Decreased SOD Activity Research Articles

Deletion of Cu/Zn Superoxide Dismutase Gene sodC Reduces Aspergillus niger Virulence on Chinese White Pear

Aspergillus niger is a common pathogenic fungus causing postharvest rot of fruit and vegetable, whereas the knowledge on virulence factors is very limited. Superoxide dismutase [SOD (EC 1.15.1.10)] is an important metal enzyme in fungal defense against oxidative damage. Thus, we try to study whether Cu/Zn-SOD is a virulence factor in A. niger. Cu/Zn-SOD encoding gene sodC was deleted in A. niger [MA70.15 (wild type)] by homologous recombination. The deletion of sodC led to decreased SOD activity in A. niger, suggesting that sodC did contribute to full enzyme activity. ΔsodC strain showed normal mycelia growth and sporulation compared with wild type. However, sodC deletion markedly increased the cell’s sensitivity to intracellular superoxide anion generator menadione. Besides, spore germination under menadione and H2O2 stresses were significantly retarded in ΔsodC mutant compared with wild type. Further results showed that sodC deletion induced higher superoxide anion production and higher content of H2O2 and malondialdehyde (MDA) compared with wild type, supporting the role of SOD in metabolism of reactive oxygen species (ROS). Furthermore, ΔsodC mutant had a reduced virulence on chinese white pear (Pyrus bretschneideri) as lesion development by ΔsodC was significantly less than wild type. The determination of superoxide anion, H2O2, and MDA in A. niger-infected pear showed that chinese white pear infected with ΔsodC accumulated less superoxide anion, H2O2, and MDA compared with that of wild type A. niger, implying that ΔsodC induced an attenuated response in chinese white pear during fruit–pathogen interaction. Our results indicate that sodC gene contributes to the full virulence of A. niger during infection on fruit. Aspergillus niger is one of the most common species found in fungal communities. It is an important fermentation industrial strain and is also known to cause the most severe symptoms in fruit during long-term storage (Pel et al., 2007). Meanwhile, plants activate their signaling pathways to trigger defense responses to limit pathogen expansion. One of the earliest host responses after pathogen attack is oxidative burst, during which large quantities of ROS are generated by different host enzyme systems, such as glucose oxidase (Govrin and Levine, 2000). ROS such as singlet oxygen, superoxide anion, hydroxyl (OH−), and H2O2 are released to hinder the advance of pathogens (Gara et al., 2003). ROS can react with and damage cellular molecules, such as DNA, protein, and lipids, which will limit fungal propagation in the host plant (Apel and Hirt, 2004).

SIRT3-SOD2-ROS pathway is involved in linalool-induced glioma cell apoptotic death.

Glioma is the most prevalent type of adult primary brain tumor and chemotherapy of glioma was limited by drug-resistance. Linalool is an acyclic monoterpene alcohol possessing various pharmacological activities. The present study was conducted to evaluate the effect of linalool on glioma cell growth. The effect of linalool on cell viability in U87-MG cells was investigated and the results showed that linalool significantly reduced cell viability in a concentration- and time-dependent manner. In addition, exposure of the cells to linalool resulted in a concentration-dependent increase of TUNEL-stained cells, indicating the occurrence of apoptotic cell death. Linalool decreased mitochondrial oxygen consumption rate, increased the expression of Bax and Bak, reduced the expression of Bcl-2 and Bcl-xl, and increased the activities of caspase 3 and caspase 9, leading to increase of apoptosis. Linalool resulted in a concentration-dependent decrease of SOD activity but had no significant effect on mRNA and protein expression of SOD2. Moreover, linalool resulted in a significant increase of the expression of acetylated SOD2. The mRNA and protein expression of SIRT3 was significantly inhibited by linalool. Immunoblot analysis showed that there was an evident protein/protein interaction between SOD2 and SIRT3 under normal condition. Linalool treatment significantly decreased the interaction between SOD2 and SIRT3. Overexpression of SIRT3 significantly inhibited linalool-induced increase of mitochondrial ROS production and apoptotic cell death, and decrease of cell viability. In summary, the data demonstrated that linalool exhibited inhibitory effect on glioma cells through regulation of SIRT3-SOD2-ROS signaling.

