Formation Of Oxygen Species Research Articles

BPM 31510 decreases doxorubicin-induced cardiomyopathy

Doxorubicin is a commonly used antineoplastic agent for treatment of cancers in adults and children. Despite its high efficacy in the treatment of various cancers, doxorubicin confers dose-limiting cardiogenic toxicity in both populations and associated with EKG changes. In a subset of patients, this effect on EKG is transient, however a significant number of patients will develop cardiomyopathy refractory to conventional treatment. Mitochondrial damage in the cardiomyocytes is the first hallmark of doxorubicin-induced cardiomyopathy. It has been suggested that lipid peroxidation may play a pivotal role in its pathophysiology by affecting the mitochondria through formation of reactive oxygen species. This theory is supported by the presence of markers of lipid peroxidation after doxorubicin administration in the blood. Coenzyme Q10 is found within the membranes of the mitochondria, lysosomes and the Golgi apparatus, and also acts as a powerful antioxidant and inhibitor of lipid peroxidation. BPM 31510 is an intravenous formulation of coenzyme Q10 in a proprietary nanodispersion formulation with ability to selectively deliver high concentrations of Coenzyme Q10 to targets tissues and organelles including the mitochondria. In this study, Sprague Dawley rats were used as model system to determine the effect of intravenous BPM31510 on doxorubicin-mediated cardiomyopathy. Lethality of doxorubicin was observed when rats were injected intraperitoneally 20 mg total dose, with a schedule of 10 mg/kg/day for two days. On day 30, signs of morbidity were present, such as cachexia and a decrease in the size of the heart was documented following necropsy. Concomitant administration of BPM31510 resulted in a modest but significant increase in lifespan in the doxorubicin treated animals (p 0.025). In contrast, pretreatment with intravenous BPM31510 followed by doxorubicin dosing was associated with significant decline in mortality, with as many as sixty percent of animals surviving past 30 days (p<0.01). The potential mechanism(s) underlying the effect of long-term use of BPM31510 on doxorubicin-induced cardiotoxicity was investigated. Rats with a protracted regimen of doxorubicin died between days 25 and 30, whereas as previously observed, BPM 31510 had a significant impact on survival (p=0.005). Increased survival in the BPM31510 treated group was associated with significant decreases in alterations of heart morphology and biochemical parameters of oxidative stress. Taken together, the data suggests that intravenous BPM31510 has the potential to mitigate doxorubicin-mediated mortality

Abstract 167: Genetic Deletion of p66Shc Adaptor Protein Leads to Increased Myocardial Infarction by Inhibiting RISK and SAFE Prosurvival Pathways

Background— Formation of reactive oxygen species (ROS) contributes to many pathophysiological processes. Although ROS production is also involved in some physiological processes, the imbalance between their generation and removal, i.e. oxidative stress, plays a major role in particular in myocardial injury caused by ischemia-reperfusion (I/R). The mammalian Shc locus encodes three Shc isoforms: p46 Shc , p52 Shc and p66 Shc . The p66 Shc is not involved in mitogenic signals as p46 Shc /p52 Shc , but it functions as a critical mediator of intracellular oxidative signal transduction. Various studies relate p66 Shc to cardiovascular disease; however, few data are available on the role of p66 Shc in myocardial I/R. Methods and Results— 8-12-week-old male p66 Shc deficient ( p66 Shc-/- ) mice and corresponding C57Bl/6 wild-type (WT) control mice were subjected in vivo to different durations of ischemia (30, 45 and 60 min) followed by 24h of reperfusion. Infarct size was assessed morphologically and by MRI. After 30 min of ischemia, p66 Shc-/- mice developed markedly larger infarcts as compared to WT. This effect was confirmed by in vivo silencing of p66 Shc prior to I/R. Both genetic deletion and silencing of p66 Shc displayed increased post-ischemic levels of serum cardiac troponin I (cTnI). However, the observed effect on infarct size was limited to 30 min of ischemia since by increasing ischemia duration to either 45 or 60 min infarct size did no longer differ between p66 Shc-/- and WT mice. Moreover, differently from WT, infarct size in p66 Shc-/- was not significantly larger with increasing duration of ischemia (from 30 to 60 min). On the molecular level the observed effect was linked to the inhibition of activation by phosphorylation of protein kinase Akt and transcription factor STAT3 - two key members of prosurvival pathways RISK and SAFE, respectively. Conclusions— Our data suggest that genetic deletion of p66 Shc leads to an increased sensitivity to myocardial infarction with larger infarcts with shorter, but not prolonged ischemia, and that RISK and SAFE prosurvival pathways are involved. Therefore, activation of p66 Shc may provide resistance to ischemia and represent a novel therapeutic target in the early phase of myocardial infarction.

