, suggesting the involvement of oxidative stress. Flow cytometric assessment on the level of intracellular peroxides using fluorescent probe 2'7'-dichlorofluorescein diacetate (DCF-DA) confirmed that STZ (46%) induced an intracellular oxidative stress in RINm5F cells, which was suppressed by SPEt (21%). In addition, SPEt also reduced (33%) the STZ-induced apoptosis (72%) in RINm5F cells indicating the mode of protection of SPEt on RIN m5Fcells, islets, and pancreatic beta-cell mass (histopathological observations). Present study thus confirms antihyperglycemic effect of SPEt and also demonstrated the consistently strong antioxidant properties of Scoparia dulcis used in the traditional medicine.     

Mitochondrial glutathione protects against cell death induced by oxidative and nitrative stress in astrocytes

The major cellular antioxidant, glutathione, is mostly localized in the cytosol but a small portion is found in mitochondria. We have recently shown that highly selective depletion of mitochondrial glutathione in astrocytes in culture markedly increased cell death induced by the peroxynitrite donor, 3-morpholino-syndnonimine. The present study was aimed at characterizing the increase in susceptibility arising from mitochondrial glutathione loss and testing the possibility that elevating this metabolite pool above normal values could be protective. The increased vulnerability of astrocytes with depleted mitochondrial glutathione to Sin-1 was confirmed. Furthermore, these cells showed marked increases in sensitivity to hydrogen peroxide and also to high concentrations of the nitric oxide donor, S-nitroso-N-acetyl-penicillamine. The increase in cell death was mostly due to necrosis as indicated by substantially increased release of lactate dehydrogenase and staining of nuclei with propidium iodide but little change in annexin V staining and caspase 3 activation. The enhanced cell loss was blocked by prior restoration of the mitochondrial glutathione content. It was also essentially fully inhibited by treatment with cyclosporin A, consistent with a role for the mitochondrial permeability transition in the development of cell death. Susceptibility to the classical apoptosis inducer, staurosporine, was only affected to a small extent in contrast to the response to the other substances tested. Incubation of normal astrocytes with glutathione monoethylester produced large and long-lasting increases in mitochondrial glutathione content with much smaller effects on the cytosolic glutathione pool. This treatment reduced cell death on exposure to 3-morpholino-syndnonimine or hydrogen peroxide but not S-nitroso-N-acetyl-pencillamine or staurosporine. These findings provide evidence for an important role for mitochondrial glutathione in preserving cell viability during periods of oxidative or nitrative stress and indicate that increases in this glutathione pool can confer protection against some of these stressors.     

Effect of chlorophyllin against oxidative stress in splenic lymphocytes in vitro and in vivo

Chlorophyllin (CHL) has been examined as an antioxidant/radioprotector in splenic lymphocytes from BALB/c mice. CHL inhibited lipid peroxidation induced by 2,2'-azobis(2-propionimidinedihydrochloride) (AAPH) in lymphocytes in vitro. It also partially prevented radiation-induced suppression of mitogenic stimulation of lymphocytes in vitro. Generation of intracellular reactive oxygen species (ROS) by radiation or AAPH was measured as oxidation of dichlorodihydrofluorescein diacetate (H(2)DCF-DA) using flow cytometry. Addition of CHL to lymphocytes in vitro significantly inhibited the increase in intracellular ROS. Further, lymphocytes from mice treated with CHL (100-400 microg/gbw i. p.) showed varying levels of ROS depending on the dose and the time (24 to 72 h) after injection. The extent of radiation-induced apoptosis and suppression of concanavalin A (con A)-induced mitogenesis ex vivo corresponded with changes in ROS levels in CHL-administered mice. Antioxidant enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX) were also estimated in lymphocytes from CHL-treated mice. CHL offered protection against whole body irradiation (WBI)-induced lipid peroxidation and apoptosis in lymphocytes at all the time points studied. These results demonstrate antioxidant effect of CHL in vivo.     

Poly(ADP-ribose) polymerase-1 protects neurons against apoptosis induced by oxidative stress

In neurons, DNA is prone to free radical damage, although repair mechanisms preserve the genomic integrity. However, activation of the DNA repair system, poly(ADP-ribose) polymerase (PARP-1), is thought to cause neuronal death through NAD+ depletion and mitochondrial membrane potential (delta psi(m)) depolarization. Here, we show that abolishing PARP-1 activity in primary cortical neurons can either enhance or prevent apoptotic death, depending on the intensity of an oxidative stress. Only in severe oxidative stress does PARP-1 activation result in NAD+ and ATP depletion and neuronal death. To investigate the role of PARP-1 in an endogenous model of oxidative stress, we used an RNA interference (RNAi) strategy to specifically knock down glutamate-cysteine ligase (GCL), the rate-limiting enzyme of glutathione biosynthesis. GCL RNAi spontaneously elicited a mild type of oxidative stress that was enough to stimulate PARP-1 in a Ca2+-calmodulin kinase II-dependent manner. GCL RNAi-mediated PARP-1 activation facilitated DNA repair, although neurons underwent delta psi(m) loss followed by some apoptotic death. PARP-1 inhibition did not prevent delta psi(m) loss, but enhanced the vulnerability of neurons to apoptosis upon GCL silencing. Conversely, mild expression of PARP-1 partially prevented to GCL RNAi-dependent apoptosis. Thus, in the mild progressive damage likely occur in neurodegenerative diseases, PARP-1 activation plays a neuroprotective role that should be taken into account when considering therapeutic strategies.