CuZnSOD Activity Research Articles

Lactulose Increases Equol Production and Improves Liver Antioxidant Status in Barrows Treated with Daidzein

Equol, one of the intestinal microflora metabolites of daidzein, has gained much attention for having greater bioactivity than its precursor (daidzein and daidzin) and seeming to be promoted by hydrogen gas. The effects of lactulose on the equol-producing capacity and liver antioxidant status of barrows treated with daidzein were investigated in this study. Male castrated piglets (barrows) of Landrace×Duroc, aged 40 days, were randomly divided into the following three groups: control group (C, n = 12, fed an isoflavones-free basic diet), daidzein group (D, n = 12, fed an isoflavones-free basic diet with 50 mg/kg of daidzein supplementation) and daidzein+lactulose group (D+L, n = 12, fed an isoflavones-free basic diet with 1% of lactulose and 50 mg/kg of daidzein supplementation). After 20 days, the profile of short-chain fatty acids in the colon digesta showed that lactulose significantly increased the fermented capacity in the gastrointestinal tract of the barrows. First-void urinary equol concentrations were significantly higher in the D+L group than in the D group (3.13±0.93 compared to 2.11±0.82 μg/ml, respectively). Furthermore, fecal equol levels were also significantly higher in the D+L group than in the D group (12.00±2.68 compared to 10.00±2.26 μg/g, respectively). The population of bacteroidetes and the percentage of bacteroidetes to bacteria in feces were higher in the D+L group than in the D group. The DGGE profiles results indicate that lactulose might shift the pathways of hydrogen utilization, and changing the profiles of SRB in feces. Moreover, the D+L group had weak enhancement of T-SOD and CuZn-SOD activities in the livers of barrows treated with daidzein.

Cloning of a putative extracellular Cu/Zn superoxide dismutase and functional differences of superoxide dismutases in invasive and indigenous whiteflies.

Superoxide dismutases (SODs) are a group of important antioxidant defense enzymes. In this study, a putative extracellular Cu/Zn superoxide dismutase (ecCuZnSOD) complementary DNA was cloned and characterized from the whitefly, Bemisia tabaci. Quantitative polymerase chain reaction analysis showed that the expression level of BtecCuZnSOD was more than 10-fold higher in the invasive Middle East Asia Minor 1 (MEAM1) than in the native Asia II 3 species of the B. tabaci species complex. After exposure to low temperature (4 °C), the expression of Bt-ecCuZnSOD gene was significantly up-regulated in MEAM1 but not in Asia II 3. Furthermore, the expression level of B. tabaci intracellular CuZnSOD (Bt-icCuZnSOD), Bt-ecCuZnSOD and mitochondrial MnSOD (Bt-mMnSOD) was compared after transferring MEAM1 and Asia II 3 whiteflies from favorable (cotton) to unfavorable host plants (tobacco). On cotton, both CuZnSOD genes were expressed at a higher level in MEAM1 compared with Asia II 3. Interestingly, after transferring onto tobacco, the expression of Bt-ecCuZnSOD was significantly induced in Asia II 3 but not in MEAM1. On the other hand, while Bt-mMnSOD was expressed equally in both species on cotton, Bt-mMnSOD messenger RNA was up-regulated in MEAM1 on tobacco. Consistently, enzymatic activity assays of CuZnSOD and MnSOD demonstrated that CuZnSOD might play an important protective role against oxidative stress in Asia II 3, whereas MnSOD activation was critical for MEAM1 whiteflies during host adaptation. Taken together, our results suggest that the successful invasion of MEAM1 is correlated with its constitutive high activity of CuZnSOD and inducible expression of MnSOD under stress conditions.