Antibacterial effect of chronic exposure of low concentration ZnO nanoparticles on E. coli

The toxicity effect due to chronic exposure of ZnO nanoparticles (NPs) was systematically studied by repeatedly treating different lower concentrations of ZnO nanoparticles with culture media of E. coli strain. The chronic exposure of ZnO NPs of concentrations below minimum inhibitory concentration (MIC) exhibited higher toxicity than the single exposure of higher concentrations. Most striking result was 57% inhibition of growth corresponding to chronic exposure of 0.06 mg/mL of ZnO NPs which was two folds more than that exhibited by single exposure of 0.30 mg/mL ZnO NPs. The toxicity of ZnO NPs in E. coli was studied in the light of formation of reactive oxygen species (ROS), measured as malondialdehyde (MDA) equivalent by thiobarbituric acid-ROS (TBARS) assay, and effect of Zn dissolution from ZnO NPs. Higher inhibition of growth for the chronic exposure batches were correlated with higher ROS generation, which subsequently contributed to cause membrane lipid peroxidation, confirmed from observation of cell wall deformation by scanning electron microscopy study and energy dispersive X-ray analysis showed adherence of ZnO NPs on cell wall. The possibility of membrane lipid peroxidation was addressed by revealing in vitro oxidation of oleic acid, which is a monounsaturated fatty acid. Further in this study we have shown that the dissolution of ZnO NPs at pH 7.4 was not significant to cause Zn-induced toxicity.

Abstract 627: Fiber-induced DNA damage response in human lung epithelial cells.

Abstract Asbestos, a group of naturally occurring mineral fibers, is a significant lung carcinogen. Exposure to asbestos fibers induces the formation of reactive oxygen species in cells and, subsequently, DNA double-strand breaks (DSBs), the most serious form of DNA damage related to transformation of cells. As a part of the DNA damage response, H2AX histone variant molecules are phosphorylated at ser139, producing γH2AX foci at the site of DSBs, and these serve as a docking site for DNA repair molecule complexes. Thereby, γH2AX foci are considered a sensitive marker for DSBs. DNA damage response has not thoroughly been investigated in particle carcinogenesis. Here, we studied fiber-induced DSB formation in two human lung epithelial cell lines, the cancer cell line A549 and the transformed cell line BEAS-2B to provide further knowledge on DNA damage associated with fiber exposure as a risk factor for lung cancer.A sub-lethal asbestos fiber concentration of 2 μg /cm2 was determined, by cell viability tests, as suitable for working with the A549 and BEAS-2B cells. Then, we studied the kinetics of fiber induced DSB formation in terms of H2AX phosphorylation. Foci of γH2AX can be detected using specific antibody in immunocytochemistry assays. A549 and BEAS-2B cells were exposed to asbestos fibers for 4, 24 and 48 h, and γH2AX foci were counted. Concurrent non-exposed control cultures were settled at each time point. At 4 h, the control group displayed remarkable endogenous DSB formation, possibly due to replication stress. In exposed cultures, the formation of γH2AX foci peaked at 24 h. We also explored whether it is possible, by using γH2AX antibody in a chromatin immunoprecipitation followed by next generation sequencing, to find out the distribution of DSBs at 24 h in the fiber-exposed A549 cells. The sequences involved in γH2AX foci were genome-widely distributed. To conclude, the kinetics of fiber-induced γH2AX foci formation in the A549 and BEAS-2B cells was partly interfered by endogenously formed DSBs. This study revealed sequences that may be implicated within γH2AX foci for further elucidation on their contribution to lung tumor progression in particle carcinogenesis. Citation Format: Eeva Kettunen, Päivi Tuominen, Satu Suhonen, Julia Catalán, Sisko L. Anttila, Penny Nymark, Hannu Norppa. Fiber-induced DNA damage response in human lung epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 627. doi:10.1158/1538-7445.AM2013-627

Abstract 2214: Genome-wide association analysis identified genetic markers associated with triptolide cellular sensitivity using HapMap LCLs as model system.

