Increased Superoxide Formation Research Articles

Abstract WP235: Hyperglycemia In The Days After Stroke Exacerbates Brain Injury By Increasing Microglial Superoxide Production

It has been recognized for decades that elevated blood glucose (hyperglycemia) during ischemia/reperfusion greatly exacerbates injury; however, little is known about the effect of hyperglycemia that begins later, over the hours to days after ischemia. This often occurs after stroke as part of a systemic stress response, even in non-diabetic patients, but neither clinical evidence nor prior basic science studies provide guidance on how post-stroke hyperglycemia should be managed. Here we investigated this issue using a mouse photothrombotic model of permanent ischemia. Normoglycemia or hyperglycemia (blood glucose of 300-400 mg/dL; 15-20 mM) was maintained between 17 - 48 hours after ischemia onset. Post-stroke hyperglycemia was found to increase the final infarct volume, increase hemorrhage formation, and exacerbate motor dysfunction. Hyperglycemia also increased superoxide formation in peri-lesional microglia/macrophages. Importantly, hyperglycemia did not have these deleterious effects in p47 phox-/- mice, which cannot form an active superoxide-producing NADPH oxidase-2 complex, or in wild-type mice treated with a peptide inhibitor of NADPH oxidase-2 (gp91-TAT peptide). These results suggest that hyperglycemia occurring many hours after ischemia can increase oxidative stress in peri-infarct tissues by fueling NADPH oxidase activity in reactive microglia/macrophages, and by this mechanism may contribute to worsened outcome.

Long-Term Effects of Aircraft Noise Exposure on Vascular Oxidative Stress, Endothelial Function and Blood Pressure: No Evidence for Adaptation or Tolerance Development.

Transportation noise is recognized as an important cardiovascular risk factor. Key mechanisms are noise-triggered vascular inflammation and oxidative stress with subsequent endothelial dysfunction. Here, we test for adaptation or tolerance mechanisms in mice in response to chronic noise exposure. C57BL/6J mice were exposed to aircraft noise for 0, 4, 7, 14 and 28d at a mean sound pressure level of 72 dB(A) and peak levels of 85 dB(A). Chronic aircraft noise exposure up to 28d caused persistent endothelial dysfunction and elevation of blood pressure. Likewise, reactive oxygen species (ROS) formation as determined by dihydroethidium (DHE) staining and HPLC-based measurement of superoxide formation in the aorta/heart/brain was time-dependently increased by noise. Oxidative burst in the whole blood showed a maximum at 4d or 7d of noise exposure. Increased superoxide formation in the brain was mirrored by a downregulation of neuronal nitric oxide synthase (Nos3) and transcription factor Foxo3 genes, whereas Vcam1 mRNA, a marker for inflammation was upregulated in all noise exposure groups. Induction of a pronounced hearing loss in the mice was excluded by auditory brainstem response audiometry. Endothelial dysfunction and inflammation were present during the entire 28d of aircraft noise exposure. ROS formation gradually increases with ongoing exposure without significant adaptation or tolerance in mice in response to chronic noise stress at moderate levels. These data further illustrate health side effects of long-term noise exposure and further strengthen a consequent implementation of the WHO noise guidelines in order to prevent the development of noise-related future cardiovascular disease.

Detection of extracellular superoxide in isolated human immune cells and in an animal model of arterial hypertension using hydropropidine probe and HPLC analysis

Superoxide formation is a hallmark of cardiovascular disease with the involvement of different tissues and cell types. Identification of the cellular sources and subcellular localization of superoxide formation is important to understand the underlying disease pathomechanisms. In the present study, we used HPLC quantification of the superoxide-specific oxidation products of hydroethidine (HE or DHE) and its derivative hydropropidine (HPr+) for measurement of intra- and extracellular superoxide formation in isolated leukocytes and tissues of hypertensive rats. Superoxide generation by isolated leukocytes from human subjects as well as tissue samples of hypertensive rats (infusion of angiotensin-II for 7 days) was investigated using HPr+ and HE fluorescent probes with HPLC or plate reader detection. Both fluorescent dyes were used to test for intra- and extracellular superoxide formation using the supernatant or cell/tissue pellet for analysis. We demonstrate the correlation of impaired functional parameters (blood pressure, vascular function, and oxidative burst) and increased superoxide formation in different organ systems of hypertensive rats using the HPr+/HPLC method. In the cell model, the differences between HE and HPr+ and especially the advantage of the extracellular specificity of HPr+, due to its cell impermeability, became evident. Plate reader-based assays showed much higher background signal and were inferior to HPLC based methods. In conclusion, the HPr+/HPLC assay for superoxide determination is highly reliable in isolated immune cells and an animal model of arterial hypertension. In particular, the cell impermeability of HPr+ made it possible to differentiate between intra- and extracellular superoxide formation.

