Manganese-containing Superoxide Dismutase Activity Research Articles

Effects of Dietary Iron Concentration on Manganese Utilization in Broilers Fed with Manganese-Lysine Chelate-Supplemented Diet.

Dietary iron (Fe) influences manganese (Mn) utilization in chickens fed with inorganic Mn-supplemented diet. This study aimed to determine if dietary Fe levels affect Mn utilization in broilers fed with organic Mn-supplemented diet. Nine hundred 8-day-old broilers were randomly assigned to 1 of 6 treatments in a 3 (Fe level) × 2 (Mn source) factorial arrangement after feeding Mn- and Fe-unsupplemented diets for 7days. The broilers were fed the basal diets (approximately 28mg Mn/kg and 60mg Fe/kg) supplemented with 0, 80, or 160mg/kg Fe (L-Fe, M-Fe, or H-Fe), and 100mg/kg Mn from Mn sulfate (MnSO4) or manganese-lysine chelate (MnLys) for 35days. The H-Fe diet decreased (P < 0.05) body weight gain and feed intake as compared with L-Fe and M-Fe diets regardless of dietary Mn sources. Dietary Fe levels did not influence (P > 0.10) serum Mn concentration in MnLys-treated broilers, but serum Mn concentration decreased (P < 0.05) with dietary Fe increasing in MnSO4-treated broilers. The Mn concentration in the duodenum and tibia decreased (P < 0.05) with increasing dietary Fe levels regardless of dietary Mn sources, and MnLys increased (P < 0.04) these indices as compared with MnSO4. Dietary Fe levels did not significantly influence (P > 0.11) Mn concentration and activity and mRNA abundance of manganese-containing superoxide dismutase (MnSOD) in the heart of MnLys-treaded broilers, but the H-Fe diet decreased (P < 0.05) these indices in MnSO4-treated broilers as compared with M-Fe and L-Fe diets. The L-Fe diet increased (P < 0.001) duodenal divalent metal transporter 1 mRNA abundance when compared with the M-Fe and H-Fe diets on day 42, regardless of dietary Mn sources. The M-Fe and H-Fe diets decreased (P < 0.001) duodenal ferroportin 1 (FPN1) mRNA level when compared with the L-Fe diet in MnSO4-treated broilers, while dietary Fe levels did not significantly influence (P > 0.40) duodenal FPN1 mRNA abundance in MnLys-treated broilers. These results indicated dietary Fe levels decreased Mn utilization in MnSO4-treated broilers, but did not influence Mn utilization in MnLys-treated broilers evaluated by Mn concentrations in the serum and heart, and the activity and mRNA expression of heart MnSOD.

Effects of Lonicera confusa and Astragali Radix extracts supplementation on egg production performance, egg quality, sensory evaluation, and antioxidative parameters of laying hens during the late laying period

Lonicera confusa (LC) and Astragali Radix (AR) extracts have been shown to have antioxidant and anti-inflammatory activities in human. To determine whether LC or/and AR extracts had similar functions to improve the egg production and egg quality, 1,440 (52-wk-old) Lohmann pink-shell hens were randomly distributed into 4 treatments with nine replicates of 40 hens. The hens were fed a basal diet (CON) or the basal diet supplemented with 0.1% LC extracts, 0.1% AR extracts, or 0.1% LC extracts plus 0.1% AR extracts (LC-AR) for 12 wk. The eggs were collected on week 6 and 12 for analysis, and the plasma and ovaries were collected at end of trial. Dietary treatment did not influence (P < 0.05) egg production, egg weight, and feed conversion ratio. However, LC-AR addition increased (P < 0.02) yolk color and sensory quality of hard-boiled eggs compared to other groups. The LC-AR supplementation increased (P = 0.02), and LC addition tended to increase (P = 0.08) Haugh unit of eggs on week 12 compared to CON. The LC-AR supplementation decreased (P < 0.001), and LC or AR individual addition tended to decrease (P < 0.10) plasma malondialdehyde concentration compared to CON. Conversely, LC supplementation increased (P = 0.02) total superoxide dismutase activity, and LC or/and AR supplementation increased the activities of manganese-containing superoxide dismutase (MnSOD) (P < 0.08) and glutathione peroxidase (GSH-Px) (P < 0.01) in plasma, and the mRNA abundance of MnSOD, GSH-Px1, and catalase in ovaries (P < 0.05) compared to CON. The LC or/and AR supplementation decreased the concentrations of interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) in plasma (P < 0.05), and the mRNA abundance of IL-6 and TNF-α in ovaries (P < 0.04) compared to CON. These results suggested dietary inclusion of LC or/and AR improved the albumen quality, and a blend of LC and AR improved yolk color, which were associated with the enhancement of antioxidant capacity and the suppression of systemic inflammation in hens.