Abstract Triptolide is a biological diterpenoid derived from the Chinese herb Tripterygium wilfordii Hook f. It has shown to have immunosuppression and anti-inflammation, and antineoplastic effect [Pan et al, Cancer Lett, 20102;3,5]. The anti-tumor effects of triptolide have been shown in wide range of solid tumor and leukemic cell lines in vitro. Present study focused on identifying the genetic markers using EBV transformed (lymphoblast cell lines as model systems). International HapMap Project LCLs derived from subjects with European ancestry (CEU; n=60 unrelated) were obtained from the Coriell Institute for Medical Research. Genotype data and gene expression data on these cell lines is available publically. The cell lines were maintained at 370C in RPMI1640 medium supplemented with 2mM Glutamine. In vitro cytotoxicity was determined by plating LCLs in a 96 well plate at the density of 250,000 cells/mL followed by treatment with six different concentration of triptolide (500, 50, 20, 10, 6.67, and 1.3 nM) 24hr after plating. Cell viability was determined 48 hr post drug treatment by MTT assays. Genome-wide gene expression and genotype data was obtained from publically available source and was evaluated for identification of SNPs and/or gene expression signatures predictive of triptolide cellular sensitivity. Interestingly we observed “clumping” of small p-values in a region on chromosome 2 consisting of SNPs with smallest p-value. SNPs within this region on chromosome 2 spanned across many biologically and functionally important genes such as: genes of importance in apoptosis caspase 8, caspase 10 and CFLR (caspase 8 and FADD like apoptosis regulator). The most significant SNP rs6759004 occurred in the 3’UTR of CFLAR gene and was associated with triptolide cytotoxicity (p=2.5E-07). SNPs in genes involved in DNA replication and repair as ORC2 (origin of replication complex subunit2); in formation of reactive oxygen species as AOX1 and AOX2P (aldehyde oxidase); proteins involved in transcription, RNA processing and splicing as protein kinase CLK1 (CDC-kine linase-1), NIF3L1 etc were also significantly associated with triptolide chemosensitivity. The two most interesting candidate markers identified by association of genome-wide expression with triptolide cytotoxicity includes JAK1, (Janus kinase 1) which is involved in JAK/STAT signaling and plays significant role in cell differentiation, growth, transformation, apoptosis, proliferation etc. and DTX1, which plays role in NOTCH1 signaling. Overall our results identified biomarkers of biological and clinical significance that predict response to triptolide and would be of clinical relevance to patients being treated by triptolide Citation Format: Jatinder K. lamba, Tanya Feldberg, Taraswi Mitra Ghosh, Neha Bhise, Brooke Fridley. Genome-wide association analysis identified genetic markers associated with triptolide cellular sensitivity using HapMap LCLs as model system. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2214. doi:10.1158/1538-7445.AM2013-2214

Abstract 1789: Effects of benzo(a)pyrene-7,8-dione and cadmium on early response to DNA damage in primary human bronchoepithelial cells.

