Trimethylamine-N-Oxide Promotes Age-Related Vascular Oxidative Stress and Endothelial Dysfunction in Mice and Healthy Humans.

Age-related vascular endothelial dysfunction is a major antecedent to cardiovascular diseases. We investigated whether increased circulating levels of the gut microbiome-generated metabolite trimethylamine-N-oxide induces endothelial dysfunction with aging. In healthy humans, plasma trimethylamine-N-oxide was higher in middle-aged/older (64±7 years) versus young (22±2 years) adults (6.5±0.7 versus 1.6±0.2 µmol/L) and inversely related to brachial artery flow-mediated dilation (r2=0.29, P<0.00001). In young mice, 6 months of dietary supplementation with trimethylamine-N-oxide induced an aging-like impairment in carotid artery endothelium-dependent dilation to acetylcholine versus control feeding (peak dilation: 79±3% versus 95±3%, P<0.01). This impairment was accompanied by increased vascular nitrotyrosine, a marker of oxidative stress, and reversed by the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl. Trimethylamine-N-oxide supplementation also reduced activation of endothelial nitric oxide synthase and impaired nitric oxide-mediated dilation, as assessed with the nitric oxide synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester). Acute incubation of carotid arteries with trimethylamine-N-oxide recapitulated these events. Next, treatment with 3,3-dimethyl-1-butanol for 8 to 10 weeks to suppress trimethylamine-N-oxide selectively improved endothelium-dependent dilation in old mice to young levels (peak: 90±2%) by normalizing vascular superoxide production, restoring nitric oxide-mediated dilation, and ameliorating superoxide-related suppression of endothelium-dependent dilation. Lastly, among healthy middle-aged/older adults, higher plasma trimethylamine-N-oxide was associated with greater nitrotyrosine abundance in biopsied endothelial cells, and infusion of the antioxidant ascorbic acid restored flow-mediated dilation to young levels, indicating tonic oxidative stress-related suppression of endothelial function with higher circulating trimethylamine-N-oxide. Using multiple experimental approaches in mice and humans, we demonstrate a clear role of trimethylamine-N-oxide in promoting age-related endothelial dysfunction via oxidative stress, which may have implications for prevention of cardiovascular diseases.

Disparate Effect of Antioxidant Supplements on the Basal Tone and Vascular Remodeling of the Aorta in Hypertensive Rats.

BACKGROUND: Oxidative stress plays an essential role in the vascular tone in hypertension; however, the mechanisms remain unclear. AIM: This study aimed to determine the antioxidant effect of tempol and vitamin C (Vit-C) on the basal tone and vascular remodeling of the aorta in nitric oxide (NO) deficiency-induced hypertensive rats. METHOD: Male Sprague-Dawley rats were induced to hypertension by Nω-nitro-L-arginine methyl ester (L-NAME). Animals were randomized as follows: vehicle (Control: CR), CR-tempol, CR-Vit-C, L-NAME, L-NAME-tempol, and L-NAME-Vit-C. After 6 weeks of treatment, the basal aortic tone was evaluated by sodium nitroprusside (SNP) and calcium-free medium. Endothelial function, NO, reduced-to-oxidized glutathione (GSH/GSSG) ratio, resting membrane potential (mP), and vascular remodeling were also measured. RESULTS: L-NAME rats showed an increased basal tone that was blunted by both SNP (-547 ± 69; n = 7 vs. CR: -7.5 ± 6.7 mg; n = 7; p < 0.001) and calcium-free medium. Tempol or Vit-C did not reverse hypertension, and the high basal tone was decreased only with tempol. In L-NAME rats, only tempol partially improved endothelial function, GSH-to-GSSG ratio, mP values, and vascular remodeling. CONCLUSIONS: Tempol decreased calcium-dependent basal aortic tone and improved vascular homeostasis in L-NAME rats. Vit-C did not lead to a similar effect, suggesting that alterations in the superoxide dismutase pathway may play a role in the basal aortic tone.

Oxidative stress impairs cGMP-dependent protein kinase activation and vasodilator-stimulated phosphoprotein serine-phosphorylation.