P 186 - Mechanisms involved in chronic effects of doxorubicin in rat hearts

Doxorubicin (DOX) is chemotherapeutic agent frequently used in treatment of many types of malignancies. Limitation of its use is toxicity on several organs. The aim of study was to investigate mechanisms involved in chronic effects of DOX on rat hearts and to search for effects of flavonoid quercetin (QCT) on DOX-induced changes. In the study, male Wistar rats were used. DOX was administered by i.p. injection of seven doses (cumulative dose 15 mg.kg-1). QCT (20 mg/kg/day) was administered 3 weeks during and 3 weeks after DOX treatment. DOX induced subcellular alterations of cardiomyocytes and disorganizations of cardiac extracellular space. Effects of DOX were associated with MMP-2 activation, decreased SOD activity, increased superoxide content, caspase-3 activation, and iNOS induction. QCT prevented the deleterious effects of DOX on ultrastructure of the tissue of left ventricle and reversed the DOX-induced effects on MMP-2 activation. QCT application also prevented effects of DOX on apoptosis induction and SOD inhibition. Moreover, QCT increased myocardial resistance to ischemia/reperfusion injury in DOX-treated rat hearts. These effects of QCT were linked to Akt kinase/GSK-3s/beta-catenin signaling and associated with connexin-43 up-regulation.

DNA damage and repair, oxidative stress and metabolism biomarker responses in lungs of rats exposed to ambient atmospheric 1-nitropyrene

1-Nitropyrene (1-NP) is a mutagenic and carcinogenic pollutant very widespread in the environment. However, the relative investigations on genotoxicity, oxidative stress and metabolic enzymes in lungs of mammalian caused by 1-NP have not been fully established. In this study, the 1-NP solutions at 3 dosages (1.0×10−5, 4.0×10−5 and 1.6×10−4mg/kg body weight) were respectively given to rats by the intratracheal instillation. The responses of 1-NP on DNA damage and repair, oxidative stress and metabolism biomarkers in rat lungs after exposure to 1-NP were measured. The results showed 1-NP at three dosages induced obvious DNA strand breaks, 8-OH-dG formation and DNA-protein cross-link in rat lungs compared with the control. Higher dosage 1-NP (4.0×10−5 and 1.6×10−4mg/kg body weight) greatly activated DNA repair gene OGG1 and inhibited MTH1 and XRCC1 expressions, and they significantly elevated the levels of GADD153, heme oxygenase-1 and malondialdehyde and decreased SOD activity, accompanied by the increases of CYP450, CYP1A1, CYP1A2 and GST levels. These results suggested the genotoxicity of 1-NP might rely on 1-NP-caused DNA damage and its combined effects on the suppression of DNA repair and the enhancement of oxidative stress and metabolic enzyme activity.

Toxicity evaluation and protective effect of Rhus longipes Engl. leaf extract in paracetamol induced oxidative stress in wister rats

Aim: Acute toxicity and protective effect of ethanol leaf extract of Rhus longipes Engl. against Paracetamol induced oxidative stress was investigated. The LD50 of the leaf extract was determined using up and down technique and the effect of 1/10th and 1/20th/ LD50 of the extract on antioxidants enzymes and non-enzymes were assessed in the serum and isolated liver of normal and Paracetamol intoxicated rats. Data obtained were analyzed by one-way analysis of variance (ANOVA) and Dunnett’s t-test was used as the test of significance. Values were considered significant at P value < 0.05. The results obtained indicated that LD50 of Rhus longipes Engl. leaf extract is greater than 5000 mg/kg /body weight. A significant (p<0.05) increase was observed in the level of hepatic (H) TBARs (81.97%), Catalase (38.42%) and serum (S) TBARs (164.44%) and catalase (64.72%) respectively but, a significant (P<0.05) decrease in hepatic activities of SOD, GPX, GR, vitamin C and E in paracetamol treated groups when compared with the serum and normal control group respectively. The extracts (250 and 500 mg/kg/body/weight) and the standard silymarin significantly (p<0.05) restored the derange antioxidants parameters to near normal in dose dependent manners. The activities of the extract at the highest concentration (500 mg/kg/b.wt) compared favourably with the standard drug. The results suggested that the leaf extract of Rhus longipes Engl. contain bioactive compounds which could protect against toxicity induced oxidative stress. The results of this study can be used as a basis for further investigations in the search for the bioactive principle.