Chitosan-Zn Chelate Increases Antioxidant Enzyme Activity and Improves Immune Function in Weaned Piglets

This experiment was performed in order to investigate the effects of chitosan-Zn chelate (CS-Zn) on activities of antioxidant enzymes and immune function in weaned piglets. One hundred and twenty weaned piglets (Duroc × Landrace × Yorkshire) with 7.12 ± 0.25 kg body weight were allotted to four treatments. A basal diet without Zn supplementation was used as control group. The other three treatments were fed the control diet supplemented with 100 mg/kg Zn as ZnSO4, 100 mg/kg Zn as CS-Zn, 100 mg/kg Zn as ZnSO4 and chitosan (the content of chitosan was the same as that of CS-Zn), respectively. The feeding trial lasted 30 days. Spleen index of pigs fed dietary CS-Zn was higher (p < 0.05) than that of control pigs. Thymus index and lymph node index did not differ among the pigs fed any diets (p > 0.05). T-AOC levels, Cu-ZnSOD, and GSH-PX activities in serum or liver of the pigs receiving CS-Zn diet were higher than those of the pigs fed CS+ZnSO4 or ZnSO4 diets (p < 0.05). These pigs fed dietary CS-Zn also showed lower MDA content in liver compared with the pigs fed other diets (p < 0.05). Serum IgA, complement 3, and complement 4 levels of pig fed dietary CS-Zn was higher than those of the pigs fed other diets (p < 0.05). Supplemental dietary Zn did not change serum IgG and IgM levels (p > 0.05). The ALP activity of pigs fed dietary CS-Zn was higher than those of the pigs fed other three diets (p < 0.05). No significant differences were founded in serum GOT or GPT activities of pigs fed dietary Zn (p > 0.05). The results of the present study indicated that chitosan-Zn chelate could increase antioxidant capacity and improve immune function in weaned piglets compared with ZnSO4 or chitosan.

Eplerenone mimics features of the alternative activation in macrophages obtained from patients with heart failure and healthy volunteers

Alternative activation of macrophages plays protective role in cardiac remodelling in heart failure and the activity of mineralocorticoid receptor may determine the phenotype of these cells. We examined the influence of eplerenone, aldosterone, and IL-4 on descriptors of alternative activation in blood monocytes collected from 19 patients with heart-failure and 20 healthy volunteers. “Heart failure” macrophages in comparison with “healthy” macrophages had increased mineralocorticoid activity, NO and reactive oxygen species production, expression of iNOS mRNA and protein, but decreased expression of arginase I and mannose receptor proteins, and activity of MnSOD and CuZnSOD. Aldosterone increased mineralocorticoid activity, NO and reactive oxygen species production, iNOS mRNA and protein expression, MnSOD and CuZnSOD activity. Eplerenone attenuated the effects of aldosterone on all but MnSOD and CuZnSOD variables. Eplerenone alone increased the production of NO, MnSOD and CuZnSOD activity, arginase I gene and protein expression, and mannose receptor gene and protein expression, but decreased mineralocorticoid activity only in “heart failure” macrophages. The latter suggests altered function of mineralocorticoid receptor in heart failure. Increased mineralocorticoid activity accounts for increased NO production, iNOS gene and protein expression but does not explain the increased basal reactive oxygen species production and decreased markers of alternative activation in “heart failure” macrophages. In the lack of change in basal mineralocorticoid activity, eplerenone increases markers of alternative activation in a mineralocorticoid receptor-independent manner. Because of changes in iNOS and NO variable, eplerenone induced qualitatively different activation of macrophages from that obtained with IL-4.

Oxidative stress markers and C-reactive protein are related to severity of heart failure in patients with dilated cardiomyopathy.