Reactive oxygen species induce vascular dysfunction and hypertension by directly interacting with nitric oxide (NO) which leads to NO inactivation. In addition to a decrease in NO bioavailability, there is evidence that oxidative stress can also modulate NO signaling during hypertension. Here, we investigated the effect of oxidative stress on NO signaling molecules cGMP-dependent protein kinase (PKG) and vasodilator-stimulated phosphoprotein (VASP) which are known to mediate vasodilatory actions of NO. Male Sprague Dawley (SD) rats were provided with tap water (control), 30 mM L-buthionine sulfoximine (BSO, a pro-oxidant), 1 mM tempol (T, an antioxidant) and BSO + T for 3 wks. BSO-treated rats exhibited high blood pressure and oxidative stress. Incubation of mesenteric arterial rings with NO donors caused concentration-dependent relaxation in control rats. However, the response to NO donors was significantly lower in BSO-treated rats with a marked decrease in pD2. In control rats, NO donors activated mesenteric PKG, increased VASP phosphorylation and its interaction with transient receptor potential channels 4 (TRPC4) and inhibited store-operated Ca2+ influx. NO failed to activate these signaling molecules in mesenteric arteries from BSO-treated rats. Supplementation of BSO-treated rats with tempol reduced oxidative stress and blood pressure and normalized the NO signaling. These data suggest that oxidative stress can reduce NO-mediated PKG activation and VASP-TRPC4 interaction which leads to failure of NO to reduce Ca2+ influx in smooth muscle cells. The increase in intracellular Ca2+ contributes to sustained vasoconstriction and subsequent hypertension. Antioxidant supplementation decreases oxidative stress, normalizes NO signaling and reduces blood pressure.

Translational repression of HIF2α expression in mice with Chuvash polycythemia reverses polycythemia.

Chuvash polycythemia is an inherited disease caused by a homozygous germline VHLR200W mutation, which leads to impaired degradation of HIF2α, elevated levels of serum erythropoietin, and erythrocytosis/polycythemia. This phenotype is recapitulated by a mouse model bearing a homozygous VhlR200W mutation. We previously showed that iron-regulatory protein 1-knockout (Irp1-knockout) mice developed erythrocytosis/polycythemia through translational derepression of Hif2α, suggesting that IRP1 could be a therapeutic target to treat Chuvash polycythemia. Here, we fed VhlR200W mice supplemented with Tempol, a small, stable nitroxide molecule and observed that Tempol decreased erythropoietin production, corrected splenomegaly, normalized hematocrit levels, and increased the lifespans of these mice. We attribute the reversal of erythrocytosis/polycythemia to translational repression of Hif2α expression by Tempol-mediated increases in the IRE-binding activity of Irp1, as reversal of polycythemia was abrogated in VhlR200W mice in which Irp1 was genetically ablated. Thus, a new approach to the treatment of patients with Chuvash polycythemia may include dietary supplementation of Tempol, which decreased Hif2α expression and markedly reduced life-threatening erythrocytosis/polycythemia in the VhlR200W mice.

Botany, Phytochemistry, Pharmacology and Toxicity of Strychnos nux-vomica L.: A Review.

Strychnos nux-vomica L. belongs to the genus Strychnos of the family Loganiaceae and grows in Sri Lanka, India and Australia. The traditional medicinal component is its seed, called Nux vomica. This study provides a relevant and comprehensive review of S. nux-vomica L., including its botany, ethnopharmacology, phytochemistry, pharmacology and toxicology, thus providing a foundation for future studies. Up to the present day, over 84 compounds, including alkaloids, iridoid glycosides, flavonoid glycosides, triterpenoids, steroids and organic acids, among others, have been isolated and identified from S. nux-vomica. These compounds possess an array of biological activities, including effects on the nervous system, analgesic and anti-inflammatory actions, antitumor effects, inhibition of the growth of pathogenic microorganisms and regulation of immune function. Furthermore, toxicity and detoxification methods are preliminarily discussed toward the end of this review. In further research on S. nux-vomica, bioactivity-guided isolation strategies should be emphasized. Its antitumor effects should be investigated further and in vivo animal experiments should be performed alongside in vitro testing. The pharmacological activity and toxicology of strychnine [Formula: see text]-oxide and brucine [Formula: see text]-oxide should be studied to explore the detoxification mechanism associated with processing more deeply.

Oxidative stress causes hypertension and activation of nuclear factor-κB after high-fructose and salt treatments.

