Zn SOD Research Articles

Identification and characterization of an intracellular Cu, Zn-superoxide dismutase (icCu/Zn-SOD) gene from clam Venerupis philippinarum

Superoxide dismutase (SOD, EC 1.15.1.1) represents one kind of enzyme involved in scavenging the high level of reactive oxygen species (ROS) into molecular oxygen and hydrogen peroxide. In the present study, the intracellular Cu/Zn-SOD gene (icCu/Zn-SOD) of Venerupis philippinarum (denoted as VpSOD) was identified from haemocytes by homology cloning and RACR approaches. The full-length cDNA of VpSOD consisted of 910 nucleotides with a canonical polyadenylation signal sequence AATAAA, a polyA tail, and an open-reading frame of 465 bp encoding 154 amino acids. The deduced amino acid of VpSOD shared high similarity with the icCu/Zn-SODs from other species, indicating that VpSOD should be a new member of icCu/Zn-SOD family. Several highly conserved motifs including Cu, Zn binding sites (H 46, H 48, H 63, H 120 for Cu binding, and H 63, H 71, H 80, D 83 for Zn binding), intracellular disulfide bond and two Cu, Zn SOD signatures were also identified in VpSOD. The temporal expression of VpSOD in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of VpSOD mRNA was up-regulated rapidly at 6 h post-infection and reached 18-fold of the control group. After a drastic decrease at 12 h, the expression level increased again and reached 22-fold to that in the control group at 96 h post-infection. All these results indicated that VpSOD was an acute-phase protein involved in the immune responses of V. philippinarum.

EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

Changes in hepatic gene expression in response to hepatoprotective levels of zinc.

Zinc (Zn) administration at non-toxic doses protects against the hepatotoxicity produced by many agents, but the underlying mechanisms remain elusive. To examine the basis of Zn-induced generalised hepatoprotective effects. Rats and mice were given Zn at known hepatoprotective levels (100 mumol ZnCl2/kg/day, s.c., for 4 days) and molecular responses were assessed. Zn treatment produced changes in 5% of the genes on custom-designed mouse liver array and Rat Toxicology-II array. Changes in gene expression were further confirmed and extended by real-time reverse transcriptase-polymerase chain reaction. Zn treatment dramatically increased the expression of the metallothionein (Mt), and modestly increased the expression of acute-phase protein genes (ceruloplasmin, Stat3, egr1, Cxc chemokines and heat-shock proteins). For genes encoding for antioxidant enzymes, some were increased (Nrf2 and Nqo1), while others remained unaltered (Cu, Zn SOD and glutathione S-transferases). Expressions of cytokine and pro-inflammatory genes were not affected, while genes related to cell proliferation (cyclin D1) were modestly upregulated. Some metabolic enzyme genes, including cytochrome P450s and UDP-glucuronosyltransferase, were modestly suppressed, perhaps to switch cellular metabolic energy to acute-phase responses. Liver Zn content was increased between 1.6- and 2.1-fold, while hepatic MT protein was increased between 50 and 200-fold. Mice typically showed greater responses than rats. Such gene expression changes, particularly the dramatic induction of MT and Nrf2 antioxidant pathway, occur in the absence of overt liver injury, and are probably important in the hepatoprotective effects of Zn against toxic insults.

Antioxidants activities and concentration of selenium, zinc and copper in preterm and IUGR human placentas

The aim of this study was to examine changes in activities of cytochrome c oxidase (CCO), glucose-6-phosphate dehydrogenase (G6PDH), Cu–Zn superoxide dismutase (Cu–Zn SOD), glutathione peroxidase (GSH-Px), glutathione (GSH) levels and copper (Cu), zinc (Zn) and selenium (Se) concentrations, and to assess the possible differences between preterm placentas, placentas from term pregnancies complicated by intrauterine growth restriction (IUGR) and full-term control placentas. The enzyme activities and the level of GSH decreased in IUGR and preterm placentas in comparison with the control group. CCO activity and GSH level in preterm placentas were markedly lower compared with the IUGR ( P<0.01; P<0.05) and control ( P<0.01; P<0.05) placentas, respectively. In IUGR placentas the level of Cu was reduced by 23% ( P<0.05) and Zn by 37%. In preterm placentas the level of Cu was reduced by 19% and Zn by 42%. Se level in IUGR and preterm placentas was higher ( P<0.05) by 28% and 32% than in control group, respectively. The strong relation was observed between birth weight and CCO activity, birth weight and Cu–Zn SOD activity, and a low level of Zn and Cu influenced the birth weight especially in IUGR cases. Moreover, the strong inverse correlation between Se level and birth weight, Se level and placental weight and Se level and CCO activity are new findings.