Background. The aim of study was to determine relationships between functional capacity (NYHA class), left ventricle ejection fraction (LVEF), hemodynamic parameters, and biomarkers of redox state and inflammation in patients with dilated cardiomyopathy (DCM). Methods. DCM patients (n = 109, aged 45.97 ± 10.82 years), NYHA class IIV, and LVEF 2.94 ± 7.1% were studied. Controls comprised age-matched healthy volunteers (n = 28). Echocardiography and right heart catheterization were performed. Serum activities of superoxide dismutase isoenzymes (MnSOD and CuZnSOD), concentrations of uric acid (UA), malondialdehyde (MDA), and C-reactive protein (hs-CRP) were measured. Results. MnSOD, UA, hs-CRP, and MDA were significantly higher in DCM patients compared to controls. Except MDA concentration, above parameters were higher in patients in III-IV NYHA class or with lower LVEF. hsCRP correlated with of MnSOD (P < 0.05) and CuZnSOD activity (P < 0.01). Both isoenzymes positively correlated with mPAP and pulmonary capillary wedge pressure (MnSOD, resp., P < 0.01 and P < 0.05 and CuZnSOD P < 0.05; P < 0.05). UA positively correlated with MnSOD (P < 0.05), mPAP (P < 0.05), and PVRI (P < 0.05). The negative correlation between LVEF and UA (P < 0.01) was detected. Conclusion. There are relationships among the severity of symptoms of heart failure, echocardiographic hemodynamic parameters, oxidative stress, and inflammatory activation. Increased MnSOD activity indicates the mitochondrial source of ROS in patients with advanced heart failure.

Biotransformation and nitroglycerin-induced effects on antioxidative defense system in rat erythrocytes and reticulocytes.

The effects of nitroglycerin (glyceryl trinitrate - GTN) are mediated by liberated nitric oxide (NO) and formed reactive nitrogen species, which induces oxidative stress during biotransformation in red blood cells (RBCs). The aim of this study was to evaluate effects of GTN on antioxidative defense system (AOS) in rat erythrocytes (without) and reticulocytes (with functional mitochondria). Rat erythrocyte and reticulocyte-rich RBC suspensions were aerobically incubated (2 h, 37°C) without (control) or in the presence of different concentrations of GTN (0.1-1.5 mM). After incubation, concentrations of non-enzymatic components of AOS, activities of antioxidative enzymes and oxidative pentose phosphate (OPP) pathway activity were followed in RBC suspensions. In rat reticulocytes, GTN decreased the activity of mitochondrial MnSOD and increased the activity of CuZnSOD. In rat RBCs, GTN induced increase of Vit E concentration (at high doses), but decreased glutathione content and activities of all glutathione-dependent antioxidative enzymes; the OPP pathway activity significantly increased. GTN biotransformation and induction of oxidative stress were followed by general disbalance of antioxidative capacities in both kinds of RBCs. We suggest that oxidative stress, MnSOD inhibition and depletion of glutathione pool in response to GTN treatment lead to decreased bioavailability of NO after GTN biotransformation in rat reticulocytes.

Effects of acupuncture on declined cerebral blood flow, impaired mitochondrial respiratory function and oxidative stress in multi-infarct dementia rats

Brain energy disorders and oxidative stress due to chronic hypoperfusion were considered to be the major risk factors in the pathogenesis of dementia. In previous studies, we have demonstrated that acupuncture treatment improved cognitive function of VaD patients and multi-infarct dementia (MID) rats. Acupuncture therapy also increased the activities of glycometabolic enzymes in the brain. But it is not clear whether acupuncture treatment compensates neuronal energy deficit after cerebral ischemic through enhancing the activities of glucose metabolic enzymes and preserving mitochondrial function, and whether acupuncture neuroprotective effect is associated with activations of mitochondrial antioxidative defense system. So, the effect of acupuncture therapy on cognitive function, cerebral blood flow (CBF), mitochondrial respiratory function and oxidative stress in the brain of MID rats was investigated in this study. The results showed that acupuncture treatment significantly improved cognitive abilities and increased regional CBF of MID rats. Acupuncture elevated the activities of total SOD, CuZnSOD and MnSOD, decreased the level of malondialdehyde (MDA) and superoxide anion, regulated the ratio of reduced glutathione (GSH) and oxidized glutathione (GSSG) in mitochondria, and raised the level of the respiratory control index (RCI) and P/O ratio and the activities of mitochondrial respiratory enzymes of MID rats. These results indicated that acupuncture treatment improved cognitive function of MID rats; and this improvement might be due to increased CBF, which ameliorated mitochondrial dysfunction induced by ischemia and endogenous oxidative stress system of brain.