There is evidence that diets rich in salt or simple sugars as fructose are associated with abnormalities in blood pressure regulation. However, the mechanisms underlying pathogenesis of salt- and fructose-induced kidney damage and/or consequent hypertension yet remain largely unexplored. Here, we tested the role of oxidative state as an essential factor along with high salt and fructose treatment in causing hypertension. Fischer male rats were supplemented with a high-fructose diet (20% in water) for 20 weeks and maintained on high-salt diet (8%) associate in the last 10 weeks. Fructose-fed rats exhibited a salt-dependent hypertension accompanied by decrease in renal superoxide dismutase activity, which is the first footprint of antioxidant inactivation by reactive oxygen species (ROS). Metabolic changes and the hypertensive effect of the combined fructose-salt diet (20 weeks) were markedly reversed by a superoxide scavenger, Tempol (10 mg/kg, gavage); moreover, Tempol (50 mM) potentially reduced ROS production and abolished nuclear factor-kappa B (NF-κB) activation in human embryonic kidney HEK293 cells incubated with L-fructose (30 mM) and NaCl (500 mosmol/kg added). Taken together, our data suggested a possible role of oxygen radicals and ROS-induced activation of NF-κB in the fructose- and salt-induced hypertension associated with the progression of the renal disease.

Effects of Tempol and Quercetin on Human Sperm Function after Cryopreservation.

　　BACKGROUND: Quercetin is a flavonoid with high reactive oxygen species (ROS) scavenging and ion chelating activity. It also enhances the activity of antioxidant enzymes and reduces enzymatic activity such as NADPH oxidase and NADH-dependent oxido-reductase. Tempol, as a superoxide dismutase mimetic agent, converts superoxide to less toxic hydrogen peroxide (H2O2), but cannot reduce highly toxic hydroxyl radicals in Fenton or Haber-Weiss reactions mediated with free iron or cupper. OBJECTIVE: The study was to compare the effect of Quercetin and Tempol in an optimized commercial cryo-protective media on ROS induced cryoinjury for the first time. MATERIALS AND METHODS: Following administration of these compounds during freezing process, sperm motility, viability, ROS production and DNA integrity were assessed before and after freezing/thawing process. RESULTS: Data showed that 10 µM Quercetin and 5 µM Tempol significantly improved sperm motility and viability, but they together had no additive effect. Supplementation with Quercetin alone or combination of Quercetin with Tempol decrease the ROS concentration, but the reduction was not significant for Tempol alone compared to control group. Quercetin and Tempol significantly decrease DNA fragmentation. CONCLUSION: The supplementation of Quercetin or Tempol, but not their combination improves the quality of cryopreserved human semen.

Targeting multiple pathways reduces renal and cardiac fibrosis in rats with subtotal nephrectomy followed by coronary ligation.

AIM: Multiple interacting pathways contribute to progression of renal and cardiac damage in chronic kidney disease followed by chronic heart failure (renocardiac syndrome). We hypothesized that simultaneous pharmacological modulation of critical pathways implicated in renocardiac syndrome would effectively reduce fibrosis in and preserve function of heart and kidney. METHODS: Rats were subjected to subtotal nephrectomy followed 9 weeks later by coronary artery ligation. From week 11 until week 16, rats received vehicle or losartan, or a combination of the NF-kB inhibitor PDTC, the NO donor molsidomine and superoxide dismutase mimetic tempol, or a combination of all four of these plus metoprolol together. At week 16, renal and cardiac structure, function and gene expression were assessed. RESULTS: Individual and combined treatments were similarly effective in limiting cardiac fibrosis and further decline in systolic function. Combined treatment with all five drugs reduced renal fibrosis and CTGF gene expression more effectively than other strategies. Combining all five drugs reduced heart rate, inotropy and mean arterial pressure (MAP). CONCLUSION: Thus, in our model of chronic renocardiac syndrome, combined treatments similarly decreased cardiac fibrosis and stabilized systolic function as losartan alone, perhaps suggesting a dominant role for a single factor such as angiotensin II type 1 (AT1) receptor activation or inflammation in the network of aberrant systems in the heart. However, tubulointerstitial fibrosis was most effectively reduced by a five-drug regimen, pointing to additive effects of multiple pathophysiological pathways in the kidney.

Novel Synthetic PEGylated Conjugate of α-Lipoic Acid and Tempol Reduces Cell Death in a Neuronal PC12 Clonal Line Subjected to Ischemia.