Effects of Dietary Iron on Manganese Utilization in Broilers Fed with Corn-Soybean Meal Diet.

To investigate the effects of dietary iron (Fe) levels on manganese (Mn) utilization, 900 8-day-old broilers were randomly assigned to 1 of 6 treatments in a 3 (Fe level) × 2 (Mn level) factorial arrangement after feeding Mn- and Fe-unsupplemented diet for 7days. The broilers were then fed with basal corn-soybean meal diets (approximately 28mg Mn/kg and 60mg Fe/kg) added with 0, 80, or 160mg/kg Fe (L-Fe, M-Fe, or H-Fe), and 0 or 100mg/kg Mn for 35days. Body weight gain was lower for H-Fe broilers than that for L-Fe and M-Fe broilers. On day 42, H-Fe broilers had lower serum Mn concentration as compared with L-Fe and M-Fe broilers, and tibia Mn concentration decreased as dietary Fe increased. In Mn-supplemented broilers, liver Mn was lower in L-Fe and H-Fe treatments than that in M-Fe treatment. H-Fe treatment decreased Mn concentration and manganese-containing superoxide dismutase (MnSOD) activity in the heart when compared with L-Fe and M-Fe treatments. Dietary Fe did not significantly influence Mn concentrations in the liver and heart, and heart MnSOD activity in Mn-unsupplemented broilers. In the duodenum, L-Fe treatment decreased divalent metal transporter 1 (DMT1) mRNA abundance when compared with M-Fe and H-Fe treatments, and ferroportin 1 (FPN1) mRNA level was higher in M-Fe treatment than that in L-Fe and H-Fe treatments. These results suggested H-Fe diet decreased Mn status in broilers evaluated by Mn concentrations in serum and heart, and heart MnSOD activity. Dietary Fe influenced Mn absorption possibly through effects on duodenal DMT1 and FPN1 expression.

The Influence of Nanoparticles of Biogenic Ferrihydrite on the Rooting of Lignified Cuttings of the Ledebour Willow

The influence of nanoparticles of biogenic ferrihydrite on the root formation of lignified cuttings of the Ledebour willow (Salix ledebouriana Trautv.) was investigated. The rooting of stem cuttings was performed in water using a phytohormone (indoyl-3-acetic acid) and nanoparticles. In comparison with the variants of incubation of cuttings in water and in a solution containing indolyl-3-acetic acid, the number and total length of the adventitious roots after 5 and 15 days of rooting were greatest in the cuttings treated with nanoparticles. In the early period of root development, increased levels of free radicals and intensities of the hyperfine structure lines of Mn2+ cleavage were recorded in the EPR spectra of plant tissues (lenticels of cuttings of treated with nanoparticles). The latter is related to the activity of manganese-containing superoxide dismutase. The response of cuttings to the treatment with nanoparticles was recorded by EPR before manifestation of adventitious root primordium initiation and development.

Integrated response of antioxidant biomarkers in the liver and white muscle of European hake (Merluccius merluccius L.) females from the Adriatic sea with respect to environmental influences

We investigated the integrated response of antioxidant defense enzymes (total superoxide dismutase (TotSOD), manganese-containing superoxide dismutase (MnSOD), copper-zinc-containing superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and phase II biotransformation enzyme, glutathione- S-transferase (GST)) in the liver and white muscle of females of European hake (Merluccius merluccius L.) from the Adriatic Sea (Montenegro) in winter and spring. The activity of GSH-Px in the liver was significantly increased, while GST activity was decreased in spring compared to the winter. In white muscle, the activities of TotSOD and CuZnSOD were increased, while the activities of MnSOD, CAT, GSH-Px, GR and GST were decreased in spring when compared to the matching values in winter. The activities of TotSOD and CuZnSOD in winter were markedly lower in the muscle than in the liver, while the activity of MnSOD in the muscle was higher when compared to the liver. Principal component analysis (PCA) revealed clear separation of the investigated antioxidant biomarkers between tissues and seasons, while the integrated biomarker response (IBR) showed that the most intensive antioxidant biomarker response was in the liver in spring. Star plots of IBR showed a dominant contribution of glutathione-dependent biomarkers (GSH-Px, GR and GST) and CAT in both tissues and seasons with respect to SOD isoenzymes. All enzyme activities (except MnSOD) were greater in the liver in comparison to the white muscle. Our results show that the liver possesses a greater capacity to establish and maintain homeostasis under changing environmental conditions in winter and spring. At the same time, seasonal effects are more pronounced in muscle tissue.