Changes in antioxidant enzyme activities in the livers and gills of three cyprinids after exposure to a cyanobacterial bloom in the Gruža Reservoir, Serbia

We examined the influence of an Aphanizomenon flos-aquae bloom on the activities of antioxidant enzymes in the livers and gills of three cyprinids, Rutilus rutilus (roach), Blicca bjoerkna (white bream) and Carassius gibelio (Prussian carp) from the Gruža Reservoir (Serbia). We also studied tissue- and species-specific differences in the antioxidant response to the cyanobacterial bloom. The activities of total, copper zinc and manganese containing superoxide dismutases (Tot SOD, CuZn SOD, Mn SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and the biotransformation phase II enzyme glutathione-S-transferase (GST) were determined before and during the cyanobacterial bloom. B. bjoerkna exhibited higher antioxidant response to the cyanobacterial bloom than R. rutilus and C. gibelio; liver Tot SOD, CuZn SOD and GST activities increased during the bloom only in this species. Liver Mn SOD activity in all three cyprinids increased during the bloom. A decrease in liver CAT and GSH-Px activities that was detected in all three species may be directly responsible for an enhanced susceptibility of fish to potential oxidative stress caused by the bloom. Tot SOD, CuZn SOD, Mn SOD, GSH-Px, GR and GST activities in gills were also inhibited during the bloom showing that the gills were affected more than the liver. The observed changes in the activities of aforementioned antioxidant enzymes during the cyanobacterial bloom suggest that these parameters could be important biomarkers for ecotoxicological risk in fish that inhabit ecosystems where A. flos-aquae blooms occur.

7th International Symposium on Enabling Technologies for Life Sciences (ETP)

7^th International Symposium on Enabling Technologies for Life Sciences (ETP)

Aspirin-induced apoptosis of yeast cells is associated with mitochondrial superoxide radical accumulation and NAD(P)H oxidation

In previous studies, we observed that aspirin, a promising cancer-preventive agent, induces apoptosis in mitochondrial manganese superoxide dismutase (MnSOD)-deficient Saccharomyces cerevisiae cells grown aerobically in ethanol medium. In this study, we show that aspirin-induced apoptosis is associated with a significant increase in mitochondrial and cytosolic O2 ·- and oxidation of mitochondrial NAD(P)H. A concomitant rise in the level of cytosolic CuZnSOD activity failed to compensate for mitochondrial MnSOD deficiency. However, an observed increase in activity of Escherichia coli FeSOD targeted to the mitochondrial matrix of the MnSOD-deficient yeast cells, markedly decreased aspirin-induced accumulation of mitochondrial O2 ·-, significantly increased the mitochondrial NAD(P)H level and rescued the apoptotic phenotype. Indeed, recombinant yeast cells expressing E. coli FeSOD behaved in a similar manner to the parent wild-type yeast cells with native mitochondrial MnSOD activity. Wild-type cells consistently showed a decrease in mitochondrial O2 ·- and an increase in mitochondrial NAD(P)H levels in the presence of aspirin in ethanol medium. In fact, in wild-type cells, our studies supported an antioxidant action of aspirin. Taken together, our results indicate that a pro-oxidant effect of aspirin occurring predominantly in cells with compromised mitochondrial redox balance may be enough to overcome antioxidant defences resulting in apoptosis, as observed in MnSOD-deficient yeast cells.

Protective Role of Aspirin, Vitamin C, and Zinc and their Effects on Zinc Status in the DMH-Induced Colon Carcinoma Model