Glutathione content and antioxidant enzyme expression of in vivo matured sheep oocytes

Retinoids play important roles in many diverse biological functions such as cell growth, morphogenesis, differentiation, and reproduction. Previous studies demonstrated that retinol administration to ewes, followed by natural service, resulted in embryos with improved competence to develop under standard in vitro conditions (5% CO 2 in air). Additional studies provided evidence that retinol may have some antioxidant effect by improving blastocyst development in cattle under atmospheric conditions (5% CO 2 in air). Glutathione is an important non-protein, sulphydryl compound found in oocytes and embryos, which acts to decrease oxidative stress. The purpose of the present study was to evaluate the effects of retinol administration to ewes on the content of glutathione and glutathione-related and antioxidant enzymes in in vivo matured sheep oocytes. Briefly, ewes were administered retinol or vehicle during superovulation, and after 60 h the oviducts were removed and mature oocytes collected. Glutathione content did not differ significantly between oocytes collected from retinol-treated ewes (6.78 ± 3.81 pmol/oocyte) and control ewes (6.38 ± 1.58 pmol/oocyte). Transcripts encoding for manganese superoxide dismutase (Mn-SOD), copper zinc superoxide dismutase (Cu–Zn SOD), glutathione synthetase (GS), and glutathione transferase pi (GSTp) were detected in single ovine oocytes; however, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis did not reveal any significant differences in transcripts between oocytes from retinol-treated ewes and those from control ewes.

Beneficial effects of apricot-feeding on myocardial ischemia-reperfusion injury in rats

The present study was undertaken to evaluate the cardio-protective potential of apricot-feeding in the ischemia-reperfusion (I/R) model of rats in vivo. Rats were divided into three groups of 12 rats each. Group 1 was fed with a standard rat chow, groups 2 and 3 were fed with a standard rat chow supplemented with 10% or 20% dried apricot during 3 months before the beginning of I/R studies. To produce I/R, the left main coronary artery was occluded for 30 min, followed by 120 min reperfusion, in anesthetized rats. Infarct sizes were found significantly decreased in 10% (55.0 ± 4.3%) and 20% (57.0 ± 2.9%) apricot-fed groups compared to control group (68.7 ± 2.0%). Light and electron microscopic evaluations of hearts also demonstrated similar beneficial effects on I/R injury in apricot-fed both groups. Total phenolic contents, DPPH radical scavenging and ferric-reducing power as in vitro antioxidant capacities of rat chows were significantly increased after supplementation with apricot for each ratio. Cu, Zn Superoxide dismutase (Cu, Zn SOD) and catalase (CAT) activities were increased, and lipid peroxidation was decreased significantly in the hearts of 20% apricot-fed group after I/R. In conclusion, we clearly demonstrated in vivo cardio-protective activity of apricot-feeding related to its antioxidant phenolic contents in rats subjected to myocardial I/R.

Superoxide dismutase activity as a measure of hepatic oxidative stress in cattle following ethionine administration

The goal of this study was to assess if oxidative stress, as measured by alterations in the concentrations of antioxidant enzymes in the liver and erythrocytes of cattle, could be induced following dl-ethionine administration. Whole blood, serum and liver biopsy samples were collected 0, 4, 7 and 10 days after intra-peritoneal ethionine administration to five cows. The activities of the antioxidant enzymes copper zinc superoxide dismutase (Cu, Zn SOD) and catalase were assessed in the liver biopsies which were also examined histopathologically. Significant increases in hepatic Cu, Zn SOD concentrations (P<0.01) were noted on days 7 and 10 post-treatment. Hepatic catalase activity decreased significantly (P<0.01) on days 4, 7 and 10 post-treatment and erythrocyte Cu, Zn SOD activity was significantly increased on day 10. Serum biochemical analysis revealed a significant increase (P<0.01) in non-esterified fatty acid concentrations on day 4 and significant decreases in total cholesterol and phospholipid levels on days 4 (P<0.05), 7 (P<0.01) and 10 (P<0.01). In this model system, dl-ethionine administration was effective in inducing oxidative stress particularly reflected in the liver.