ZYZ451 protects cardiomyocytes from hypoxia-induced apoptosis via enhancing MnSOD and STAT3 interaction

3,5-dimethoxy-4-(2-amino-3-prop-2-ynylsulfanyl-propionyl)-benzoic acid 4-guanidino-butyl ester (ZYZ451) was found to be an excellent cardio-protective agent in the previous research in our lab. However, its potent therapeutic effects on myocardial infarction and the underlying mechanism remain elusive. In the present study, we demonstrate that ZYZ451 protects neonatal rat ventricular cardiomyocytes (NRVCs) from hypoxia-induced apoptosis via increasing manganese-containing superoxide dismutase (MnSOD) activity and inhibiting mitochondrial reactive oxidative species (mitoROS) production. MnSOD knockdown impairs the anti-apoptotic effects of ZYZ451. We report here for the first time that signal transducer and activator of transcription 3 (STAT3), an important nuclear transcriptional factor also identified in mitochondria, co-localizes with MnSOD and interacts with it, as determined by using methods of co-immunofluorescence and co-immunoprecipitation. Knockdown of STAT3 rather than inhibition of STAT3 phosphorylation results in a significant reduction in MnSOD activity. Furthermore, interaction between MnSOD and STAT3 is diminished in STAT3 deficient H9C2 cells. Its novel subcellular localization and interaction with MnSOD suggest that STAT3 may be involved in regulation of MnSOD activity beyond its transcriptional potential. Consistent with the results in vitro, ZYZ451 reduces myocardial infarct size as well as cardiomyocytes apoptosis, inhibits lactate dehydrogenase (LDH) and malondialchehyche (MDA) release, and restores MnSOD activity in peri-infarct hearts. These benefits appear to be attributed to the enhanced interaction between STAT3 and MnSOD. These findings shed a light on a new role of STAT3 in oxidative stress and suggest that ZYZ451 is likely an effective cardio-protective agent.

Factors Affecting the Induction of Lignin Peroxidase in Manganese-Deficient Cultures of the White Rot Fungus &amp;lt;i&amp;gt;Phanerochaete chrysosporium&amp;lt;/i&amp;gt;

The lignin peroxidase (LIP) production and regulation, in manganese ions (Mn2+) deficient cultures of the white rot fungus Phanerochaete chrysosporium, is still not clearly understood. Mn2+ deficiency is correlated to low levels of manganese containing superoxide dismutase (MnSOD). In this work, we show that despite the low activity level of MnSOD in Mn2+-deficient cultures, the presence of H2O2 is essential for the expression of the lip-H2 gene, which encodes for the major LIP isoenzyme produced (LIP-H2). Thus, the H2O2 present in Mn2+-deficient cultures is probably produced by other mechanisms rather than dismutation of superoxide ions by MnSOD. Glyoxal oxidase gene (glox) expression was significantly higher than MnSOD (MnSOD1) and cellobiose dehydrogenase (cdh1) expression in Mn2+-deficient cultures, indicating its clear involvement in H2O2 production in those cultures. Glyoxal oxidase may compensate the absence of MnSOD activity in Mn2+-deficient cultures. The high levels of reactive oxygen species (ROS) needed for the enhancement of LIP expression in Mn2+-deficient cultures were not directly correlated to the protein kinase C (PKC) activity involved in signal transduction pathway. High level of oxidative stress was observed in MnSOD silenced mutants, grown in the presence of Mn2+, indicating that oxidative stress in Mn2+-deficient cultures was caused by low levels of MnSOD rather than the deficiency in Mn2+. The results of this work can further contribute to the understanding of LIP regulation in Mn2+-deficient cultures.