Unnecessary hesitancy on human vaccine tests-Response.

20-Hydroxyeicosatetraenoic acid (20-HETE) is formed by the ω-hydroxylation of arachidonic acid by cytochrome P450 4A and 4F enzymes, and it induces angiogenic responses in vivo. To test the hypothesis that 20-HETE increases endothelial cell (EC) proliferation via vascular endothelial growth factor (VEGF), we studied the effects of WIT003 [20-hydroxyeicosa-5(Z),14(Z)-dienoic acid], a 20-HETE analog on human macrovascular or microvascular EC. WIT003, as well as pure 20-HETE, stimulated EC proliferation by ∼40%. These proliferative effects were accompanied by increased VEGF expression and release that were observed as early as 4 h after 20-HETE agonist addition. This was accompanied by increased phosphorylation of the VEGF receptor 2. The proliferative effects of 20-HETE were markedly inhibited by a VEGF-neutralizing antibody. Polyethylene glycol-superoxide dismutase (PEG-SOD) markedly inhibited both the increases in VEGF expression and the proliferative effects of 20-HETE. In contrast, administration of the NAD(P)H oxidase inhibitor apocynin had no effect to the proliferative response to 20-HETE. The 20-HETE agonist markedly increased superoxide formation as reflected by an increase in dihydroethidium staining of EC, and this increase was inhibited by PEG-SOD but not by apocynin. 20-HETE also increased the phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) in EC, whereas an inhibitor of MAPK [U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] suppressed the proliferative and the VEGF changes but not the pro-oxidant effects of 20-HETE. These data suggest that 20-HETE stimulates superoxide formation by pathways other than apocynin-sensitive NAD(P)H oxidase, thereby activating MAPK and then enhancing VEGF synthesis that drives EC proliferation. Thus, 20-HETE may be involved in the regulation of EC functions, such as angiogenesis.

Evaluation of physiological and biochemical aberration linked to effect of sodium dodecyl sulphate on barley seedlings

Sodium dodecyl sulphate (SDS) amount in domestic and industrial effluents is consistently increasing since SDS is available in almost all cleaning products. Therefore, it has become one of the potential destructive factors on habitats such as agricultural areas and aquatic environments. This study was carried out to evaluate the SDS-induced changes on barley (Hordeum vulgare L. cv. Olgun) seedlings. Barley seedlings were hydroponically cultivated for nine days at optimal conditions and, then, exposed to five different doses of SDS. After 72 h from SDS addition, root and coleoptile lengths were seriously reduced as compared to control. Also, SDS decreased soluble protein, chlorophyll a, chlorophyll b and carotenoid contents of the barley leaves. Furthermore, the SDS-induced reductive effects on protein profiles of barley leaves were also detected with electrophoresis. SDS caused serious decreases in activities of antioxidant enzymes, including superoxide dismutase, catalase, and ascorbate peroxidase and glutathione reductase except for peroxidase. Moreover, the SDS led to the emergence of oxidative damage symptoms that were manifested by the increased superoxide anion formation, hydrogen peroxide content and lipid peroxidation level of barley seedlings. Eventually, the results of the present study revealed that SDS cause to a severe reduction in the growth of barley seedlings.

The depletion of p38alpha kinase upregulates NADPH oxidase 2/NOX2/gp91 expression and the production of superoxide in mouse embryonic stem cells

P38alpha kinase plays an important role in the regulation of both cell stress response and cell fate. In this study, we report that p38alpha kinase-deficient embryonic stem cells exhibit a higher production of reactive oxygen species (ROS) in contrast to their wild-type counterpart. Analysis of the expressions of NADPH oxidases (NOXs) and dual oxidases, crucial enzymes involved in intracellular ROS formation, shows NOX2/gp91phox is over-expressed in p38alpha deficient cells. The particular increase in superoxide formation was confirmed by the specific detection of hydroethidine derivate 2-hydroxyethidium. ROS formation decreased when the level of NOX2 was silenced by siRNA in p38alpha deficient cells. These data suggest the importance of p38alpha kinase in the regulation of ROS metabolism in embryonic stem cells and the significance of the observed phenomena of cancer cell-like phenotypes, which is discussed.