Chemoprotection refers to the use of specific natural or synthetic chemical agents to suppress or prevent the progression to cancer. The purpose of this study is to assess the protective effect of aspirin, vitamin C or zinc in a dimethyl hydrazine (DMH) colon carcinoma model in rats and to investigate the effect of these supplements on changes associated with colonic zinc status. Rats were randomly divided into three groups, group 1 (aspirin), group 2 (vitamin C) and group 3 (zinc), each being subdivided into two groups and given subcutaneous injection of DMH (30 mg/kg body wt) twice a week for 3 months and sacrificed at 4 months (A-precancer model) and 6 months (B-cancer model). Groups 1, 2, 3 were simultaneously given aspirin, vitamin C, or zinc supplement respectively from the beginning till the end of the study. It was observed that 87.5% of rats co-treated with aspirin or vitamin C showed normal colonic histology, along with a significant decrease in colonic tissue zinc at both time points. Rats co-treated with zinc showed 100% reduction in tumor incidence with no significant change in colonic tissue zinc. Plasma zinc, colonic CuZnSOD (copper-zinc superoxide dismutase) and alkaline phosphatase activity showed no significant changes in all 3 cotreated groups. These results suggest that aspirin, vitamin C or zinc given separately, exert a chemoprotective effect against chemically induced DMH colonic preneoplastic progression and colonic carcinogenesis in rats. The inhibitory effects are associated with maintaining the colonic tissue zinc levels and zinc enzymes at near normal without significant changes.

Bradykinin preconditioning affects the number of degenerated neurons and the level of antioxidant enzymes in spinal cord ischemia in rabbits

Bradykinin preconditioning has been used for acquisition of tolerance after spinal cord ischemia. Rabbits were preconditioned intraperitoneally with bradykinin 48h prior to 20min of abdominal aorta ligation followed by 24 and 48h of reperfusion. The activities of SOD and catalase were measured and Fluoro Jade B (FJB)-positive degenerated neurons were evaluated. The outcomes of Tarlov scoring system used to assess neurological functions showed significant improvement in bradykinin groups compared to the ischemic group. The number of FJB-positive degenerated neurons was decreased in ventral horns of both bradykinin groups. Significantly decreased activities of total SOD and mitochondrial Mn-SOD were also detected in both bradykinin groups versus ischemic group while CuZn-SOD and catalase activities were significantly decreased only in the bradykinin group after 24h of reperfusion versus ischemic group. These findings suggest that one of the possibilities of the neuroprotective effect of delayed bradykinin preconditioning against spinal cord ischemic injury could be realized by mitochondrial protection and decreased synthesis of Mn-SOD as well as by promotion of neuronal survival.

Protective effect of Hsp70i against chronic social isolation stress in the rat hippocampus

Stress-related glucocorticoids and glutamate release has been implicated in depression. Glutamate neurotoxicity is mediated, in part, by the production of nitric oxide via nitric oxide synthase (NOS) isoforms and mitochondrial damage. We previously reported that chronic social isolation stress triggers proapoptotic signaling in the rat prefrontal cortex, but not in the hippocampus. Given that the hippocampus is highly sensitive to stress, we examined signaling cascades underlying the hippocampal cellular protection through the NOS pathway, antioxidant capacity and heat shock protein (Hsp) expression. We investigated neuronal (nNOS) and inducible (iNOS) protein levels, subcellular protein distributions of nuclear factor-κB (NF-κB), CuZnSOD and MnSOD activity, reduced glutathione (GSH), stress-inducible Hsp70 (Hsp70i) protein expression and serum corticosterone (CORT) levels of rats exposed to 21 days of chronic social isolation, an animal model of depression, alone or in combination with 2 h of acute immobilization or cold stress (combined stress). Both acute stressors elevated CORT, with lesser magnitude increase in chronically isolated rats exposed to novel acute stress as compared to acute stressors alone, indicating compromised HPA axis activity. Acute cold decreased nuclear CuZnSOD activity and stimulated NF-κB nuclear translocation. Chronic social isolation resulted in no activation of NF-κB, but led to decreased GSH, iNOS and increased nNOS and Hsp70i levels, alterations that remained following combined stressors. Decreased mitochondrial MnSOD activity after combined stressors suggests compromised detoxifying capacity. These data indicate that Hsp70i upregulation may provide hippocampal cellular protection against chronic social isolation stress mediated by downregulation of iNOS protein expression through suppression of NF-κB activation.