Nitrogen or phosphorus repletion strategies for enhancing lipid or carotenoid production from Tetraselmis marina

The objective of this study was to investigate the accumulation of lipid and photosynthetic pigments from Tetraselmis marina. When the cells were grown in F/2-medium for seven days in the first stage, the carotenoid and lipid contents, and productivity were 44g/kg (DW), 27% and 31mg/L/d, respectively. After second stage of cultivation of T. marina for further 3-days under N-replete condition (4.41mM NaNO3) increased biomass concentration of 1900mg/L and lipid content of 50% were observed, with an enhanced lipid productivity of 86.36mg/L/d and SFA and MUFA fractions of 70.76 and 13.14%, respectively. However, under P-repletion (2.08mM NaH2PO4), its carotenoid content increased to 89.23g/kg and its PUFA for 65% of total lipids. Results showed that N and P-replete conditions decreased SOD activity and increased H2O2 and TBARS levels of T. marina. Thus, this native microalga strain could be a potent candidate for feed, food or biofuel production.

MG53 anchored by dysferlin to cell membrane reduces hepatocyte apoptosis which induced by ischaemia/reperfusion injury in vivo and in vitro

Hepatic ischaemia/reperfusion (HIR) induces severe damage on hepatocyte cell membrane, which leads to hepatocyte death and the subsequent HIR injury. In this study, we investigated the role and the mechanism of mitsugumin‐53 (MG53), a novel cell membrane repair protein, in protecting the liver against HIR injury. Rats were subjected to sham operation or 70% warm HIR with or without recombined MG53 (rhMG53), caudal vein‐injected 2 hrs before inducing HIR. In vitro, cultured hepatocyte AML12 cells were subjected to hypoxia/reoxygenation (H/R) in the presence of rhMG53 and/or dysferlin gene shRNAs or adenovirus transfection. HIR resulted in severe liver injury manifested as severe liver histological changes and increased AST and ALT release. Post‐ischaemic hepatic oxidative stress was significantly enhanced demonstrated by elevated dihydroethidium level, increased 4‐hydroxynonenal, enhanced 15‐F2t‐isoprostane and decreased SOD activity. rhMG53 administration attenuated post‐HIR liver injury, decreased liver oxidative stress and further enhanced dysferlin protein expression and its colocalization with MG53. Similarly, H/R induced AML12 cell injury and oxidative stress, which were abolished by either rhMG53 or dysferlin overexpression but were exacerbated by dysferlin gene knockdown. Dysferlin overexpression further increased H/R‐induced increased colocalization of MG53 and dysferlin. In conclusion, MG53 was anchored by dysferlin to reduce oxidative stress and cell death and attenuate HIR injury.

Ecophysiological responses of juvenile seabass (Dicentrarchus labrax) exposed to increased temperature and dietary methylmercury

The ecotoxicological effects of methylmercury (MeHg) exposure have been intensively described in literature. Yet, it is still unclear how marine biota will respond to the presence of MeHg under climate change, namely ocean warming. The present study aimed to investigate, for the first time, fish condition [Fulton's K index (K), hepatosomatic index (HIS) and brain-to-body mass ratio (BB-ratio)] and several stress-related responses in an ecologically and commercially important fish species (Dicentrachus labrax) exposed for 28days to dietary MeHg (8.0mg kg-1 dw) and temperature increase (+4°C). Results showed significant impairments on fish condition, i.e. up to 34% decrease on K, >100% increase on HIS and 44% decrease on BB-ratio, compared to control conditions. Significant changes on tissue biochemical responses were observed in fish exposed to both stressors, acting alone or combined, evidencing the relevance of assessing possible interactions between different environmental stressors in ecotoxicological studies. For instance, muscle showed to be the least affected tissue, only revealing significant alterations in GST activity of MeHg-enriched fish. On the other hand, liver exhibited a significant induction of GST (>100%) and CAT (up to 74%) in MeHg-enriched fish, regardless of temperature exposure, as well as decreased SOD activity (19%) and increased HSP70/HSC70 content (87%) in fish exposed to warming alone. Brain showed to be affected by temperature (69% of GST inhibition and >100% of increased CAT activity), MeHg (>100% of increased CAT activity, 47% of SOD inhibition and 55% of AChE inhibition), as well as by the combination of both (GST, SOD and AChE inhibition, 17%, 48% and 53%, respectively). Hence, our data provides evidences that the toxicological aspects of MeHg ca be potentiated by warmer temperatures, thus, evidencing the need for further research combining contaminants exposure and climate change effects, to better forecast ecological impacts in the ocean of tomorrow.