Abstract Polycyclic aromatic hydrocarbons (PAHs) and heavy metals often co-exist in the environment, but despite their significant toxicities little is known about their combined effects. Cadmium (Cd), a known human carcinogen, interferes with cellular responses to DNA damage, through inhibition of DNA repair systems, disturbance of tumor suppressor functions, or increased resistance to apoptosis. Cd has been linked to lung cancer in humans and the most common exposure scenarios in humans involve co-exposure with PAHs. However, studies on combined effects of Cd and PAHs are mostly limited to marine invertebrates or soil bacteria. Both PAHs and Cd induce formation of reactive oxygen species (ROS) in the cell. Mitochondria are the main target for oxidative DNA damage and also play an important role in programmed cell death. Cytochrome c is an important component of apoptosis and its translocation from mitochondria to the cytosol is an indicator of apoptotic activity in intact cells. Another indicator of the early response to apoptotic signals is the phosphorylation of H2AX, which also reflects DNA damage. Here, we propose that the exposure of primary human bronchoepithelial cells (hBEC) to the mixture of benzo(a)pyrene-7,8-dione (BaPD) and cadmium chloride (CdCl2) will significantly augment DNA damage and disrupt mitochondrial functions involving apoptosis. Methods: Cytotoxicity was analyzed by the WST-8 assay. hBEC were exposed to CdCl2 (5 μg/ml) and BaPD (10 μg/ml) for 24h. Levels of 8-hydroxy-2’-deoxyguanosine (8-OHdG) and cytochrome c were quantified using ELISA. Quantification of H2AX phosphorylation (Ser139, γ-H2AX) was performed by western blotting. Statistical tests were conducted using GraphPad Prism software at a significance level of 0.05. Mean levels across treatment groups were compared using analysis of variance (ANOVA). Results: IC50 values at 24h for CdCl2 and BaPD were 8.7 and 12.5 μg/ml, respectively. Levels of 8-OHdG in nuclear DNA were significantly higher in hBEC exposed to CdCl2 or BaPD when compared to controls. We observed an increased release of cytochrome c during the first few hours after exposure to CdCl2 and BaPD, followed by significant reductions at 24h. Furthermore, cytochrome c levels in cells exposed to the combination of BaPD and CdCl2 were substantially lower than those observed with either treatment alone. Phosphorylation of H2AX increased in hBEC exposed to CdCl2 or BaPD, with higher levels observed in cells treated with BaPD. Conclusion: Our results suggest increased DNA damage in hBEC after 24h of exposure to Cd and BaPD. We also observed decreased cytochrome c levels after exposure to BaPD and/or Cd, suggesting disruption of mitochondrial functions. This effect was more pronounced in cells exposed to the mixture. Citation Format: Jibanananda Mishra, Berrin Serdar. Effects of benzo(a)pyrene-7,8-dione and cadmium on early response to DNA damage in primary human bronchoepithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1789. doi:10.1158/1538-7445.AM2013-1789

Abstract 4481: Myeloperoxidase as a determinant for activity of etoposide (VP-16) and other phenolic and non-phenolic anticancer agents: implications for drug-induced leukemogenesis.

Abstract Clinical utilization of the phenolic anticancer agent etoposide (VP-16) is limited in selected malignancies due to its ability to induce acute myeloid leukemias causally linked to MLL gene rearrangements. Previously, we demonstrated that myeloperoxidase (MPO) found in myeloid precursors converts VP-16 to its phenoxyl radical [Mol. Pharm. 79: 479-87, 2011], which can redox cycle leading to enhanced DNA topoisomerase II (topo II)-mediated strand cleavage, and resultant MLL translocations. In the present study, we utilized MPO shRNA in myeloid leukemia HL-60 cells to further examine MPO dependency for VP-16-induced: 1) inhibition/poisoning of topo II isoforms; 2) DNA damage; 3) formation of reactive oxygen species; 4) generation of a VP-16 ortho-quinone GSH adduct; 5) antiproliferative/cytotoxic activity. In MPO knockdown cells, mature MPO expression was reduced to 6% and enzyme activity was reduced to 18% of the level found in shRNA controls. Topo II alpha and beta, and DNA topoisomerase I (topo I) levels were similar in MPO knockdown and control cells. VP-16 (0-100 μM) induced a progressive increase in DNA double strand breaks in MPO+ HL-60 cells which was attenuated in MPO-knockdowns. Direct VP-16-induced topo II alpha and beta complexes with genomic DNA were reduced in MPO knockdown cells compared to shRNA controls. Using 3’-(p-hydroxyphenyl) fluorescein (HPF), pro-oxidant activity of VP-16 was demonstrated in MPO+ HL-60 cells which converted to anti-oxidant effects in MPO knockdowns. Mass spectrometric analysis indicated that the level of inactive VP-16 ortho-quinone GSH adducts in MPO knockdown cells was reduced to 33% of that in control cells. Paradoxically, cytotoxicity of VP-16 and additional topo II poisons mAMSA, and mitoxantrone was decreased in MPO+ cells compared to MPO knockdown cells. Podophyllotoxin, the non-phenolic natural product related to VP-16, and camptothecin were equally cytotoxic in MPO knockdown and control cells. Hydroquinone and the dietary flavonol quercetin were more cytotoxic in MPO+ cells compared to knockdowns. Together results suggest that MPO conversion of VP-16 to a pro-oxidant, which leads to genotoxicity/carcinogenicity, may simultaneously protect cells from cytotoxicity, in part due to subsequent production of inactive GSH conjugates. The consequences of MPO activity on mAMSA and mitoxantrone metabolism, topo II poisoning, and MLL gene rearrangements are currently under investigation as are the redox-related activities of dietary flavonoids dependent on MPO activity. Support: NIH CA090787. Citation Format: Jack C. Yalowich, Anna Skwarska, Andrew J. Rabovsky, Yuan Zhao, Mitch A. Phelps, Billy W. Day, Ragu Kanagasabai. Myeloperoxidase as a determinant for activity of etoposide (VP-16) and other phenolic and non-phenolic anticancer agents: implications for drug-induced leukemogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4481. doi:10.1158/1538-7445.AM2013-4481