Fragmentation of kidney epithelial cell primary cilia occurs by cisplatin and these cilia fragments are excreted into the urine

The primary cilium, which protrudes from the cell surface, is associated with the pathogenesis of various diseases, including acute kidney injury (AKI). Primary cilium length dynamically changes during the progression of diseases. However, its relevance in disease and the underlying mechanism are largely unknown. In this study, we investigated the role of primary cilia in AKI induced by cisplatin, an effective anticancer drug, and the underlying mechanisms. In addition, we evaluated the usefulness of length alteration and deciliation of primary cilia into the urine for the diagnosis of AKI. Cisplatin induced shortening, elongation, and normalization of the primary cilia in kidney epithelial cells over time. During shortening, primary cilia fragments and ciliary proteins were excreted into the urine. During deciliation, cell proliferation and the expression of cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen were not significantly changed. Shortening and deciliation of primary cilia were observed before significant increases in plasma creatinine and blood urea nitrogen concentration occurred. Pretreatment with Mito-Tempo, a mitochondria-targeted antioxidant, prevented cisplatin-induced primary cilium shortening and inhibited the increases in superoxide formation, lipid peroxidation, blood urea nitrogen, and tissue damage. In contrast, isocitrate dehydrogenase 2 (Idh2) gene deletion, which results in defect of the NADPH-associated mitochondrial antioxidant system, exacerbated cisplatin-induced changes in mice. Taken together, our findings demonstrate that cisplatin induces deciliation into the urine and antioxidant treatment prevents this deciliation, renal dysfunction, and tissue damage after cisplatin injection. These results suggest that cisplatin-induced AKI is associated with primary cilia and urine primary cilia proteins might be a non-invasive biomarker of kidney injury.

Exposure of Breast and Lung Cancer Cells to a Novel Estrone Analog Prior to Radiation Enhances Bcl-2-Mediated Cell Death.

Following exposure of cells to gamma-radiation, a cascade of intracellular consequences may be observed in a semitemporal manner. This includes deoxyribonucleic acid (DNA) damage and reactive oxygen species (ROS) accumulation initially, with consequent signaling for DNA repair and facilitative regulation of the cell cycle. Failure to rectify the damage or ROS levels leads to induction of senescence or apoptosis. 2-Ethyl-3-O-sulfamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol), a 2-methoxyestradiole analog designed in silico for superior pharmacokinetics, was investigated for its potential to enhance apoptotic signaling and decrease the long-term survival of cells exposed to radiation. Sequential early intracellular effects within radiation-treated MCF-7 breast- and A549 lung cancer cells pre-exposed to low-dose ESE-15-ol were investigated using various flow cytometric protocols, spectrophotometry, and microscopy. Long-term cellular survival and proliferation was examined using clonogenic studies, which demonstrated a significant decrease in the presensitized cells. Combination-treated cells exhibited increased superoxide formation, and decreased Bcl-2 expression and -phosphorylation. Induction of apoptosis and elevation of the sub-G1 phase was evident in the pre-exposed MCF-7 cells, although only minimally in the A549 cells at 48-h. These results indicate that low-dose ESE-15-ol may increase tumor response to radiation. Future studies will investigate the effect of ESE-15-ol pre-exposure on radiation-induced DNA damage and repair mechanisms.

Hydrogen sulfide-producing cystathionine γ-lyase is critical in the progression of kidney fibrosis

Cystathionine γ-lyase (CSE), the last key enzyme of the transsulfuration pathway, is involved in the production of hydrogen sulfide (H2S) and glutathione (GSH), which regulate redox balance and act as important antioxidant molecules. Impairment of the H2S- and GSH-mediated antioxidant system is associated with the progression of chronic kidney disease (CKD), characterized by kidney fibrosis and dysfunction. Here, we evaluated the role of CSE in the progression of kidney fibrosis after unilateral ureteral obstruction (UUO) using mice deficient in the Cse gene. UUO of wild-type mice reduced the expression of H2S-producing enzymes, CSE, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase in the obstructed kidneys, resulting in decreased H2S and GSH levels. Cse gene deletion lowered H2S and GSH levels in the kidneys. Deleting the Cse gene exacerbated the decrease in H2S and GSH levels and increase in superoxide formation and oxidative damage to proteins, lipids, and DNA in the kidneys after UUO, which were accompanied by greater kidney fibrosis, deposition of extracellular matrixes, expression of α-smooth muscle actin, tubular damage, and infiltration of inflammatory cells. Furthermore, Cse gene deletion exacerbated mitochondrial fragmentation and apoptosis of renal tubule cells after UUO. The data provided herein constitute in vivo evidence that Cse deficiency impairs renal the H2S- and GSH-producing activity and exacerbates UUO-induced kidney fibrosis. These data propose a novel therapeutic approach against CKD by regulating CSE and the transsulfuration pathway.