Increased renal oxidative stress in salt-sensitive human GRK4γ486V transgenic mice

We tested the hypothesis that salt-sensitive hypertension is caused by renal oxidative stress by measuring the blood pressure and reactive oxygen species-related proteins in the kidneys of human G protein-coupled receptor kinase 4γ (hGRK4γ) 486V transgenic mice and non-transgenic (Non-T) littermates on normal and high salt diets. High salt diet increased the blood pressure, associated with impaired sodium excretion, in hGRK4γ486V mice. Renal expressions of NOX isoforms were similar in both strains on normal salt diet but NOX2 was decreased by high salt diet to a greater extent in Non-T than hGRK4γ486V mice. Renal HO-2, but not HO-1, protein was greater in hGRK4γ486V than Non-T mice on normal salt diet and normalized by high salt diet. On normal salt diet, renal CuZnSOD and ECSOD proteins were similar but renal MnSOD was lower in hGRK4γ486V than Non-T mice and remained low on high salt diet. High salt diet decreased renal CuZnSOD in hGRK4γ486V but not Non-T mice and decreased renal ECSOD to a greater extent in hGRK4γ486V than Non-T mice. Renal SOD activity, superoxide production, and NOS3 protein were similar in two strains on normal salt diet. However, high salt diet decreased SOD activity and NOS3 protein and increased superoxide production in hGRK4γ486V mice but not in Non-T mice. High salt diet also increased urinary 8-isoprostane and 8-hydroxydeoxyguanosine to a greater extent in hGRK4γ486V than Non-T mice. hGRK4γwild-type mice were normotensive and hGRK4γ142V mice were hypertensive but both were salt-resistant and in normal redox balance. Chronic tempol treatment partially prevented the salt-sensitivity of hGRK4γ486V mice. Thus, hGRK4γ486V causes salt-sensitive hypertension due, in part, to defective renal antioxidant mechanisms.

Induction of Apoptosis and Cell Cycle Arrest by Flavokawain C on HT-29 Human Colon Adenocarcinoma via Enhancement of Reactive Oxygen Species Generation, Upregulation of p21, p27, and GADD153, and Inactivation of Inhibitor of Apoptosis Proteins.