Cloning and characterization of a differentially expressed mitochondrial manganese superoxide dismutase gene from Pleurotus ostreatus

Manganese-containing superoxide dismutase (Mn-SOD) is one of the most important superoxide dismutases found in many eukaryotes and bacteria. In this study, the full-length cDNA of mitochondrial manganese superoxide dismutase from Pleurotus ostreatus (PoMn-SOD) was obtained. It contained 776 nucleotides with an open reading frame (ORF) of 663 bp, encoding 220 amino acid residues. The deduced amino acid sequence showed high identity with the sequences of other basidiomycetous Mn-SODs from Trametes versicolor (71 %) to Laccaria bicolor (77 %). Quantitative real-time PCR (RT-PCR) analysis revealed that PoMn-SOD gene transcripts were abundant in the stage of young fruit bodies and mature fruit bodies. The up-regulation of the PoMn-SOD gene suggested that it was developmental regulated and could play an important role in antagonizing environmental stresses. In addition, another isoform of Mn-SOD was detected by a non-denaturing polyacrylamide gel electrophoresis (PAGE) approach and the highest total Mn-SOD activity (203.9 U/mg) was observed in the stage of mature fruit bodies. These data could provide important reference for functional study of the PoMn-SOD gene and may benefit biotechnological production of Mn-SOD in the future.

Manganese Regulates Manganese-Containing Superoxide Dismutase (MnSOD) Expression in the Primary Broiler Myocardial Cells

Previous studies showed that dietary manganese can increase the MnSOD mRNA expression in a dose-dependent manner in the heart of broilers. In order to explore the specific mechanism of the MnSOD expression induced by manganese, a model of MnSOD expression was developed with primary cultured broiler myocardial cells. The objective of the present study was to investigate whether the model was working or not and to determine how manganese affects the expression of the enzyme in broiler myocardial cells in vitro. In experiment 1, various amount of manganese (0, 0.25, 0.5, 1, 2, and 4mM) were added into the cultures for 24-h incubation to investigate MnSOD expression and for 0-, 6-, 12-, 24-, 36-, and 48-h incubation to measure the cell viability. In experiment 2, the most suitable Mn supplementation based on the results of experiment 1 was added into cultures for 6-, 12-, 24-, and 48-h incubation. The results showed that MnSOD mRNA, MnSOD protein, and MnSOD activity were induced by manganese in dose- and time-dependent manner. Manganese regulates MnSOD expression not only at transcriptional level but also at translational and/or posttranslational levels.

An Estimation of the Manganese Requirement for Broilers from 1 to 21 Days of Age

A dose-response experiment was conducted to find the sensitive and consistent biomarker for the estimation of dietary manganese (Mn) requirement and establish the optimal Mn level for broilers fed a practical corn-soybean meal diet from 1 to 21days of age post-hatching. A total of 480 1-day-old Arbor Acres male chicks were randomly allotted to one of eight treatments with five replicates of 12 birds each and fed diets supplemented with 0, 20, 40, 60, 80, 100, 120, or 140mg Mn/kg from reagent grade Mn sulfate. Tissue Mn concentrations, manganese-containing superoxide dismutase (MnSOD) activity, and MnSOD mRNA concentration within heart tissue were analyzed at 7, 14, and 21days of age. Tissue Mn concentrations and heart MnSOD activity showed significant quadratic responses, and heart MnSOD mRNA concentration showed an asymptotic response to dietary supplemental Mn level, respectively. The estimate of dietary Mn for chicks from 1 to 21days of age was 122-128 for heart Mn concentration, 141-159 for pancreas Mn concentration, 127-138 for liver Mn concentration, and 135-156mg/kg for heart MnSOD activity, respectively. Heart MnSOD mRNA concentration was a consistent index for the estimation of the Mn requirement of broilers. Based on this index, the estimate of dietary Mn requirement for broilers from 1 to 21days of age post-hatching was about 130mg/kg, which was a little more than two times of the current NRC (1994) requirement.

Effects of oxygen, growth state, and senescence on the antioxidant responses of WI-38 fibroblasts