P 159 - Role of NADPH oxidase on TNFα-induced intestinal permeabilization

Increased intestinal barrier permeability is a major event in the pathophysiology of diseases with an inflammatory background. Tumor necrosis factor alpha (TNFα) plays a central role promoting barrier dysfunction. NADPH oxidase (NOX) is co-activated by TNFα and increased superoxide anion formation could be involved in barrier disruption. In human Caco-2 cells differentiated into intestinal epithelial monolayers we investigated: i- the relative abundance of NOX family members, ii) the effects of TNFα on NOX activation/expression, iii) the effects of epicatechin (EC) and cyanidin (CN) on NOX activity and Caco-2 monolayer protection. Assessment of NOX1, NOX2, NOX3, NOX4 and NOX5 mRNA levels showed NOX1 and NOX4 as predominant. TNFα treatment for 6 h increased (circa 32% and 30%) NOX1 and NOX4 expression; and for 10 min increased (circa 49%) NOX activity. From a group of flavonoids tested, EC and CN were the most effective inhibiting basal and TNFα-stimulated NOX activity. The extent of inhibition was different between basal and stimulated conditions suggesting differential effects on different NOX members. EC- and CN-mediated NOX inhibition was associated with their capacity to protect barrier permeability. NOX can play a major role in the development of inflammation-mediated intestinal permeabilization, suggesting a beneficial effect of EC/CN-rich diets on intestinal pathologies.

Decreased bioavailability of nitric oxide in aorta from ovariectomized senescent mice. Role of cyclooxygenase

This study investigates the effects of aging and/or ovariectomy on vascular reactivity to thromboxane A2 (TXA2) receptor stimulation with U46619, and the modulation by nitric oxide (NO) and cyclooxygenase (COX) in aorta from female senescence-accelerated mice (SAMP8) and from senescence resistant mice (SAMR1). Five-month-old female SAMR1 and SAMP8 were divided into three groups: sham-operated, ovariectomized and ovariectomized plus estradiol. Twenty-eight days after surgery, thoracic aortic rings were mounted for isometric recording of tension and concentration–response curves for U46619 (10−10–3×10−7M) were performed in the absence and in the presence of the NO synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME, 10−4M) and/or COX inhibitor indomethacin (10−5M). Vascular superoxide production was detected by dihydroethidium staining on sections of thoracic aorta. NO bioavailability in response to U46619 was suppressed by estrogen withdrawn in young and senescent mice and was restored by the administration of estradiol. In the presence of indomethacin, contractions to U46619 decreased in all groups indicating an aging- and estrogen-dependent modulation of contractile prostanoids. The simultaneous incubation of L-NAME and indomethacin did not change the maximal responses and sensitivities to TXA2 in any group in comparison with untreated aortic segments. The superoxide generation induced by TXA2 was greater in aorta from SAMP8 than in SAMR1. Moreover, in ovariectomized groups superoxide production was further increased and treatment with 17β-estradiol reverted the effects of the ovariectomy. Inhibition of COX with indomethacin prevented the U46619-induced increase in superoxide formation. Our results indicate that NO bioavailability in response to TP receptor activation is both estrogen- and aging-dependent. TXA2 induced contractions are partially mediated by COX activation. Both aging and ovariectomy enhanced COX-dependent component of the TXA2-induced contraction. It is noteworthy that in the absence of estrogen, COX inhibition induces an increase of NO bioavailability. Therefore, in senescent female mice with an experimental menopause, TP-receptor stimulation is responsible for COX activation and enhanced superoxide generation, which may result in reduced NO bioavailability. These effects were reversed by estrogen administration.