Chalcones have been shown to exhibit anti-cancer properties by targeting multiple molecular pathways. It was, therefore, of interest to investigate flavokawain C (FKC), a naturally occurring chalcone, which can be isolated from Kava (Piper methysticum Forst) root extract. The aim of this study was to investigate the inhibitory effect of FKC on the growth of HT-29 cells and its underlying mechanism of action. Cell viability of HT-29 cells was assessed by Sulforhodamine B assay after FKC treatment. Induction of apoptosis was examined by established morphological and biochemical assays. ROS generation was determined by dichlorofluorescein fluorescence staining, and superoxide dismutase activity was measured using the spectrophotometric method. Western blotting was used to examine the changes in the protein levels. FKC markedly decreased the cell viability of HT-29 cells and the cells showed dramatic changes in cellular and nuclear morphologies with typical apoptotic features. The induction of apoptosis correlated well with the externalization of phosphatidylserine, DNA fragmentation, decreased mitochondrial membrane potential, activation of caspases, and PARP cleavage. This was associated with an increase in reactive oxygen species and a decrease in SOD activity. The protein levels of XIAP, c-IAP1, and c-IAP2 were downregulated, whereas the GADD153 was upregulated after FKC treatment. FKC induced cell cycle arrest at the G1 and G2/M phases via upregulation of p21 and p27 in a p53-independent manner. Our results provide evidence that FKC has the potential to be developed into chemotherapeutic drug for the treatment of colon adenocarcinoma.SUMMARY: Flavokawain C inhibited the growth of HT-29 human colon adenocarcinoma cellsFlavokawain C induced apoptosis in HT-29 cells, associated with an increase in reactive oxygen species and a decrease in SOD activityFlavokawain C induced cell cycle arrest at the G1 and G2/M phases via upregulation of p21 and p27 in HT-29 cellsHT-29 cells treated with flavokawain C caused downregulation of XIAP, c-IAP1, and c-IAP2, and upregulation of GADD153. Abbreviations used: FKC: Flavokawain C; SRB: Sulforhodamine B; ROS: Reactive oxygen species; SOD: Superoxide dismutase; PARP: Poly(ADP-ribose) polymerase; ER: Endoplasmic reticulum; IAPs: Inhibitor of apoptosis proteins; TUNEL: Transferase dUTP nick end labeling; Annexin V-FITC: Annexin V conjugated with fluorescein isothicyanate

The effects of cadmium on the biochemical and physiological parameters of Eruca sativa.

In this study, Eruca sativa (Rocket) seedlings were treated with different cadmium (Cd) concentrations (0, 150, 300 and 450 μg ∙ g-1). The effects of Cd on lipid peroxidation, enzymatic (APx, CAT, GPX, SOD) and non-enzymatic antioxidants (total ascorbate, dehydroascorbate, ascorbate, non-protein thiol), fresh and dry masses, water content were determined. Also, Cd content of the leaves and the roots were analysed. The highest cadmium accumulation of leaves was at 450 μg ∙ g-1 Cd treatment and the accumulation was 2.62 times greater than those in the roots. The translocation factor was 3.89 at 300 μg ∙ g-1 Cd treatment. Cd treatments caused decreases of fresh, dry mass and water content of leaves and roots. Malondialdehyde content, which is an index of lipid peroxidation, was increased in proportion with the increase in Cd. While there was not change in the activity of GPX according to control, a decrease in activities of SOD, CAT and APX were observed with the increase of cadmium concentration. Although a significant increase in the amounts of non-protein thiol groups and proline were observed in 450 μg ∙ g-1 Cd treated plants, Cd did not lead to a significant change in AsA, DHA and total AsA contents. According to the results of the research, E. sativa may be a Cd hyperaccumulator plant and we suggest that the plant may be a candidate plant for remediation of Cd-contaminated soil.

Perinatal hypothyroidism modulates antioxidant defence status in the developing rat liver and heart.

In the present study, we investigated oxidative stress parameters and antioxidant defence status in perinatal hypothyroid rat liver and heart. We found that the proteincarbonyl content did not differ significantly between the three groups both in the pup liver and in the heart. The OH˙ level was significantly decreased in the hypothyroid heart but not in the liver compared with controls. A slight but not significant decrease in SOD activity was observed in both perinatal hypothyroid liver and heart. A significantly increased activity of CAT was observed in the liver but not in the heart of hypothyroid pups. The GPx activity was considerably increased compared with controls in the perinatal hypothyroid heart and was unaltered in the liver of hypothyroid pups. We also found that vitamin E levels in the liver decreased significantly in hypothyroidism and were unaltered in the heart of perinatal hypothyroid rats. The GSH content was elevated significantly in both hypothyroid liver and heart. The total antioxidant capacity was higher in the liver of the hypothyroid group but not in the hypothyroid heart. Thyroxine replacement could not repair the above changes to normal. In conclusion, perinatal hypothyroidism modulates the oxidative stress status of the perinatal liver and heart.