Association between enzymatic and non-enzymatic antioxidant defense mechanism with apolipoprotein E genotypes in Alzheimer disease

Objective There are evidence suggesting that APOE-ɛ4 allele play an important role in the pathogenesis of Alzheimer's disease (AD) by reducing peripheral levels and activities of a broad spectrum of nonenzymatic and enzymatic antioxidants systems. However, the link between APOE genotype, oxidative stress, and AD has yet to be established. In this study we examined whether antioxidant defense mechanism exacerbates the risk of AD in individual carrying APOE-ɛ4 allele in a population from Tehran, Iran. Method We determined the enzymatic activities of the erythrocyte Cu–Zn superoxide dismutase (Cu–Zn SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and serum level of total antioxidant status(TAS) in various APOE genotypes in 91 patients with AD and 91 healthy subjects as control group (age and sex-matched). Result The results showed that the TAS level and the activities of enzymatic antioxidants CAT and GSH-Px were significantly lower and the SOD activity was significantly higher in AD patients compared to controls. The AD patients with APOE-ɛ4 allele genotype had significantly lower serum TAS concentration and lower erythrocytes GSH-Px and CAT activities ( p = 0.001) but significantly higher erythrocytes Cu–Zn SOD activity ( p = 0.001) than the non-APOE-ɛ4 carrier AD and the control group. In addition, the association observed between the factors involved in an antioxidant defense mechanism and APOE-ɛ4 allele in AD increased with age of the subjects. Conclusion These data indicate that the reduced serum level of TAS and activity of CAT, GSH-Px and increased SOD exacerbate the risk of AD in individuals carrying APOE-ɛ4 allele. The reduced antioxidants defense in APOE–ɛ4 allele carrier may contribute to beta-amyloidosis. This effect, however, is more pronounced in the AD patients older than 75 years of age. This suggests that a therapeutic modality should be considered for these subjects.

Physical vapor deposited zinc oxide nanoparticles for direct electron transfer of superoxide dismutase

This communication describes a novel and facile approach to direct electron transfer of copper, zinc–superoxide dismutase (Cu, Zn–SOD) at a physical vapor deposited zinc oxide (ZnO) nanoparticles surface. SOD exhibits quasi-reversible electrochemical behavior in phosphate buffer solution (PBS, pH 7.25), with apparent formal potential of 195.2 ± 4.6 mV vs. Ag|AgCl and high heterogeneous electron rate constant of 10.4 ± 1.8 s −1. Electrochemical results indicate that SOD stably immobilizes on the nanostructured ZnO film and processes its intrinsic enzymatic activity after adsorbed on ZnO nanoparticles. An electrostatic interaction between SOD with low isoelectric point (p I = 4.9) and ZnO nanoparticles with surface p K a of 9.35 is suggested to be the main force for the adsorption of SOD molecules on the nanostructured ZnO surface, and as a consequence, ZnO nanoparticles surface is favor for direct electron transfer of SOD. The enhanced electron transfer of SOD, together with intrinsic bidirectional catalytic activity of SOD toward superoxide anion ( O 2 - ) dismutation paves a way for the development of a third-generation O 2 - biosensor.

TGF β‐1 mediated increase in Nox‐4 expression enhances hypoxic pulmonary vasoconstriction in bovine pulmonary arteries