Amelioration of nicotinamide adenine dinucleotide phosphate–oxidase mediated stress reduces cell death after blast-induced traumatic brain injury

A total of 1.7 million traumatic brain injuries (TBIs) occur each year in the United States, but available pharmacologic options for the treatment of acute neurotrauma are limited. Oxidative stress is an important secondary mechanism of injury that can lead to neuronal apoptosis and subsequent behavioral changes. Using a clinically relevant and validated rodent blast model, we investigated how nicotinamide adenine dinucleotide phosphate oxidase (Nox) expression and associated oxidative stress contribute to cellular apoptosis after single and repeat blast injuries. Nox4 forms a complex with p22phox after injury, forming free radicals at neuronal membranes. Using immunohistochemical-staining methods, we found a visible increase in Nox4 after single blast injury in Sprague Dawley rats. Interestingly, Nox4 was also increased in postmortem human samples obtained from athletes diagnosed with chronic traumatic encephalopathy. Nox4 activity correlated with an increase in superoxide formation. Alpha-lipoic acid, an oxidative stress inhibitor, prevented the development of superoxide acutely and increased antiapoptotic markers B-cell lymphoma 2 (t = 3.079, P < 0.05) and heme oxygenase 1 (t = 8.169, P < 0.001) after single blast. Subacutely, alpha-lipoic acid treatment reduced proapoptotic markers Bax (t = 4.483, P < 0.05), caspase 12 (t = 6.157, P < 0.001), and caspase 3 (t = 4.573, P < 0.01) after repetitive blast, and reduced tau hyperphosphorylation indicated by decreased CP-13 and paired helical filament staining. Alpha-lipoic acid ameliorated impulsive-like behavior 7 days after repetitive blast injury (t = 3.573, P < 0.05) compared with blast exposed animals without treatment. TBI can cause debilitating symptoms and psychiatric disorders. Oxidative stress is an ideal target for neuropharmacologic intervention, and alpha-lipoic acid warrants further investigation as a therapeutic for prevention of chronic neurodegeneration.

Reducing NADPH Oxidative Stress following Acute Neurotrauma

1.7 million traumatic brain injuries occur each year in the USA. Utilizing a validated rodent blast model, we investigated how NADPH oxidase expression and associated oxidative stress contributes to cellular apoptosis following blast injuries. We found a visible increase in NADPH Nox4 following single blast injury in Sprague Dawley rats. Interestingly, Nox4 was also increased in post‐mortem human samples obtained from deceased athletes. Nox4 activity led to an increase in superoxide formation. Lipoic acid decreased Nox4 when given i.p. 10mg/kg 5‐minutes after blast exposure. Lipoic acid also significantly decreased the subsequent formation of superoxide. Lipoic acid increased the anti‐apoptotic markers Bcl‐2 (t = 3.079, p&lt;0.05) and heme oxygenase 1 (t = 8.169, p&lt;0.001) compared to blast injured animals. Two weeks following the last blast exposure (6 blasts total over 2 weeks), the lipoic acid treatment group had significantly reduced pro‐apoptotic markers Bax (t=4.483, p&lt;0.05), caspase 12 (t=6.157, p&lt;0.001), and caspase 3 (t=4.573, p&lt;0.01) compared to blast injured animals. Lipoic acid treated animals also had significantly reduced tau hyperphosphorylation. Tissue from the NFL player had increased Bax expression (t=5.424, p&lt;0.01) compared to control. A significant difference in impulsive‐like behavior between experimental animal groups was seen 7 days after repetitive blast injury (F(2,21)=4.327, p&lt;0.05). Rats exposed to repetitive blast displayed increased time in the open arm compared to control (t=3.632, p&lt;0.05), but lipoic acid ameliorated this impulsive‐like behavior (t=3.573, p&lt;0.05). Lipoic acid warrants further investigation as a therapeutic for the treatment of acute neurotrauma.