Tetrasulfanes as Selective Modulators of the Cellular Thiolstat

Naturally occurring polysulfanes often exhibit a wide spectrum of biological activity, which results from their ability to react with, and hence modify cysteine residues in key proteins and enzymes of the cellular thiolstat. Such interactions frequently proceed via S-thiolation of cysteine residues and subsequent formation of Reactive Oxygen Species. Polysulfanes are highly effective, yet also surprisingly selective for cysteine residues and enable the control of numerous cellular processes ranging from an activation of antioxidant defenses to the induction of programed cell death. Unfortunately, the arsenal of natural tri- and tetrasulfanes is limited. Here, we showcase the synthesis of asymmetric tetrasulfanes, which exhibit an interesting nematocidal activity and represent a new generation of tailor-made polysulfanes with potential applications in the field of agriculture and medicine. Supplemental materials are available for this article. Go to the publisher's online edition of Phosphorus Sulfur and Silicon and the Related Elements.

Donor Alcohol Abuse Increases the Risk for Graft Dysfunction in Lung Transplant Recipients

Purpose Primary graft dysfunction (PGD) is a form of ischemia reperfusion lung injury that is a major cause of early morbidity and mortality after lung transplantation. Alcohol abuse is known to alter pulmonary immunity, promote alveolar epithelial dysfunction and increase the risk of acute lung injury. The effects of alcohol abuse in donors of lung allografts on transplant outcomes is unknown, and our aim was to examine the incidence of donor alcohol abuse and early effects on graft function following transplantation. Methods and Materials We performed a single center cohort study of lung transplant recipients from 2007-2011, reviewing patient charts for donor information and lung transplant outcome data. Statistical analysis included student’s t-test and Mann-Whitney U test. Results 195 lung transplant recipients were included in the cohort and 20.5% (N=40) of the recipients had donors with significant alcohol use. Recipients of allografts from alcoholic donors spent more time on mechanical ventilation following transplant when compared to recipients whose donors had no reported alcohol abuse, mean 6.9±12.1 days versus 3.9±8.1 days on the ventilator, (p=0.07). Gas exchange, represented as PaO2/FiO2, was significantly worse in recipients of donors with alcohol abuse following transplant compared to recipients with no donor alcohol abuse ( Fig 1 ). Conclusions Lung transplant recipients who received allografts from alcoholic donors had evidence of graft dysfunction following transplant. We speculate that alcoholic donor allografts may be susceptible to damage from formation of reactive oxygen species, which is exaggerated by the ischemia-reperfusion of transplantation, resulting in an increased rate of PGD.

P.1.002 Pharmacological characterisation of positive allosteric modulators acting on the metabotropic glutamate receptor 2

3NP treated cells). After 12 h of 3NP addition, we observed Bax mitochondrial translocation, (26±5 Bax traslocation under control conditions vs 41±2 after 3NP treatment). Finally, scavengers of reactive oxygen species failed to prevent mitochondrial alterations, while cyclosporine A, but not Mdivi-1, prevented the generation of ROS. Data shown are mean±SEM, unless otherwise stated. Statistical significance of differences between groups was determined by ANOVA followed by a Newman–Keuls post hoc analysis. The level of statistical significance was set at P< 0.05. Conclusions: There was a direct correlation between formation of mitochondrial permeability transition pore and autophagy induced by 3NP treatment. Activation of autophagy preceded the apoptotic process and was mediated, at least partly, by formation of reactive oxygen species and mitochondrial permeability transition pore. 3NP induces mitochondrial swelling at short time, thus Drp1 is not able to migrate to the mitochondria. Moreover, cell apoptosis is activated through the intrinsic pathway.