Mitotically active, growth-arrested cells and proliferatively senescent cultures of human fetal lung fibroblasts (WI-38) were exposed to six different oxygen tensions for various lengths of time and then analyzed to determine the responses of their antioxidant defense system. Glutathione (GSH) concentration increased as a function of ambient oxygen tension in early passage cultures; the effect was larger in exponentially growing cultures than in those in a state of contact-inhibited growth arrest, but was absent in senescent cells. Conversely, the activity of glutathione disulfide reductase was greater in growth-arrested cultures than in mitotically active cells irrespective of oxygen tension. Glucose-6-phosphate dehydrogenase was lowest in log-phase cells exposed to different oxygen tensions for 24 h and in senescent cells. Both hypoxia and hyperoxia depressed selenium-dependent glutathione peroxidase activity in early passage cultures, while the activity of the enzyme progressively declined with increasing oxygen in senescent cells. The GSH S-transferase activity was unresponsive to changes in ambient oxygen tension in either young or senescent cultures. Manganese-containing superoxide dismutase (MnSOD) activity was unaffected by oxygen tension, but was elevated in young confluent cultures as compared with cultures in log-phase growth. MnSOD activity was significantly higher in senescent cultures than in early passage cultures and was also responsive to increased oxygen tension in senescent cultures. Copper–zinc-containing superoxide dismutases activity was not affected by oxygen tension or the passage of time, but it declined in senescent cultures.

Enzymatic Activity Is Necessary for the Tumor-Suppressive Effects of MnSOD

The antioxidant protein manganese-containing superoxide dismutase (MnSOD) has been found to be a new type of tumor-suppressor protein. Overexpression of the cDNA for this gene in various types of cancer via plasmid transfection or adenovirus transduction leads to growth suppression both in vitro and in vivo. The growth-suppressive effect of MnSOD overexpression has been presumed to be due to the enzymatic activity of the MnSOD protein, but could be due to a number of other mechanisms, including a regulatory effect of the RNA or protein produced. To examine this question, we used site-directed mutagenesis to produce a mutant form of human MnSOD that has a leucine at amino acid 26 in the active site rather than the usual histidine. We demonstrate that plasmid transfection or adenoviral transduction of this mutant MnSOD cDNA leads to a large increase in immunoreactive MnSOD protein, but little or no increase in enzymatic activity. In contrast, overexpression of wild-type MnSOD leads to cells with both increased MnSOD protein and activity. Overexpression of wild-type, but not mutant, MnSOD leads to decreased plating efficiency and growth. These results clearly demonstrate that the tumor-suppressive effect of MnSOD protein is largely due to its enzymatic activity.

Antioxidant N-acetylcysteine restores myocardial Mn-SOD activity and attenuates myocardial dysfunction in diabetic rats

Manganese-containing superoxide dismutase (Mn-SOD) plays a critical role in guarding against mitochondrial oxidative stress. Abnormal myocardial mitochondrial metabolism of reactive oxygen species plays an important role in the pathogenesis of diabetic cardiac dysfunction. We hypothesised that chronic treatment with N-acetylcysteine, an antioxidant and glutathione (GSH) precursor, would normalize hyperglycemia induced inactivation of Mn-SOD and attenuate myocardial dysfunction. Control and streptozotozin-induced diabetic rats were treated or untreated with N-acetylcysteine in drinking water for 8 weeks, initiated 1 week after streptozotozin injection. Myocardial performance was determined using the isolated perfused working heart preparation. Myocardial Mn-SOD activity, but not Mn-SOD protein expression, in diabetic rats was significantly reduced while levels of oxidative stress as determined by myocardial free 15-F 2t-isoprostane were increased in diabetic rats and were normalized by N-acetylcysteine treatment. However, compensatory increases in myocardial Cu/Zn-SOD and GSH content were seen in diabetic rats accompanied by an increase in tissue antioxidant capacity as compared to control. N-acetylcysteine abolished the compensatory increase in myocardial Cu/Zn-SOD. The left ventricular developed pressure and rates of left ventricular pressure development and relaxation were decreased in diabetic rats as compared to control. These effects were attenuated, but not prevented by N-acetylcysteine treatment. N-acetylcysteine attenuation of diabetic myocardial dysfunction could be attributed to the restoration of myocardial Mn-SOD activity.

Antioxidant N‐acetylcysteine restores myocardial Mn‐SOD activity and attenuates left ventricular diastolic dysfunction in diabetic rats