Lipopolysaccharide (LPS)-mediated Angiopoietin-2-dependent Autocrine Angiogenesis Is Regulated by NADPH Oxidase 2 (Nox2) in Human Pulmonary Microvascular Endothelial Cells

Sepsis-mediated endothelial Angiopoeitin-2 (Ang2) signaling may contribute to microvascular remodeling in the developing lung. The mechanisms by which bacterial cell wall components such as LPS mediate Ang2 signaling in human pulmonary microvascular endothelial cells (HPMECs) remain understudied. In HPMEC, LPS-induced Ang2, Tie2, and VEGF-A protein expression was preceded by increased superoxide formation. NADPH oxidase 2 (Nox2) inhibition, but not Nox4 or Nox1 inhibition, attenuated LPS-induced superoxide formation and Ang2, Tie2, and VEGF-A expression. Nox2 silencing, but not Nox4 or Nox1 silencing, inhibited LPS-mediated inhibitor of κ-B kinase β (IKKβ) and p38 phosphorylation and nuclear translocation of NF-κB and AP-1. In HPMECs, LPS increased the number of angiogenic tube and network formations in Matrigel by >3-fold. Conditioned media from LPS-treated cells also induced angiogenic tube and network formation in the presence of Toll-like receptor 4 blockade but not in the presence of Ang2 and VEGF blockade. Nox2 inhibition or conditioned media from Nox2-silenced cells attenuated LPS-induced tube and network formation. Ang2 and VEGF-A treatment rescued angiogenesis in Nox2-silenced cells. We propose that Nox2 regulates LPS-mediated Ang2-dependent autocrine angiogenesis in HPMECs through the IKKβ/NF-κB and MAPK/AP-1 pathways.

Physiological and molecular level studies on the toxicity of silver nanoparticles in germinating seedlings of mung bean (Vigna radiata L.)

In the present study, the toxic effect of silver nanoparticles was studied in seedlings of economically important food crop mung bean (Vigna radiata L.). The plants were grown in 1/2 MS agar medium containing 0, 5, 10, 20, and 50 mg/L of silver nanoparticles for 21 days. The toxic effects were studied using different parameters such as growth, chlorophyll, and proline contents, reactive oxygen species (ROS) generation, lipid peroxidation, changes in mitochondrial membrane potential (ΔΨm), and oxidative stress responsive gene expression. The shoot length and weight was significantly reduced upon exposure to 50 mg/L of silver nanoparticles. Significant reduction in root elongation and weight was observed upon exposure to 20 and 50 mg/L of silver nanoparticles. The total chlorophyll content significantly reduced after exposure to 50 mg/L of silver nanoparticles. However, proline content was increased significantly upon exposure to 20 and 50 mg/L of silver nanoparticles. The hydrogen peroxide level and lipid peroxidation levels increased significantly in roots after exposure to 20 and 50 mg/L of silver nanoparticles. Histochemical staining with nitroblue tetrazolium showed a concentration-dependant increase in superoxide formation in roots. Roots treated with 2′, 7′-dichloro-dihydro-fluorescein diacetate and 3, 3′-diaminobenzidine showed a concentration-dependant increase in ROS generation. Exposure to increasing concentrations of silver nanoparticles has resulted in changes in ΔΨm in roots as revealed by increased Rhodamine 123 fluorescence. Gene expression analysis using real-time polymerase chain reaction showed changes in the relative mRNA expression of CuZn-SOD, CAT, and APX genes indicating the plants’ antioxidative defense responses under silver nanoparticle stress.

Linking alpha-synuclein phosphorylation to reactive oxygen species formation and mitochondrial dysfunction in SH-SY5Y cells.

Alpha-synuclein (α-syn) is a soluble protein highly enriched in presynaptic terminals of neurons. Accumulation of α-syn as intracellular filamentous aggregates is a pathological feature of sporadic and familial forms of Parkinson's disease (PD). Changes in α-syn post-translational modifications, as well as mitochondrial dysfunction and oxidative stress constitute key pathogenic events of this disorder. Here we assessed the correlation between α-syn phosphorylation at serine 129 (Ser129), the formation of reactive oxygen species (ROS) and mitochondrial dysfunction in SH-SY5Y cells expressing A53T mutant or wild-type (WT) α-syn, exposed to ferrous iron (FeSO4) and rotenone (complex I inhibitor). Under basal conditions, prolonged expression of A53T mutant α-syn altered mitochondria morphology, increased superoxide formation and phosphorylation at Ser129, which was linked to decreased activity of protein phosphatase 2A (PP2A). Exposure to FeSO4 or rotenone enhanced intracellular ROS levels, including superoxide anions, in both types of cells, along with α-syn Ser129 phosphorylation and mitochondrial depolarization. Most of these changes were largely evident in A53T mutant α-syn expressing cells. Overall, the data suggest that stimuli that promote ROS formation and mitochondrial alterations highly correlate with mutant α-syn phosphorylation at Ser129, which may precede cell degeneration in PD.