Efficiency of Different Respiratory Protective Devices for Removal of Particulate and Gaseous Reactive Oxygen Species From Welding Fumes

ABSTRACT Ultraviolet (UV) light inherent to welding processes generates ozone (O3) with subsequent formation of reactive oxygen species (ROS) through photochemical reactions when UV light is present with O3. This study aimed to determine the performance of filters used as respiratory protective devices by welding personnel to simultaneously mitigate particulate and gaseous inhalation hazards. Four respiratory protective devices were selected for this study, including a surgical facemask, a cotton-fabric facemask, an activated-carbon facemask, and an N95 respirator. The removal efficiencies for the particulates in welding fumes were all above 98%. For particulate-phase ROS, the removal efficiencies of the different respiratory protective devices ranged from 83.5% to 94.1%; however, the removal efficiencies for gaseous ROS were only 1.3% (active carbon facemask) to 21.1% (N95 respirator). The data indicated that the respiratory protective devices commercially available cannot block the passage of the gas-phase ROS found in welding fumes.

Metabolic effects of ganglioside GM1 on PC12 cells in oxidative stress depend on modulation of activity of tyrosine kinase Trk of receptors

Evidence has been obtained that only GM1, but also other main brain gangliosides (GD1a, GD1b, and GT1b) increase viability of ells of the PC12 neuronal line submitted to action of H2O2. By the example of GM1 and GD1a, gangliosides have been shown to induce a protective effect when acting on PC12 cells under conditions of oxidative stress both at micro- and nanomolar concentrations that are physiological concentrations of gangliosides in cerebrospinal fluid. It has been shown for the first time that GM1 at nanomolar concentrations decrease the H2O2-induced formation of reactive oxygen species (ROS). It was found that in the presence of K-252a, an inhibitor of tyrosine kinase of Trk receptors, GM1 at concentrations of 10 microM and 10 nM lost the ability to increase viability of these cells under conditions of oxidative stress. The dependence of protective and metabolic effects of ganglioside GM 1 in PC 12 cells at action on them of H2O2 on modulation of activity of tyrosine kinase of Trk receptors (i. e., on the same signal system) agrees with concept of the essential role of the GM1 antioxidant effect in its increase of cell viability.

Free Radicals and Antioxidants in Human Diseases: A Review

Intensive oxidative processes occurring in human organisms lead to formation of reactive oxygen species (ROS), which can damage systemic cells and tissues. ROS are well recognised for playing a dual role, being both deleterious and beneficial species. They are mainly derived from oxygen (ROS) and generated in our body through various endogenous systems, exposure to different physic-chemical conditions or pathophysiological states. ROS can adversely alter lipids, proteins and DNA and are implicated in ageing and in several human diseases. Antioxidants appear to be the first line of defence against free radical damage, and are vital for maintaining optimum health and well-being. This review describes the ROS-linked pathogenesis of cancer, heart diseases, ischaemia/reperfusion injury, diabetes mellitus, neurodegenerative diseases (Alzheimer's disease and Parkinson's disease) and ageing.

Cardiac myocyte-specific transgenic ecSOD targets mitochondria to protect against Ca2+ induced permeability transition

ecSOD function has prototypically been associated with the extracellular space due to its secretion and localization to the extracellular matrix. A myocyte-specific ecSOD transgenic mouse has shown that it can also be localized to the myocyte intracellular compartment and is capable of attenuating Reactive oxygen species (ROS) formation and increasing NO bioavailability after ischemia reperfusion. Here, the subcellular localization of transgenic ecSOD was further defined by subcellular fractionation, immunofluorescent confocal microscopy, and Western analysis. Its impact on mitochondrial function was assessed by mitochondrial permeability transition (MPT). ecSOD was found to exist in cytosolic and nuclear fractions in addition to membrane. Colocalization of ecSOD with myocardial mitochondria was further demonstrated by confocal microscopy and subcellular fractionation of mitochondria and Western analysis. Isolated ventricular myocytes from cardiac-specific transgenic ecSOD mice were protected from hypoxia reoxygenation injury. Increased ecSOD colocalization to myocardial mitochondria in ecSOD Tg hearts limited MPT in response to Ca2+ challenge. These results demonstrate that ecSOD is not restricted to the extracellular space and can alter MPT response to Ca2+ suggesting mitochondrial localization of ecSOD can affect key mitochondrial functions such as MPT which are integral to cell survival.