Hypoxic pulmonary vasoconstriction (HPV) in de‐endothelialized bovine pulmonary arteries (BPA) is potentially mediated through lowering of superoxide derived peroxide and removal of its basal relaxing effect. Simultaneous force and peroxide measurements showed decreased peroxide levels preceding HPV. Peroxide scavenging with 0.1mM ebselen increased 25mM KCl force and attenuated HPV. BPA pretreated with copper chelator 10mM diethyldithiocarbamate (DETCA), for inactivating Cu Zn SOD, increased 5μM lucigenin‐detectable superoxide and decreased horseradish peroxidase‐luminol detectable peroxide. DETCA also increased 20mM KCl force and attenuated HPV. 48 hr culture of BPA with 10nM Transforming growth factor β‐1 (TGF β‐1) increased NAD(P)H oxidase‐4 (Nox‐4) expression along with superoxide and peroxide levels. 10μM plumbagin (Nox‐4 inhibitor) reversed the increase in superoxide and peroxide with TGF β‐1, however 100μM apocynin (Nox‐2 inhibitor) did not reverse the increased superoxide level. Also TGF β‐1 decreased 20mM KCl force and increased HPV in BPA. Thus, TGF β‐1 mediated increase in Nox‐4 resulted in higher levels of superoxide derived peroxide in BPA which increased HPV.NIH grants (HL31069/HL66331).

Biochemical Characterization of G93A Mutant human Cu, Zn Superoxide Dismutase Purified from Transgenic Rat Spinal cord

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder with no cure and mutations to Cu‐Zn superoxide dismutase (SOD1) remain the only proven cause of the disease. SOD1, a 32‐kDa homodimer, is a ubiquitously expressed cytosolic antioxidant enzyme with one Cu and one Zn per monomer stabilized by an intra‐subunit disulfide (Cys 57 and Cys 146) bond. It also contains two more cysteines (Cys 6 and Cys 111) thought to be present as reduced thiols. To identify the potential mechanisms by which the mutant SOD1 (mSOD1) mediates neurotoxicity, we purified the mSOD1 from pre‐symptomatic and end stage ALS‐associated G93A mutant rat spinal cords (a model for ALS) using simple reverse‐phase preparative HPLC. The purified mSOD1 was characterized in terms of Cu and Zn levels, SOD activity, and thiol content using chromophoric chelation assay, SOD activity assay and thiol assay respectively. Our results indicate that Zn levels were significantly higher in mSOD1 purified from end stage cord to that of pre‐symptomatic cord, however no significant differences were observed in Cu levels and specific activities. Interestingly, thiol assay revealed that Cys 111 exists primarily in an oxidized state with Cys 6 primarily in the reduced state. These results suggest that biochemical impact of altered Zn affinity and cysteine modifications on SOD1 could have important implications in disease progression.

Antioxidant Activity of Bol D'Air Jacquier® Breathing Sessions in Wistar Rats — First Studies

The Bol d'Air Jacquier is used to create a molecule able to deliver oxygen at the cellular level to manage hypoxia due to environmental pollution, ageing, or inflammatory disease. This study was designed to determine, firstly, whether the device generated oxidative stress and, secondly, whether it might induce an antioxidant effect. Over a period of 62 weeks, 10 male Wistar rats were randomized into two groups: the Bol d'Air group (BA) regularly breathed peroxidizing terpens delivered by the device and the control group breathed water vapour during 9-min sessions, at the frequency of 1-12 per month. Several antioxidant compounds and KRL levels were determined in the blood and major organs. The results showed that the two groups did not differ with respect to the organ concentrations of Cu, Zn SOD, GPx, GSH, GSSG and TBARS. The device might have a weak slimming effect over time. The BA group presented a significantly higher GR level in plasma throughout the experiment, and in the muscle at the end of the study. In the BA group, the plasma Cu, Zn SOD level was related to the number of breathing sessions per week before blood collection. The BA group also had a higher KRLantioxidant status at two different time-points: at the onset of the study, in the blood of young rats; and after three breathing sessions per week, in the blood and RBCs of old rats. The device did not generate oxidative stress and seemed to produce global antioxidant effect depending on the number of sessions per week, especially in old rats.