Su1387 F1F0-ATPase Modulation Is Efficacious in Models in IBD and Induces Apoptosis of Human CCR9+ Gut-Tropic T-cells

Background: Targeting immune metabolism may represent a novel approach for therapeutic intervention in autoimmunity. The mitochondrial F1F0-ATPase is the final complex in the electron transport chain. Small molecule, allosteric modulators of this complex cause increase in superoxide formation and apoptosis of susceptible cells. Because of their distinctive metabolic phenotype, chronically activated lymphocytes are preferentially sensitive to ATPase Modulator-induced apoptosis vs resting or acutely activated lymphocytes. In inflammatory bowel disease (IBD), mucosal lymphocytes are chronically activated, overexpress anti-apoptotic proteins and show resistance to activation-induced cell death (AICD). Thus, restoring lymphocyte AICD may be a potential therapeutic strategy. Here a set of F1F0-ATPase Modulators was evalulated in murine IBD models and in gut-tropic T cells from IBD patients. Methods: Acute IBD was induced by sensitizing mice with topical TNBS followed 7 days later by intracolonic TNBS challenge. ATPase Modulators were dosed orally on Day1 and Day2 after TNBS challenge. On Day4 mice were sacrificed and the colon was collected for histological analysis or isolation of lamina propria mononuclear cells which were analyzed for viability and inflammatory molecules by flow cytometry or RT-PCR. Peripheral blood mononuclear cells (PBMCs) from healthy donors or IBD patients were treated with ATPase Modulators for 20 hours. Cell death of activated, gut-tropic T cells (CD3+CCR9+) vs other T cells (CD3+ CCR9-) was detected by 7ADD uptake and Annexin V staining. Statistical analyses used unpaired Student's T test. Results: In acute murine TNBS-induced colitis, oral dosing at of ATPase Modulators attenuated weight loss, improved histologic assessments, decreased IFNγ and TNF production and significantly increased LP lymphocyte cell death (%viability: vehicle 15.8±3.3 vs LYC-51194 27.3±6.6, p<0.05). In PBMC from healthy subjects (n=24), ATPase Modulators were able to induce cell death but only in CCR9+ gut-tropic T cells (%cell death: CD3+CCR9+ vehicle 22.9±11.7 vs LYC-53661 56.6±11.7, p<0.0001; CD3+CCR9vehicle 0.47±0.51 vs LYC-53661 3.2±2.97). The induction of cell death in gut-tropic T cells was preceded by an increase in reactive oxygen species and was prevented by the ROS scavenger vitamin E. Similar experiments were performed with PBMC from IBD patients (CD n=15, UC n=15). LYC-51661, and similarly LYC-51194, induced cell death in CCR9+ T cells (%cell death: CD vehicle 17.9±13 vs LYC-51661 61.3±16.6 p< 0.0001; UC vehicle 18.8±9.5 vs LYC-51661 55.2±16.1 p< 0.0001) while sparing CD3+CCR9negative cells. Conclusions: The apparently selective effect on chronically activated lymphocytes may provide an advantage over current immunosuppressive treatments and suggest that ATPase Modulators may be a promising approach for treating IBD.

Chlorine gas exposure disrupts nitric oxide homeostasis in the pulmonary vasculature

Exposure to chlorine (Cl2) gas during industrial accidents or chemical warfare leads to significant airway and distal lung epithelial injury that continues post exposure. While lung epithelial injury is prevalent, relatively little is known about whether Cl2 gas also promotes injury to the pulmonary vasculature. To determine this, rats were subjected to a sub-lethal Cl2 gas exposure (400ppm, 30min) and then brought back to room air. Pulmonary arteries (PA) were isolated from rats at various times post-exposure and contractile (phenylephrine) and nitric oxide (NO)-dependent vasodilation (acetylcholine and mahmanonoate) responses measured ex vivo. PA contractility did not change, however significant inhibition of NO-dependent vasodilation was observed that was maximal at 24–48h post exposure. Superoxide dismutase restored NO-dependent vasodilation suggesting a role for increased superoxide formation. This was supported by ∼2-fold increase in superoxide formation (measured using 2-hydroethidine oxidation to 2-OH-E+) from PA isolated from Cl2 exposed rats. We next measured PA pressures in anesthetized rats. Surprisingly, PA pressures were significantly (∼4mmHg) lower in rats that had been exposed to Cl2 gas 24h earlier suggesting that deficit in NO-signaling observed in isolated PA experiments did not manifest as increased PA pressures in vivo. Administration of the iNOS selective inhibitor 1400W, restored PA pressures to normal in Cl2 exposed, but not control rats suggesting that any deficit in NO-signaling due to increased superoxide formation in the PA, is offset by increased NO-formation from iNOS. These data indicate that disruption of endogenous NO-signaling mechanisms that maintain PA tone is an important aspect of post-Cl2 gas exposure toxicity.