7b, a novel amonafide analog, inhibited proliferation and phorbol 12-myristate 13-acetate/phytohemagglutinin-induced inflammatory responses of Jurkat T cells via p73-dependent pathway and decrease of nuclear factor-κB DNA-binding, respectively

7b, a novel amonafide analog, has shown high antitumor activity against Raji B-cell lymphoma. We report here that 7b also shows high cytotoxicity against various T lymphoma cells, with the highest IC50 (concentration for 50% cytotoxicity) value in Jurkat cells. In a previous study, p53-mutant Raji cells were sensitive to 7b treatment. In the present study, the Jurkat T lymphoma cells were characterized as p53-null. Additional assays showed that 7b could induce G1/S phase arrest and mitochondrial apoptosis in Jurkat cells, suggesting 7b as a potential drug candidate for treatment of T-cell lymphoma. This action was not affected by p53 status. Further analysis of molecular mechanisms revealed that up-regulation of p21 and the Bak/Bcl-2 ratio and down-regulation of UHRF1 and c-Myc were attributed to p73 activation. In turn, up-regulation of p73 was initiated by DNA damage-induced reactive oxygen species (ROS) formation. Interestingly, at non-toxic drug concentrations, 7b could also inhibit phorbol 12-myristate 13-acetate/phytohemagglutinin (PMA/PHA)-induced inflammatory responses of Jurkat T cells owing to the suppression of nuclear factor-κB (NF-κB) DNA-binding. Indeed, electrophoretic mobility shift assay and NF-κB binding assay showed that NF-κB DNA-binding was inhibited by 7b, and correspondingly, proinflammatory cytokine production was also decreased. In conclusion, 7b exhibits both antiproliferative and anti-inflammatory activities in T lymphoma cells.

Study of Superoxide Dysmutase Isozyme Activities in Hepatitis C Virus-Positive Cirrhotic Liver Transplant Recipients

p S h 1 t THE FORMATION OF reactive oxygen species following ischemia-reperfusion causes hepatocellular injury during transplantation. It has been suggested that hepatitis C virus may cause oxidative stress in infected cells. Oxidative stress develops when reactive oxygen forms are produced in excess and factors preventing their harmful effects fail to occur. Enzymatic antioxidant defense mechanism in the organism include superoxide dysmutase (SOD), which protects cells from toxic effects of superoxide radicals. Superoxide dysmutase activity has been related to the pathogenesis of hepatitis C virus by excessive superoxide radical production by NADPHoxidase. Three types of SOD isozymes—Cu,=Zn-SOD, Mn-SOD, and extracellular SOD (EC-SOD)— can catalyze dysmutation of superoxide radicals. Whereas MnSOD and EC-SOD are localized in the mitochondrial matrix and on the outer cell membrane surface respectively, Cu, Zn-SOD resides in the cytosol. In the present tudy, we studied perioperative SOD isozymes Cu, n-SOD and Mn-SOD activity in 22 patients with hepaitis C virus who received liver transplants.

Increased organ levels of angiotensin II in ren2 rats leads to the formation of reactive oxygen species and DNA damage

Increased organ levels of angiotensin II in ren2 rats leads to the formation of reactive oxygen species and DNA damage

Abstract 322: Imbalance Between MMP-2 and TIMP-4 Associated With Oxidative Stress Contributes to Temporal Renovascular Hypertension-Induced Cardiac Hypertrophy