Dietary copper requirements of juvenile grouper, Epinephelus malabaricus

To quantify dietary copper (Cu) requirements in grouper, Epinephelus malabaricus, copper sulfate was added to the basal diet at 0, 1, 2, 4, 6, 8, 10 and 20 mg Cu/kg diet providing the actual dietary value of 0.11, 1.66, 2.41, 4.37, 6.56, 8.97, 11.03 and 20.05 mg Cu/kg diet, respectively. Each diet was fed to triplicate groups of grouper (initial body weight 13.35±0.01 g) in a recirculated rearing system for 8 weeks. The Cu concentration in rearing water was monitored during the feeding period and ranged from 1.0–1.5 μg/l. Weight gain (WG) and feed efficiency (FE) were higher (P<0.05) in fish fed diets with 4.37 and 6.56 mg Cu/kg diet than those in fish fed diets with ≥11.03 and ≤1.66 mg Cu/kg diet. Hepatic thiobarbituric acid reactive substances (TBARS) value was lowest in fish fed diets with 4.37 and 6.56 mg Cu/kg diet, intermediate in fish fed diets with 11.03 and ≤1.66 mg Cu/kg diet, and highest in fish fed the diet with 20.05 mg Cu/kg diet. Differences between each of the three groups were statistically significant (P<0.05). Both hepatic copper–zinc superoxide dismutase (Cu–Zn SOD) and plasma ceruloplasmin activities in fish fell into four groups. Cu–Zn SOD value was highest in fish fed the diet with 6.56 mg Cu/kg diet, second highest in fish fed the diet with 8.97 mg Cu/kg diet, intermediate in fish fed the diet with 20.05 mg Cu/kg diet, and lowest in fish fed the diet with 0.11 mg Cu/kg diet; plasma ceruloplasmin was highest in fish fed the diet with 6.56 mg Cu/kg diet, second highest in fish fed diets with 2.41, 4.37 and 8.97 mg Cu/kg diet, intermediate in fish fed diets with ≥11.03 mg Cu/kg diet, and lowest in fish fed diets with ≤1.66 mg Cu/kg diet. All values in each of the four groups were significantly different from the values of three other groups (P<0.05). Analysis by broken-line regression of WG, hepatic SOD activity and TBARS value and linear regression of whole-body Cu retention of the fish indicate that the adequate dietary Cu concentration in growing grouper is about 4–6 mg Cu/kg diet.

Inactivation of Copper, Zinc superoxide dismutase by H 2O 2 : Mechanism of protection

Cu,Zn SOD is known to be inactivated by HO 2 − and to be protected against that inactivation by a number of small molecules including formate, imidazole, and urate. This inactivation has been shown to be due to oxidation of a ligand field histidine residue by a bound oxidant formed by reaction of the active site Cu(II) with HO 2 −. We now report that protective actions of both formate and NADH increase as the pH was raised in the range 8.0–9.5. This is taken to indicate increased accessibility of the Cu site with rising pH and/or increased reactivity of the bound oxidant toward exogeneous substrates at high pH. Formate appears to act as a sacrificial substrate that protects by competing with the endogenous histidine residue for reaction with the bound oxidant, or that repairs the damage by reducing the histidyl radical intermediate. The same is likely also true of NADH.

Curcumin attenuates gentamicin-induced renal oxidative damage in rats

The present investigation reports the effect of curcumin, an antioxidant, on gentamicin-induced-renal oxidative damage in rats. Curcumin (200 mg/kg p.o.) was administered for 2 weeks before and 1 week simultaneously with gentamicin (100 mg/kg i.p.). Saline treated rats served as control. Serum creatinine, blood urea (BUN), urinary protein, glucose, urine gamma glutamyl transferase and urine volume increased in rats treated with gentamicin while creatinine clearance decreased compared to controls P < 0.001. Renal histological examination revealed tubular necrosis. Curcumin significantly normalized the above parameters. Gentamicin decreased the activities of catalase (CAT), gutathione peroxidase (GSHPx) and the level of glutathione (GSH) but the activity of copper, zinc–superoxide dismutase (Cu, Zn–SOD) was unaltered compared to control. Curcumin attenuated the gentamicin-induced reduction in the activities of CAT, GSHPx and level of GSH by 31%, 55% and 74%, respectively. Curcumin attenuated the gentamicin-induced increases in both plasma malondialdehyde (MDA) and kidney MDA by 57% and 62%, respectively, as well as lipid hydroperoxide (LOOH) formation by 52% and 56% in rat plasma and kidney, respectively. However, Curcumin did not reduce gentamicin-induced formation of LOOH, both in the plasma and kidney, in the presence of exogenous oxidants (1 mM FeSO 4, 1 mM ascorbate, 0.2 mM H 2O 2). Our data indicate that the natural antioxidant curcumin can be a potent protective agent against renal oxidative damage mediated by gentamicin.