Limb remote ischemic preconditioning attenuates liver ischemia reperfusion injury by activating autophagy via modulating PPAR-γ pathway

To investigate the effect of limb remote ischemic preconditioning (RIPC) on hepatic ischemia/reperfusion (IR) injury and the underlying mechanisms. Rats were subjected to partial hepatic IR (60 min ischemia followed by 24 hours reperfusion) with or without RIPC, which was achieved by 3 cycles of 10 min-occlusion and 10 min- reperfusion at the bilateral femoral arteries interval 30 min before ischemia. Some rats were treated with a new PPAR-γ inhibitor, T0070907, before RIPC. At the end of reperfusion, liver injury was significantly increased (increases in Suzike's injury score, AST and ALT release), concomitant with elevated oxidative stress (increases in MDA formation, MPO activity, as well as the decrease in SOD activity) and inflammation (increases in TNF-α and IL-6 levels, decrease in IL-10 content). RIPC improved liver function and reduced histologic damage, accompanied by the increased PPAR-γ activation and autophagosome formation as well as the reduced autophagosome clearance. The beneficial effects of RIPC were markedly attenuated by T0070907, an inhibitor of PPAR-γ. RIPC exerts the protective effects on liver by activation of autophagy via PPAR-γ.

Porcine placenta hydrolysates enhance osteoblast differentiation through their antioxidant activity and effects on ER stress

BackgroundOsteoporosis is a disease characterized by decreased bone strength, decreased bone mass, and bone deterioration. Oxidative damage is an important contributor to functional changes in the development of osteoporosis. Here we found that porcine placenta hydrolysates (PPHs) protect MC3T3-E1 osteoblastic cells against hydrogen peroxide (H2O2)-induced oxidative damage.MethodsIn vitro cell viability was determined using trypan blue dye exclusion. ER stress and apoptosis were evaluated using immunoblotting and a commercially available caspase kit. ALP, osteocalcin, Runx2, and osterix expression levels were evaluated by RT-PCR using isolated RNA. ROS, NADPH oxidase, and SOD activity levels were also measured.ResultsWe investigated the mechanisms underlying PPH-mediated inhibition of H2O2-induced ER stress and ROS production. PPHs also regulated osteoblast differentiation via the upregulation of alkaline phosphatase (ALP) expression in MC3T3-E1 osteoblastic cells. Also, treatment with PPHs enhanced the transcription of osteocalcin, Runx2, and osterix. These effects were all associated with the antioxidant actions of PPHs. Moreover, PPHs reversed the decrease in SOD activity, decreased ROS release, and inhibited NADPH oxidase activity in H2O2-treated MC3T3-E1 osteoblastic cells.ConclusionsPPHs protect cells against H2O2-induced cell damage when ER stress is involved. In addition, PPHs enhance osteoblast differentiation. This enhancement likely explains the regulatory effect of PPHs on bone metabolism disturbances, i.e. PPHs control ER stress and the related ROS production in osteoblasts.

Carbon allocation, osmotic adjustment, antioxidant capacity and growth in cotton under long-term soil drought during flowering and boll-forming period

Responses of plant to drought largely depend on the intensity, duration and developmental stage at which water stress occurs. The purpose of this study was to analyze the dynamic of cotton physiology response to different levels sustained soil water deficit during reproductive growth stage at leaf basis. Three levels of steady-state water regimes [soil relative water content (SRWC) maintained at (75 ± 5)%, (60 ± 5)% and (45 ± 5)%] were imposed when the white flowers had opened on the first fruiting position of the 6–7th fruiting branches (FB6-7), which was the first day post anthesis (i.e. 1 DPA) and lasted to 50 DPA. Results showed decreasing SRWC slowed cotton growth on the base of biomass and leaf area. However, carbon metabolites levels were globally increased under drought despite of notably inhibited photosynthesis throughout the treatment period. Clear diurnal pattern of sucrose and starch concentrations was obtained and sucrose levels were evaluated while starch concentration was reduced with decreasing soil water content during a 24-h cycle. Osmotic adjustment (OA) was observed at most of the sampling dates throughout the drought period. K+ was the main contributor to osmotic adjustment (OA) at 10 and 24 DPA then turned out to be amino acid at 38 and 50 DPA. The stressed cotton gradually failed to scavenge reactive oxygen species (ROS) with increasing days post anthesis, primarily due to the permanent decrease in SOD activity. Elevated carbohydrates levels suggest cotton growth was more inhibited by other factors than carbon assimilation. OA and antioxidant could be important protective mechanisms against soil water deficit in this species, and transition of these mechanisms was observed with drought intensity and duration increased.