Manganese-containing superoxide dismutase (Mn-SOD) plays a critical role in guarding against mitochondrial oxidative stress (OS). Abnormal myocardial mitochondrial metabolism of reactive oxygen species plays an important role in the pathogenesis of diabetic cardiac dysfunction. We hypothesized that chronic treatment with N-acetylcysteine (NAC), an antioxidant, would normalize hyperglycemia induced inactivation of Mn-SOD and attenuate myocardial dysfunction. Control and streptozotozin-induced diabetic rats were treated (CT, DT) or untreated (C, D) with NAC in drinking water for 8 weeks. Myocardial Mn-SOD activity, but not Mn-SOD protein expression, in D rats was significantly reduced while levels of OS as determined by myocardial free 15-F2t-isoprostane was increased as compared to all other groups. This was accompanied by increased membrane translocation of p67phox, a cytosolic subunit of the NAD(P)H oxidase whose activation may contribute to the elevated OS in diabetes. A decrease in left ventricular maximum relaxation (-dP/dT) and an increase in the t1/2 to maximum relaxation were found in D rats. NAC normalized or attenuated the changes seen in D group. The results suggest that OS-mediated inactivation of myocardial Mn-SOD may contribute to the development of myocardial diastolic dysfunction in diabetes. This study was funded by GIA from Canadian Diabetes Association and a Program Grant from the Heart & Stroke Foundation of BC and Yukon, Canada. ZX and JJ are supported by CIHR/Rx &D HRF.

Cerebral Oxidative Stress and Mitochondrial Dysfunction in Oxonate-Induced Hyperuricemic Mice

Relation between blood uric acid levels and brain cell functions were examined using potassium oxonateinduced hyperuricemic mice and allopurinol-induced hypouricemic mice. In hyperuricemic mice and hypouricemic mice, erythrocyte catalase activity was significantly lower and higher, respectively, than that in the control group. No significant change of superoxide dismutase (SOD) activity or glutathione peroxidase activity was observed in either group. Cerebral lipid peroxide levels expressed as thiobarbituric acid reactive substances (TBARS) were significantly higher in hyperuricemic mice, and cerebral mitochondrial ATP synthesis and manganese-containing SOD (Mn-SOD) activity were significantly diminished in the same mice. In hypouricemic mice, no significant change was observed for TBARS levels, mitochondrial ATP synthesis and Mn-SOD activity. Then significant negative correlation between erythrocyte catalase activity and cerebral TBARS levels, and significant positive correlation between catalase activity and mitochondrial ATP synthesis were confirmed. Furthermore, when mouse erythrocytes were treated with uric acid, their catalase activities were particularly diminished. These results suggest that the reduction of erythrocyte catalase activity resulted from the elevation of blood uric acid levels is a major cause of cerebral oxidative stress and mitochondrial dysfunction in hyperuricemic mice. Therefore, it is possible that hyperuricemia and gout are risk factors of cerebral disorders.

Reversible modulation of cell cycle kinetics in NIH/3T3 mouse fibroblasts by inducible overexpression of mitochondrial manganese superoxide dismutase.

To study the mechanism(s) by which manganese-containing superoxide dismutase (MnSOD) mediates cellular growth inhibition, an inducible retroviral vector system regulated by the lac repressor was used to overexpress MnSOD protein in NIH/3T3 cells. Increased MnSOD activity led to decreased cell growth due to prolonged cell cycle transition times in G(1) and S phases without significant changes in G(2)/M phase. Changes in cell cycle transition time were reversible and tightly correlated with MnSOD levels. A transient increase of reactive oxygen species and concomitant decrease in mitochondrial membrane potential were documented following MnSOD induction. N-Acetyl-L-cysteine prevented growth inhibition by MnSOD. Our data suggest that MnSOD may serve a physiological function of regulating cell cycle progression through its prooxidant activity of generating hydrogen peroxide, resulting in coordination of mitochondrial redox state and cellular proliferation.

The plasma superoxide scavenging activity in canine cancer and hepatic disease.

To clarify the relationship between plasma antioxidant activity and diseases in dogs, plasma samples were collected from 6 healthy dogs and 16 diseased dogs (6 dogs with cancer, 5 dogs with hepatic disease, and 5 dogs with inflammation ), and measured superoxide anion scavenging activities. Antioxidant activities of canine plasma were evaluated by measuring their superoxide anion (O(2)(-.)) scavenging activities with electron spin response spectroscopy combined with spin trapping reagent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Total O(2)(-.) scavenging activities in the presence of plasma of diseased dogs tended to be higher than those in healthy controls, especially significant higher activities in the presence of canine plasma of hepatic disease and inflammation were observed. In diseased dogs, KCN-insensitive activities, suggesting the activity of manganese-containing superoxide dismutase (Mn-SOD), were significantly higher than those in healthy controls. Therefore, it seems that there is a possibility of utilizing of plasma O(2)(-.) scavenging activity as one of clinical indicators for oxidative-related diseases such as cancer, hepatic disease and inflammation in dogs.