Reactive oxygen species metabolism in veins and arteries from rat: why is it different?

Oxidative stress plays an important pathophysiological role in the development of cardiovascular diseases. The imbalance of pro and antioxidant factors, defining oxidative stress, occurs when production of reactive oxygen species (ROS) by generators (NADPH oxidase, xanthine oxidase, uncoupled NO synthase, lipoxygenases, cyclooxygenases or the mitochondrial electron transport chain) overcomes cellular protection provided by defense systems (superoxide dismutase, catalase, glutathione and nonenzymatic scavengers). Our laboratory is researching the role of veins in the development of hypertension. We investigated ROS generation and some of its major determinants in venous and arterial blood vessels from normal rats. The superior mesenteric vein (SMV) and artery (SMA), as well as the inferior vena cava (VC) and the thoracic aorta (Ao) were compared in terms of their basal superoxide production relative to tissue weight, using a lucigenin chemiluminiscence based assay. In both cases, the vein generated more superoxide than its arterial counterpart (VC = 210 ± 42% Ao; SMV = 267 ± 48% SMA). Western analysis of homogenates from rat VC and Ao revealed that xanthine oxidase expression was significantly increased in the VC compared to the Ao (280 ± 31%), as well as catalase expression (VC = 135 ± 11% Ao) while p47phox had decreased expression in the VC compared to Ao (70 ± 11%). No significant change was observed for eNOS, RAC1 and Cu Zn SOD. These data suggest that veins differ from arteries with respect to ROS metabolism. However, the molecular mechanisms behind the different ROS handling by venous and arterial vascular tissue have yet to be uncovered.

Immunoproteomic characterization of Brucella abortus 1119-3 preparations used for the serodiagnosis of Brucella infections

The diagnosis of brucellosis is mainly based on the detection of anti-LPS antibodies. Due to substantial similarity of the O-polysaccharide of Brucella LPS to that of various other Gram-negative bacteria, serological tests of samples containing high amounts of LPS lack specificity. Hence, the development of assays based on more specific protein antigens is an essential subject in brucellosis research. The aim of this study was proteomic characterization of various antigen preparations of the diagnostic reference strain Brucella abortus 1119-3 and the identification of immunogenic proteins suitable for serological assays. Seventeen out of 383 protein spots of B. abortus 1119-3 were identified to be immunogenic by 2-D immunoblotting. These immunogenic spots were assigned to 6 proteins by MALDI-MS and nLC-ESI-MS/MS: Cu–Zn SOD, BCSP31, L7/L12, GroEL, GroES, and DnaK. All immunogenic proteins were present in three different antigen preparations investigated, i.e. native antigen, standard agglutination and commercially available agglutination antigen. 2-D immunoblotting of bacteria cross-reacting with Brucellae in agglutination tests proved that cross-reactivity of proteins is negligible. Surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) spectra also differentiated B. abortus clearly from cross-reacting bacteria. The combination of SELDI-MS analysis with the specificity of antibody binding will improve the identification of Brucella specific immunogenic proteins.

Cu–Zn super oxide dismutase as a potential antifibrotic drug for hepatitis C related fibrosis

Liver fibrosis plays a pivotal role in liver function impairment and is a feature of chronic infection by hepatitis C virus (HCV). Ten to 20% of patients progress to cirrhosis leading to an increased risk of liver failure or hepatocellular carcinoma (HCC). Recent advances have culminated in clear evidence that fibrosis can be reversible, and in expectation that effective anti-fibrotic therapy will improve the prognosis. Among the different cellular pathways involved in fibrosis, the transforming growth factor-β1 (TGF-β1) signaling plays a major role. Increases of TGF-β1 expression is associated with fibrotic diseases and this cytokine could be a target for an antifibrotic drug. Clinical results on radiation-induced fibrosis have brought some evidences that Cu–Zn SOD (SOD1) could be an anti-fibrotic drug. Its therapeutic effect could be related to a down-regulation of TGF-β1 as proved in a well characterized pig model of radiation induced fibrosis, where the efficacy of Cu–Zn SOD has been shown in reversing fibrosis. Using 3-D skin co-culture of fibroblasts from this pig model, it was showed that SOD significantly reduces the expression of the TGFβ1, both at the mRNA and protein level. An experimental work could be undertaken to validate the Cu–Zn SOD as an anti-fibrotic drug, using HCV core protein expressing transgenic mice. As the current anti-HCV therapy with pegylated interferon combined with ribavirin can eradicate virus and stop the progression of fibrosis, but near 50% of HCV infected patients are non responders, a controlled trial is planned for HCV infected patients non responders to this therapy with a Metavir fibrosis score reaching F=2 or >2.