α-Lipoic acid (α-LA), a natural thiol antioxidant, and Tempol, a synthetic free radical scavenger, are known to confer neuroprotection following ischemic insults in both in vivo and in vitro models. The aim of this study was to synthesize and characterize a conjugate of α-LA and Tempol linked by polyethylene glycol (PEG) in order to generate a more efficacious neuroprotectant molecule. AD3 (α-Tempol ester-ω-lipo ester PEG) was synthesized, purified, and characterized by flash chromatography and reverse phase high pressure liquid chromatography and by 1H nuclear magnetic resonance, infrared spectroscopy, and mass spectrometry. AD3 conferred neuroprotection in a PC12 pheochromocytoma cell line of dopaminergic origin, exposed to oxygen and glucose deprivation (OGD) insult measured by LDH release. AD3 exhibited EC50 at 10 µM and showed a 2-3-fold higher efficacy compared to the precursor moieties, indicating an intrinsic potent neuroprotective activity. AD3 attenuated by 25% the intracellular redox potential, by 54% lipid peroxidation and prevented phosphorylation of ERK, JNK, and p38 by 57%, 22%, and 21%, respectively. Cumulatively, these findings indicate that AD3 is a novel conjugate that confers neuroprotection by attenuation of MAPK phosphorylation and by modulation of the redox potential of the cells.

Early generation of nitric oxide contributes to copper tolerance through reducing oxidative stress and cell death in hulless barley roots.

The objective of this study was to investigate the specific role of nitric oxide (NO) in the early response of hulless barley roots to copper (Cu) stress. We used the fluorescent probe diaminofluorescein-FM diacetate to establish NO localization, and hydrogen peroxide (H2O2)-special labeling and histochemical procedures for the detection of reactive oxygen species (ROS) in the root apex. An early production of NO was observed in Cu-treated root tips of hulless barley, but the detection of NO levels was decreased by supplementation with a NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO). Application of sodium nitroprusside (a NO donor) relieved Cu-induced root inhibition, ROS accumulation and oxidative damage, while c-PTIO treatment had a synergistic effect with Cu and further enhanced ROS levels and oxidative stress. In addition, the Cu-dependent increase in activities of superoxide dismutase, peroxidase and ascorbate peroxidase were further enhanced by exogenous NO, but application of c-PTIO decreased the activities of catalase and ascorbate peroxidase in Cu-treated roots. Subsequently, cell death was observed in root tips and was identified as a type of programed cell death (PCD) by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The addition of NO prevented the increase of cell death in root tips, whereas inhibiting NO accumulation further increased the number of cells undergoing PCD. These results revealed that NO production is an early response of hulless barley roots to Cu stress and that NO contributes to Cu tolerance in hulless barley possibly by modulating antioxidant defense, subsequently reducing oxidative stress and PCD in root tips.

Contribution of Cyclooxygenase End Products and Oxidative Stress to Intrahepatic Endothelial Dysfunction in Early Non-Alcoholic Fatty Liver Disease.

INTRODUCTION: Metabolic syndrome induces endothelial dysfunction, a surrogate marker of cardiovascular disease. In parallel, metabolic syndrome is frequently associated with non-alcoholic fatty liver disease (NAFLD), which may progress to cirrhosis. The aim of the present study was to evaluate intrahepatic endothelial dysfunction related to cyclooxygenase end products and oxidative stress as possible mechanisms involved in the pathophysiology of NAFLD. MATERIALS AND METHODS: Sprague-Dawley rats were fed standard diet (control-diet, CD) or high-fat-diet (HFD) for 6 weeks. Metabolic syndrome was assessed by recording arterial pressure, lipids, glycemia and rat body weight. Splanchnic hemodynamics were measured, and endothelial dysfunction was evaluated using concentration-effect curves to acetylcholine. Response was assessed with either vehicle, L-NG-Nitroarginine (L-NNA), indomethacin, tempol, or a thromboxane receptor antagonist, SQ 29548. We quantified inflammation, fibrosis, oxidative stress, nitric oxide (NO) bioavailability and thromboxane B2 levels. RESULTS: HFD rats exhibited metabolic syndrome together with the presence of NAFLD. Compared to control-diet livers, HFD livers showed increased hepatic vascular resistance unrelated to inflammation or fibrosis, but with decreased NO activity and increased oxidative stress. Endothelial dysfunction was observed in HFD livers compared with CD rats and improved after cyclooxygenase inhibition or tempol pre-incubation. However, pre-incubation with SQ 29548 did not modify acetylcholine response. CONCLUSIONS: Our study provides evidence that endothelial dysfunction at an early stage of NAFLD is associated with reduced NO bioavailability together with increased cyclooxygenase end products and oxidative stress, which suggests that both pathways are involved in the pathophysiology and may be worth exploring as therapeutic targets to prevent progression of the disease.