In-vitro effects of PDE5 inhibitor and statin treatment on the contractile responses of experimental MetS rabbit's cavernous smooth muscle

Hypercholesterolaemia promotes erectile dysfunction through increased superoxide formation and decreased nitric oxide bioactivity in cavernosal tissue. The role of nitric oxide on erectile function is well known. Statins have lipid lowering properties and can modulate endothelial nitric oxide bioavailability. Sildenafil, enhances smooth muscle relaxation in corpus cavernosum. We investigated in-vitro effects of sildenafil and rosuvastatin on nonadrenergic, non-cholinergic and nitric oxide mediated cavernosal smooth muscle relaxation in metabolic syndrome rabbits, since alterations in this pathway are recognised in diabetic and hypercholesterolemic erectile dysfunction. Ten male rabbits were fed a standard diet as control group, fourty male rabbits were fed a hypercholesterolemic diet for 12 weeks. Hypercholesterolemic group were divided for without treatment, rosuvastatin treatment, sildenafil treatment, and rosuvastatin + sildenafil treatment (N = 10 per groups). Serum levels of cholesterol and glucose were significantly higher in the experimental group than in the control group (p < 0.05). After therapy no differences were found among the groups in relaxation responses to sodium nitroprusside. The relaxation responses to carbachol and EFS were significantly reduced in metabolic syndrome group to control group (p < 0.05), but there were no differences between the other groups and control group. There was a significantly lower in-vitro relaxation response in the metabolic syndrome rabbits than in controls and the others (p < 0.05). Both agents improve in-vitro relaxation responses of erectile tissue from metabolic syndrome rabbits to endothelial non-adrenergic, non-cholinergic and nitric oxide. This finding supports to the results of other clinical studies with these drugs.

Bypassing the compromised mitochondrial electron transport with methylene blue alleviates efavirenz/isoniazid-induced oxidant stress and mitochondria-mediated cell death in mouse hepatocytes

Efavirenz (EFV) is an anti-retroviral drug frequently combined with isoniazid (INH) to treat HIV-1/tuberculosis co-infected patients. Both drugs have been associated with idiosyncratic liver injury (DILI), but combined anti-retroviral and anti-tubercular therapy can increase the risk for DILI as compared to either drug class alone. Because both EFV and INH have been implicated in targeting mitochondria, we aimed at exploring whether the two drugs might cause synergistic effects on the electron transport chain. We found that EFV inhibited complex I activity in isolated mouse liver mitochondria (IC50 ˜30 μM), whereas hydrazine, a major metabolite of INH generated by acylamidase-mediated hydrolytic cleavage, inhibited complex II activity (IC50 ˜30 μM). Neither INH alone (≤1000 μM) nor EFV alone (≤30 μM) was able to induce cell injury in cultured mouse hepatocytes. However, combined EFV/INH exposure resulted in increased superoxide formation and peroxynitrite stress, leading to the opening of the cyclosporine A-insensitive mode of the mitochondrial permeability transition (mPT), and necrotic cell death. The peroxynitrite scavengers, CBA or Fe-TMPyP, protected against mPT induction and alleviated cell injury. The acylamidase inhibitor bis-p-nitrophenyl phosphate prevented cell injury, suggesting that hydrazine greatly contributed to the toxicity. Methylene blue, a redox-active alternative electron acceptor/donor that bypasses complex I/II, effectively protected against EFV/INH-induced toxicity. These data demonstrate that, in murine hepatocytes, the mitochondrial electron transport chain is a critical target of combined EFV/INH exposure, and that this drug combination can lead to peroxynitrite stress-induced mPT and hepatocellular necrosis. These results are compatible with the concept that underlying silent mitochondrial dysfunction may be a key susceptibility factor contributing to idiosyncratic drug-induced liver injury.