Effect of Evodiae fructus extracts on gene expressions related with alcohol metabolism and antioxidation in ethanol-loaded mice

The effects of Evodiae fructus on alcohol concentration in blood plasma and the relative expression of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), Cu–Zn superoxide dismutase (Cu–Zn SOD), glutathione peroxidase type 5 (GPX5), and catalase (CAT) in mice were assessed 1, 2, 3, and 4 h after acute alcohol consumption. Evodiae fructus extracts (2.5, 5, and 10 mL/kg-bw), a commercial hangover removal drug (2.5, 5, and 10 mL/kg-bw), and saline solution, referred to as treatment, positive control, and negative control group, respectively, were orally given at the same time as alcohol administered to mice (20% and 40% concentration). Alcohol concentrations were the lowest in all of the Evodiae fructus treatment groups, as well as one (10 mL/kg-bw) of the positive control groups, 4 h after ethanol exposure (0–5 mg/dL and 60–110 mg/dL for 20% and 40% ethanol-loaded mice, respectively). Overall, the relative expression of ADH and Zn–Cu SOD were higher in treatment groups than in positive controls; whereas, the relative expression of GPX5 was higher in positive control groups than in treatment groups. The relative expression of ALDH and CAT was unchanged between treatments and positive controls. This study suggests that Evodiae fructus extracts can be used as a possible therapy for alcohol-induced hangover symptoms by stimulating the expression of hepatic alcohol metabolizing and antioxidant enzymes.

Impaired insulin-induced vasodilation in small coronary arteries of Zucker obese rats is mediated by reactive oxygen species

Insulin resistance (IR) and associated hyperinsulinemia are major risk factors for coronary artery disease. Mechanisms linking hyperinsulinemia to coronary vascular dysfunction in IR are unclear. We evaluated insulin-induced vasodilation in isolated small coronary arteries (SCA; approximately 225 microm) of Zucker obese (ZO) and control Zucker lean (ZL) rats. Vascular responses to insulin (0.1-100 ng/ml), ACh (10(-9)-10(-5) mol/l), and sodium nitroprusside (10(-8)-10(-4) mol/l) were assessed in SCA by measurement of intraluminal diameter using videomicroscopy. Insulin-induced dilation was decreased in ZO compared with ZL rats, whereas ACh and sodium nitroprusside elicited similar vasodilations. Pretreatment of arteries with SOD (200 U/ml), a scavenger of reactive oxygen species (ROS), restored the vasorelaxation response to insulin in ZO arteries, whereas ZL arteries were unaffected. Pretreatment of SCA with N-nitro-L-arginine methyl ester (100 micromol/l), an inhibitor of endothelial nitric oxide (NO) synthase (eNOS), elicited a vasoconstrictor response to insulin that was greater in ZO than in ZL rats. This vasoconstrictor response was reversed to vasodilation in ZO and ZL rats by cotreatment of the SCA with SOD or apocynin (10 micromol/l), a specific inhibitor of vascular NADPH oxidase. Lucigenin-enhanced chemiluminescence showed increased basal ROS levels as well as insulin (330 ng/ml)-stimulated production of ROS in ZO arteries that was sensitive to inhibition by apocynin. Western blot analysis revealed increased eNOS expression in ZO rats, whereas Mn SOD and Cu,Zn SOD expression were similar to ZL rats. Thus IR in ZO rats leads to decreased insulin-induced vasodilation, probably as a result of increased production of ROS by vascular NADPH oxidase, leading to decreased NO bioavailability, despite a compensatory increase in eNOS expression.