Tempol, a Membrane-Permeable Radical Scavenger, Exhibits Anti-Inflammatory and Cardioprotective Effects in the Cerulein-Induced Pancreatitis Rat Model.

To date, it remains unclear whether mild form of acute pancreatitis (AP) may cause myocardial damage which may be asymptomatic for a long time. Pathogenesis of AP-related cardiac injury may be attributed in part to ROS/RNS overproduction. The aim of the present study was to evaluate the oxidative stress changes in both the pancreas and the heart and to estimate the protective effects of 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (tempol) at the early phase of AP. Cerulein-induced AP led to the development of acute edematous pancreatitis with a significant decrease in the level of sulfhydryl (-SH) groups (oxidation marker) both in heart and in pancreatic tissues as well as a substantial increase in plasma creatine kinase isoenzyme (CK-MB) activity (marker of the heart muscle lesion) which confirmed the role of oxidative stress in the pathogenesis of cardiac damage. The tempol treatment significantly reduced the intensity of inflammation and oxidative damage and decreased the morphological evidence of pancreas injury at early AP stages. Moreover, it markedly attenuated AP-induced cardiac damage revealed by normalization of the -SH group levels and CK-MB activity. On the basis of these studies, it is possible to conclude that tempol has a profound protective effect against cardiac and pancreatic damage induced by AP.

Endothelial Dysfunction in Rheumatoid Arthritis: Mechanistic Insights and Correlation with Circulating Markers of Systemic Inflammation.

OBJECTIVES: To determine mechanisms involved in endothelial dysfunction (ED) during the course of arthritis and to investigate the link between cytokines, chemokines and osteoprotegerin. APPROACH AND RESULTS: Experiments were conducted on aortic rings at day 4 (preclinical), day 11 (onset of disease), day 33 (acute disease) and day 90 (chronic disease) after adjuvant-induced arthritis (AIA) in Lewis rats. At day 4, the unique vascular abnormality was a reduced norepinephrine-induced constriction. At day 11, endothelial function assessed by the relaxation to acetylcholine was normal despite increased cyclo-oxygenase-2 activity (COX-2) and overproduction of superoxide anions that was compensated by increased nitric oxide synthase (NOS) activity. At day 33, ED apparition coincides with the normalization of NOS activity. At day 90, ED was only observed in rats with a persisting imbalance between endothelial NOS and COX-2 pathways and higher plasma levels of IL-1ß and TNFα. Plasma levels of IL-1ß, TNFα and MIP-1α negatively correlated with Ach-induced relaxation throughout the course of AIA. CONCLUSIONS: Our data identified increased endothelial NOS activity as an important compensatory response that opposes the ED in the early arthritis. Thereafter, a cross-talk between endothelial COX-2/NOS pathways appears as an important element for the occurrence of ED. Our results encourage determining the clinical value of IL-1ß, TNFα and MIP-1α as biomarkers of ED in RA.

Effects of Tempol on Experimental Acute Necrotizing Pancreatitis Model in Rats.

AIM: We aimed to investigate the effects of Tempol on local organ damage in an experimental acute pancreatitis model. METHODS: This experimental study was conducted on 40 male Wistar- albino rats. The animals were randomly allocated into four groups: (i) Sham-operated group, laparotomies and cannulations of the pancreatic duct without acute necrotizing pancreatitis (ANP) (n=10); (ii) Sham + Tempol group, identical to group 1 except for intravenous tempol treatment for 4 hours (n = 10); (iii) ANP group, glycodeoxycholic acid was infused into the pancreatic duct and cerulein was infused intravenously for 6 hours for development of ANP (n=10); and (iv) ANP + Tempol treated group, in addition to the procedure in group 3, rats were administered tempol intravenously for 4 hours (n = 10). Injury of the pancreas was evaluated histopathologically. Malondialdehyde and myeloperoxidase levels of the pancreatic tissue, blood gas analysis, leukocyte and hematocrit levels were measured. Wet/dry weight of pancreatic tissue was also measured. RESULTS: Serum amylase levels, pancreatic tissue malondialdehyde and myeloperoxidase levels, wet/dry weight ratio, pancreatic edema, acinar necrosis, fat necrosis and hemorrhage, inflammation and perivascular infiltration were significantly lower in the ANP + Tempol group compared with the ANP group. CONCLUSION: Tempol infusion reduced local organ damage due to acute necrotizing pancreatitis in this experimental study. These findings demonstrate that tempol has protective effects on local organ damage due to acute necrotizing pancreatitis in rats.