Imbalanced matrix metalloproteinase (MMP) activity is associated with left ventricular hypertrophy (LVH). Increased reactive oxygen species (ROS) formation enhances MMP activity and decrease tissue inhibitor of MMP (TIMP) activity. We examined the temporal relationship between ROS, MMP-2, and TIMP-4 levels in the heart during the progression of LVH in two-kidney, one-clip (2K1C) hypertension. Sham or 2K1C hypertensive rats were studied after 15, 30, and 75 days of hypertension, and the results are expressed as % of values found in corresponding sham animals at each time point. Systolic blood pressure increased with time in 2K1C rats: 167±3, 201±1, and 203±2 mmHg after 15, 30, and 75 days of hypertension (all P&lt;0.05). Hypertension induced LVH in a time-dependent manner (P&lt;0.05). Collagen deposition increased by 250±23%, 213±20% and 235±16% in 2K1C rats, respectively, after 15, 30, and 75 days of hypertension; all P&lt;0.05 vs. respective sham). Cardiac MMP-2 levels increased from 13±1 (sham) to 19±2, 17±1, and 20±2 arbitrary units (AU) after 15, 30, and 75 days of hypertension, respectively (all P&lt;0.05 vs. sham). Cardiac TIMP-4 levels increased by 325±68% (P&lt;0.05) only after 75 days of hypertension. Cardiac gelatinolytic activity increased by 129±4%, 150±6%, and 152±6% after 15, 30, and 75 of hypertension, respectively (all P&lt;0.05 vs. sham). Cardiac ROS levels increased by 137±5%, 154±15%, and 130 ±10% after 15, 30, and 75 days of hypertension, respectively (all P&lt;0.05 vs. sham). These results indicate that LVH in is an early process associated with imbalanced MMP-2 and TIMP-4 levels in renovascular hypertension, thus resulting in increased gelatinolytic activity and fibrosis, which may be due to oxidative stress.

Biological aspects of cadmium and lead uptake by Phragmites australis (cav. Trin ex steudel) in natural water ecosystems

In natural environment plants are exposed to many different stress factors, including heavy metals, whose elevated concentration causes oxidative stress, connected with formation of reactive oxygen species (ROS). Therefore, plants have developed defence systems, including enzymatic antioxidant system, able to remove ROS. The work concerns the accumulation of two heavy metals, cadmium (Cd) and lead (Pb), as well as the phenomenon of oxidative stress caused by increased concentration of these metals in common reed (Phragmites australis), a dominant species in the littoral zone of many water reservoirs. The plants were obtained from four water bodies situated in Poznan: Kierskie Lake, Rusa³ka Lake, Strzeszynskie Lake and So³acki Pond. The aim of the study was to examined the accumulation of heavy metals and the relation between activity of antioxidant enzymes in rhizome, stem and reed leaves during the vegetative period. Three antioxidant enzymes were analyzed: ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and superoxide dismutase (SOD). The statistical analysis was done to determine the influence of the heavy metals on the activity of the antioxidant enzymes, involved in limiting and removing results of oxidative stress. Heavy metals were accumulated in common reed in all the four water reservoirs, but the activity of enzymes was variable during the observation period. Statistical analyses suggest that there are some correlations among concentration of metals and the activity of antioxidative enzymes. However, the results do not provide an unambiguous determination of the effect of heavy metals on enzymatic activity. Summing up, the contamination

Is there NO help for leptin?

Since the initial identification of leptin as the product of the ob gene in 1994, the signaling pathways by which this hormone alters cell physiology have been the subject of extensive investigations. The fact that leptin can induce nitric oxide (NO) production was first demonstrated in studies of the pituitary gland and pancreatic islets. A large number of additional studies further showed that this adipokine stimulates NO synthesis in multiple tissues. This review article discusses the role of leptin in NO production and its pathophysiological consequences. The role of this gaseous messenger in cell physiology depends on the cell type, the concentration of NO and the duration of exposure. It can be either a potent oxidant or a protector of cell integrity against the formation of reactive oxygen species. Leptin plays two opposing roles on arterial pressure. It exerts a hypertensive effect due to sympathetic activation and a vasorelaxant effect due to NO production. This adipokine acts via NO to produce pro-inflammatory factors in cartilage pathology, potentially contributing to an increased risk for osteoarthritis. Another well-documented role of leptin-induced NO, acting either directly or via the hypothalamus, concerns lipid metabolism in muscle and adipose tissue. In adipocytes, the direct and rapid action of leptin is to activate the nitric oxide synthase III, which favors lipolysis. In contrast, in the long-term, leptin reduces lipolysis. However, both in the short-term and in the long-term, glyceroneogenesis and its key enzyme, the cytosolic phosphoenolpyruvatecarboxykinase (PEPCK-C), are down-regulated by the adipokine, thus favoring fatty acid release. Hence, leptin-induced NO production plays a crucial role in fatty acid metabolism in adipose tissue. The resulting effects are to prevent lipid storage and to improve energy expenditure, with possible improvements of the obese state and its associated diseases.