Involvement of endogenous salicylic acid content, lipoxygenase and antioxidant enzyme activities in the response of tomato cell suspension cultures to NaCl.

• The effects of salt stress and adaptation on salicylic acid (SA) content and on antioxidant and lipoxygenase (LOX) enzyme activities were studied in tomato (Lycopersicon esculentum cv. Pera) cells. • NaCl-adapted cells were obtained from calli adapted to 100mm NaCl by successive subcultures in medium supplemented with 100mm NaCl. Salt stress treatments consisted of the addition of 100mm NaCl to cells. • Adapted cells contained a lower concentration of SA than unadapted cells. The lower manganese-containing superoxide dismutase (Mn-SOD) and LOX activities as well as the higher glutathione reductase (GR) and ascorbate peroxidase (APX) activities in adapted cells than in unadapted cells could be correlated with the development of salt adaptation. Salt stress increased APX and LOX activities as well as lipid peroxidation in unadapted cells and increased Mn-SOD activity in both types of cells. Application of 200µm SA+100mm NaCl inhibited APX activity in both unadapted and adapted cells, induced the Mn-SOD in adapted cells and increased lipid peroxidation in unadapted cells. • Our data indicate that adaptation of tomato cells to NaCl results in a higher tolerance to NaCl-induced oxidative stress and suggest a role for SA in this response.

Correlation between antioxidant status, tumorigenicity and radiosensitivity in sister rat cell lines.

Tumorigenicity and radiosensitivity of related cell lines expressing distinct p53 mutants were analyzed in parallel with key components of the antioxidant metabolic pathway. Six sublines deriving from the same parental cell population and expressing either the mutant p53K130R or p53V270F were investigated. Both mutations abrogate the transcriptional activity of p53 as well as its ability to induce apoptosis. The cells expressing p53K130R showed a higher tumorigenicity and a higher radiosensitivity than those expressing p53V270F. An increase in tumorigenicity was associated with a decrease in manganese-containing superoxide dismutase activity, and with further decreases in the glutathione content and glutathione peroxidase (GPX) activity. A positive correlation was found between GPX activity, glutathione content and cell survival following ionizing irradiation. The fact that sister cell lines exhibit different tumorigenicity and radiosensitivity while expressing a mutant p53 further supports the notion that knowledge of p53 status is not sufficient to predict tumor outcome, especially the response to irradiation. A better understanding of antioxidant defenses might be more informative.

Redox regulation of adenovirus-induced AP-1 activation by overexpression of manganese-containing superoxide dismutase.

Adenovirus gene therapy is a promising tool in the clinical treatment of many genetic and acquired diseases. However, it has also caused pathogenic effects in organs such as the liver. The redox-sensitive transcription factors AP-1 and NF-kappaB have been implicated in these effects. To study the mechanisms of adenovirus-mediated AP-1 and NF-kappaB activation and the possible involvement of oxidative stress in adenovirus transduction, rats were injected with either replication-defective recombinant adenovirus with DNA containing the cytomegalovirus promoter region only (AdCMV), adenovirus containing human manganese-containing superoxide dismutase (MnSOD) cDNA (AdMnSOD), or vehicle. Compared to vehicle and AdCMV transduction, MnSOD gene transfer yielded a fivefold increase in liver MnSOD activity 7 days postinjection. Gel shift assay showed that AdCMV transduction induced DNA binding activity for AP-1 but not NF-kappaB. MnSOD overexpression abolished this activation. Western blotting analysis of c-Fos and c-Jun suggested that up-regulation of c-fos and c-jun gene expression does not directly contribute to the induction of AP-1 activation. Glutathione/glutathione disulfide ratios were decreased by adenovirus transduction and restored by MnSOD overexpression. The AP-1 binding activity that was induced by AdCMV was decreased by immunoprecipitation of Ref-1 protein. Ref-1 involvement was confirmed by restoration of AP-1 binding activity after the immunoprecipitated Ref-1 protein had been added back. AP-1 DNA binding activity was also elevated in control and AdMnSOD-injected rats after addition of the immunoprecipitated Ref-1 protein. These data indicate that cellular transduction by recombinant adenovirus stimulates AP-1 DNA binding activity. Furthermore, our results suggest that MnSOD overexpression decreases AP-1 DNA binding activity by regulating intracellular redox status, with the possible involvement of Ref-1 in this redox-sensitive pathway.