Antioxidant Supplementation Modulates Age-Related Placental Bed Morphology and Reproductive Outcome in Mice.

The number of women who delay their first childbirth is increasing. This demographic shift is an important health issue because advanced maternal age is a risk factor for reproductive capacity loss and the occurrence of placental bed disorders that may lead to placenta abruption, preeclampsia, and placenta insufficiency. A redox imbalance status, resulting from the enhanced production of reactive oxygen species or their deficient neutralization, is proposed to occur in this setting. Thus, uterine redox status was evaluated in young (8- to 12-wk-old) and reproductively aged (38- to 42-wk-old) mice. In addition, it was hypothesized that specific dietary antioxidant supplementation would restore the balance and improve the reproductive outcome of aging female mice. To test this hypothesis, two different antioxidants, the nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor apocynin and the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL), were added to the drinking water of female mice prior to and during pregnancy. Compared to younger females, uteri from reproductively aged nonpregnant mice exhibited areas of endometrial cystic dilation, increased level of NOX1 expression, and enhanced protein carbonylation, especially in the apical surface of the luminal epithelium. Both antioxidants decreased protein carbonylation level in the uterus of reproductively aged mice. When reproductively aged females became pregnant, the litter size was smaller and fetuses were heavier. The change was accompanied by a significant decrease in decidua thickness. Provision of apocynin significantly increased litter size and restored decidua thickness. Reproductively aged mice provided with TEMPOL did not evidence such benefits, but whereas apocynin normalized fetal birth weight, TEMPOL further increased it. These findings emphasize that uterine redox balance is important for reproductive success and suggest that age-related redox imbalance might be compensated by specific antioxidant supplementation.

Koumine, Humantenine, and Yohimbane Alkaloids from Gelsemium elegans.

Nine new alkaloids of the koumine (1-4), humantenine (5-7), and yohimbane (8, 9) types as well as 12 known analogues were isolated from the leaves and vine stems of Gelsemium elegans. Compound 1 is the first N-4-demethyl alkaloid of the koumine type, compound 7 is the first nor-humantenine alkaloid, and compounds 8 and 9 are the first N-1-oxide and the first seco-E-ring alkaloids, respectively, of the yohimbane type. Compounds 1 and 7 exhibited moderate cytotoxicity against five human tumor cell lines with IC50 values in the range 4.6-9.3 µM.

Antiproliferative effects of mitochondria-targeted cationic antioxidants and analogs: Role of mitochondrial bioenergetics and energy-sensing mechanism.

One of the proposed mechanisms for tumor proliferation involves redox signaling mediated by reactive oxygen species such as superoxide and hydrogen peroxide generated at moderate levels. Thus, the antiproliferative and anti-tumor effects of certain antioxidants were attributed to their ability to mitigate intracellular reactive oxygen species (ROS). Recent reports support a role for mitochondrial ROS in stimulating tumor cell proliferation. In this study, we compared the antiproliferative effects and the effects on mitochondrial bioenergetic functions of a mitochondria-targeted cationic carboxyproxyl nitroxide (Mito-CP), exhibiting superoxide dismutase (SOD)-like activity and a synthetic cationic acetamide analog (Mito-CP-Ac) lacking the nitroxide moiety responsible for the SOD activity. Results indicate that both Mito-CP and Mito-CP-Ac potently inhibited tumor cell proliferation. Both compounds altered mitochondrial and glycolytic functions, and intracellular citrate levels. Both Mito-CP and Mito-CP-Ac synergized with 2-deoxy-glucose (2-DG) to deplete intracellular ATP, inhibit cell proliferation and induce apoptosis in pancreatic cancer cells. We conclude that mitochondria-targeted cationic agents inhibit tumor proliferation via modification of mitochondrial bioenergetics pathways rather than by dismutating and detoxifying mitochondrial superoxide.

On the morphology of cellulose nanofibrils obtained by TEMPO-mediated oxidation and mechanical treatment.