Impaired Corpus Cavernosum Relaxation Is Accompanied by Increased Oxidative Stress and Up-Regulation of the Rho-Kinase Pathway in Diabetic (Db/Db) Mice.

Basal release of nitric oxide from endothelial cells modulates contractile activity in the corpus cavernosum via inhibition of the RhoA/Rho-kinase signaling pathway. We aimed to investigate nitric oxide bioavailability, oxidative stress and the Rho-kinase pathway in the relaxation of the corpus cavernosum of an obese and diabetic model of mice (db/db mice). We hypothesized that in db/db mice impaired relaxation induced by Rho-kinase inhibitor is accompanied by diminished NO bioavailability, increased oxidative stress and upregulation of the RhoA/Rho-kinase signalling pathway. Cavernosal strips from male lean and non-diabetic db/+ and db/db mice were mounted in myographs and isometric force in response to Rho-kinase inhibitor Y-27632 was recorded. Enzyme activity and protein expression of oxidative stress markers and key molecules of the RhoA/Rho-kinase pathway were analyzed. The Rho-kinase inhibitor Y-27632 concentration-dependently caused corpus cavernosum relaxation and inhibited cavernosal contractions. Nonetheless, a rightward shift in the curves obtained in corpus cavernosum of db/db mice was observed. Compared to db/+, this strain presented increased active RhoA, higher MYPT-1 phosphorylation stimulated by phenylephrine, and increased expression of ROKα and Rho-GEFs. Further, we observed normal expression of endothelial and neuronal NOS in corpus cavernosum of db/db mice. However, nitrate/nitrate (NOx) levels were diminished, suggesting decreased NO bioavailability. We measured the oxidant status and observed increased lipid peroxidation, with decreased SOD activity and expression. In conclusion, our data demonstrate that in db/db mice, upregulation of the RhoA/Rho-kinase signalling pathway was accompanied by decreased NO bioavailability and increased oxidative stress contributing to impaired relaxation of the corpus cavermosum of db/db mice.

MnSOD and GPx1 polymorphism relationship with coronary heart disease risk and severity.

BackgroundDisturbance of the equilibrium between reactive oxygen species (ROS) and anti-oxidants (AOX) has been implicated in various diseases, including atherosclerosis, the most common pathologic process underlying coronary heart disease (CHD). Thus, the defense systems against ROS are critical protecting blood vessel walls against oxidative damage. In this study, we investigate whether Ala16Val MnSOD and Pro198Leu GPx polymorphisms are associated with CHD susceptibility and/or severity.MethodsBoth polymorphisms were genotyped in a sample of 203 controls and 164 patients. CHD risk and severity, antioxidant status (enzymatic and/or non enzymatic) and biochemical parameters were assessed and analysed by genotype.ResultsA significant association of MnSOD variant to CHD risk was revealed in males. Males harboring the Val/Val genotype were approximately at twofold increased risk of CHD compared to controls (Ala carriers vs Val/Val, adjusted OR 1.89; 95 % CI 1.18‒3.42, p = 0.03). Significant decreases in SOD activity and total antioxidant status (TAS) were observed in Val carriers and by CHD status. Whereas, no association of GPx variant genotype (Leu/Leu) and activity to cardiopathy events was discerned. CHD severity, as demonstrated by the number of vessel stenosis, was associated with significantly higher frequency of Val allele and LDL levels in CHD subjects.ConclusionsOur results showed a lack of association of Pro198Leu GPx polymorphism to CHD risk and severity. However, they suggest that Ala16Val MnSOD polymorphism and decreased antioxidant defences are likely contributed to CHD risk in Tunisian men. Furthermore, the Val encoding MnSOD allele and decreased SOD activity were significantly correlated with CHD stenosis progression.