The morphological properties of cellulose nanofibrils obtained from eucalyptus pulp fibres were assessed. Two samples were produced with the same chemical treatment (NaClO/NaBr/TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical) oxidation), but distinct mechanical treatment intensities during homogenization. It was shown that the nanofibrils production yield increases with the mechanical energy. The effect of mechanical treatment on the yield was confirmed by laser profilometry of air-dried nanocellulose films. However, no significant differences were detected regarding the nanofibrils width as measured by atomic force microscopy (AFM) of air-dried films. On the other hand, differences in size were found either by laser diffraction spectroscopy or by dynamic light scattering (DLS) of the cellulose nanofibrils suspensions as a consequence of the differences in the length distribution of both samples. The nanofibrils length of the more nanofibrillated sample was calculated based on the width measured by AFM and the hydrodynamic diameter obtained by DLS. A length value of ca. 600 nm was estimated. The DLS hydrodynamic diameter, as an equivalent spherical diameter, was used to estimate the nanofibrils length assuming a cylinder with the same volume and with the diameter (width) assessed by AFM. A simple method is thus proposed to evaluate the cellulose nanofibrils length combining microscopy and light scattering methods.

Novel roles of hydrogen peroxide (H2O2) in regulating pectin synthesis and demethylesterification in the cell wall of rice (Oryza sativa) root tips.

Hydrogen peroxide (H2O2) has been reported to increase lignin formation, enhance cell wall rigidification, restrict cell expansion and inhibit root elongation. However, our results showed that it not only inhibited rice (Oryza sativa) root elongation, but also increased root diameter. No study has reported how and why H2O2 increases cell expansion and root diameter. Exogenous H2O2 and its scavenger 4-hydroxy-Tempo were applied to confirm the roles of H2O2. Immunofluorescence, fluorescence probe, ruthenium red staining, histological section and spectrophotometry were used to monitor changes in the degree of pectin methylesterification, pectin content, pectin methylesterase (PME) activity and H2O2 content. Exogenous H2O2 inhibited root elongation, but increased cell expansion and root diameter significantly. H2O2 not only increased the region of pectin synthesis and pectin content in root tips, but also increased PME activity and pectin demethylesterification. The scavenger 4-hydroxy-Tempo reduced root H2O2 content and recovered H2O2-induced increases in cell expansion and root diameter by inhibiting pectin synthesis, PME activity and pectin demethylesterification. H2O2 plays a novel role in the regulation of pectin synthesis, PME activity and pectin demethylesterification. H2O2 increases cell expansion and root diameter by increasing pectin content and demethylesterification.

Cytokine and radical inhibition in septic intestinal barrier failure.

BACKGROUND: Breakdown of the intestinal barrier is a driving force of sepsis and multiple organ failure. Radical scavengers or cytokine inhibitors may have a therapeutic impact on intestinal failure. Therapeutic effects on different sites of small intestine and colon have not been compared. Therefore, we investigated time-dependent intestinal permeability changes and their therapeutic inhibition in colon and small intestine with an ex vivo model. METHODS: Male Sprague-Dawley rats were either pretreated for 24 h with lipopolysaccharide (LPS) intraperitoneally alone or in combination with a radical scavenger (pyruvate or Tempol) or a cytokine inhibitor (parecoxib or vasoactive intestinal peptide). The gastrointestinal permeability was measured by time-dependent fluorescein isothiocyanate inulin diffusion using washed and everted tube-like gut segments. Blood and tissue samples were taken to investigate the development of inflammatory cytokine level (interleukin 6) in the context of cytokine inhibition and reactive oxygen species level via nicotinamide adenine dinucleotide phosphate oxidase activity in radical scavenger groups. RESULTS: After LPS treatment, mucosal permeability was enhanced up to 170% in small intestine and colon. In the small intestine the most significant reduction in permeability was found for pyruvate and parecoxib. Treatment with vasoactive intestinal peptide and parecoxib resulted in the most pronounced reduction of permeability in the colon. CONCLUSIONS: Our data suggest that cytokine inhibitors and radical scavengers have pronounced effects in LPS-induced disrupted intestinal barrier of the colon and small intestine. Our novel model comparing different anatomic sites and different points in time after the onset of sepsis may contribute to gain new insight into mechanisms and treatment options of sepsis-related gut mucosal breakdown.