Abstract P140: Association Between Physical Activity and Oxidative Stress Biomarkers in Generally Healthy Women: The Nurses’ Health Study

Background: The associations between physical activity and oxidative stress biomarkers are still controversial, and few large human studies have comprehensively investigated the relationship between physical activity and oxidative stress biomarkers. The purpose of this study was to examine the association between physical activity and oxidative stress biomarkers in a large sample of women by measuring biomarkers of both oxidation and antioxidant defense. Methods: We conducted a cross-sectional study among 1,144 generally healthy women ages 43-70 years, who were included in a prospective nested case-control study of coronary heart disease in the Nurses’ Health Study. Fluorescent oxidation products (FlOPs) are oxidation markers reflecting global oxidation burden. Antioxidant defense was quantified by the activities of three major antioxidant enzymes in erythrocyte (superoxide dismutase [SOD], glutathione peroxidase [GPx] and catalase [CAT]). Self-reported physical activity was estimated in metabolic equivalents per week. The associations of physical activity with the levels of FlOPs and the activities of SOD, GPx, CAT were analyzed with linear regression models. Results: Physical activity was not associated with FlOP levels, or GPx and CAT activities after adjusting for covariates (all Ptrend > 0.15). Higher levels of physical activity were associated with decreased SOD activity (Ptrend < 0.01). We then conducted subgroup analysis of participants with and without any vigorous physical activity. Greater levels of physical activity were associated with lower SOD activity among participants with any vigorous physical activity (Ptrend = 0.02). Conclusions: Greater physical activity was associated with lower SOD activity, but not with higher plasma FlOPs in generally healthy women.

Prognostic value of the parameters of free radical oxidation in traumatic brain injury

The dynamics of lipoperoxides content and activity of antioxidant (glutathione peroxidase, superoxide dismutase, catalase) and prooxidant (xanthine oxidase) enzymes were investigated in the blood and cerebrospinal fluid of patients with traumatic brain injury of various severity depending on the left- or right-hemisphere localization of injuries. Reciprocal relationship between lipid peroxidation and oxidative modification of proteins from first to 14th day, increase of the level of total antioxidant activity, accompanied with the growth of GP and catalase activity, against the background of decrease in SOD activity from 1 to 7 day have been revealed. Were set lower "average" content of lipid peroxides in the blood and cerebrospinal fluid of patients with the subsequent development of lethal results in compare with cases of favorable outcomes, decrease of geptanofilic lipid peroxides in serum below the reference level, as well as the reduction of antioxidant activity in the blood and cerebrospinal fluid, associated with a sharp falling in superoxide dismutase activity and a significant increase of xanthine oxidase activity, which preceded the lethal results.

Redox balance influences differentiation status of neuroblastoma in the presence of all-trans retinoic acid

Neuroblastoma is the most common extra-cranial solid tumor in childhood; and patients in stage IV of the disease have a high propensity for tumor recurrence. Retinoid therapy has been utilized as a means to induce differentiation of tumor cells and to inhibit relapse. In this study, the expression of a common neuronal differentiation marker [neurofilament M (NF-M)] in human SK-N-SH neuroblastoma cells treated with 10μM all-trans retinoic acid (ATRA) showed significantly increased expression in accordance with reduced cell number. This was accompanied by an increase in MitoSOX and DCFH2 oxidation that could be indicative of increased steady-state levels of reactive oxygen species (ROS) such as O2•− and H2O2, which correlated with increased levels of MnSOD activity and immuno-reactive protein. Furthermore PEG-catalase inhibited the DCFH2 oxidation signal to a greater extent in the ATRA-treated cells (relative to controls) at 96h indicating that as the cells became more differentiated, steady-state levels of H2O2 increased in the absence of increases in peroxide-scavenging antioxidants (i.e., glutathione, glutathione peroxidase, and catalase). In addition, ATRA-induced stimulation of NF-M at 48 and 72h was enhanced by decreasing SOD activity using siRNA directed at MnSOD. Finally, treatment with ATRA for 96h in the presence of MnSOD siRNA or PEG-catalase inhibited ATRA induced increases in NF-M expression. These results provide strong support for the hypothesis that changes in steady-state levels of O2•− and H2O2 significantly contribute to the process of ATRA-induced differentiation in neuroblastoma, and suggest that retinoid therapy for neuroblastoma could potentially be enhanced by redox-based manipulations of superoxide metabolism to improve patient outcome.