Oxidative stress status in patients with acute urticaria

Background: The conflicting information related to oxidative stress status in patients with chronic idiopathic urticaria has been reported in several studies. However, the association between acute urticaria (AU) and oxidative stress has not been investigated exhaustively.Objectives: To evaluate the role of the oxidative stress in the patients with AU by determining the oxidant/antioxidant activity in AU and to establish its clinical significance.Methods: About 50 patients with AU, (10 males, 40 females) and 30 unrelated healthy controls (4 males, 26 females) were enrolled into the study. The activity of the antioxidant enzymes copper-zinc superoxide dismutase (Cu-Zn-SOD), glutathione peroxidase (GSH-Px) and catalase, and the levels of malondialdehyde (MDA), serum NO and protein carbonyls levels in the plasma were measured spectrophotometrically at samples.Results: A statistically significant increase was observed in serum Cu-ZnSOD activities of the patients when compared with that of the controls (p < 0.001), while a statistically significant decrease was observed in GSH-Px activities of the patients according to the controls (p = 0.002). Serum MDA and NO levels were significantly higher in patients with AU when compared with control group (p < 0.001 for both of them). The levels of protein carbonyls were significantly lower in patients with AU when compared with control group (p < 0.001).Conclusion: It seems there is an oxidative burden in the patients with AU. Cutaneous oxidative stress may play a role in pathogenesis of the disease.

Chronic social isolation induces NF-κB activation and upregulation of iNOS protein expression in rat prefrontal cortex

Exposure of an organism to stress, results in oxidative stress and increased nitric oxide (NO) production in the brain. The role of the processes caused by chronic stress in the prefrontal cortex has not been fully investigated. Considering that chronic stress increases NO production by the enzyme nitric oxide synthase (NOS), we examined the cytosolic neuronal (nNOS) or inducible (iNOS) protein levels in the prefrontal cortex of rats exposed to 21d of chronic social isolation stress, an animal model of depression, alone or in combination with 2h of acute immobilization or cold (4°C) stress (combined stress). Antioxidative status via cytosolic CuZnSOD and mitochondrial MnSOD activity, cytosolic redox status via reduced glutathione (GSH) concentration were determined. Furthermore, cytosolic inducible heat shock protein 70 (Hsp70i), cytosolic/nuclear distributions of NF-κB and serum corticosterone (CORT) were also investigated to elucidate the possible mechanism involved in the cellular NOS pathway. Our results showed that both acute stressors led to increases of CORT and nNOS protein while iNOS protein expression was unaffected. In contrast to the acute stress, chronic social isolation compromised hypothalamic–pituitary-adrenal axis functioning such that the normal stress response was impaired following subsequent acute stressors. Downregulated redox GSH status as well as decreased activity of CuZnSOD and MnSOD suggests the existence of oxidative stress which remained as such following combined stressors. Changes in redox-status associated with decreased Hsp70i protein expression enabled NF-κB translocation into the nucleus, causing increased cytosolic nNOS and iNOS protein expression. Results suggest that NOS signaling pathway plays a differential role between acute and chronic stress whereby state of oxidative/nitrosative stress after chronic social isolation is caused, at least in part, by NF-κB activation and increased iNOS protein expression.

The characterization, expression and activity analysis of superoxide dismutases (SODs) from Procambarus clarkii

The superoxide dismutases (SODs) are important antioxidant enzymes to remove excess biologically reactive oxygen intermediates, and thus avoid adverse conditions. In this study, an extracellular copper–zinc superoxide dismutase (ecCuZnSOD) gene and a mitochondrial manganese superoxide dismutase (mtMnSOD) gene were cloned from hemocytes of the freshwater crayfish, Procambarus clarkii. The open reading frame (ORF) of ecCuZnSOD is 612bp encoding a 204 amino acid (aa) protein with a 24 aa signal peptide, whereas the ORF of mtMnSOD is 654bp and encodes a 218 aa protein with a 20 aa mitochondrial-targeting sequence in the N-terminus. P. clarkii ecCuZnSOD and mtMnSOD proteins showed high similarity with ecCuZnSODs and mtMnSODs from other crustaceans, respectively. Both ecCuZnSOD and mtMnSOD of P. clarkii were highly expressed in hepatopancreas, hemocytes and gill; lower expression levels were seen in intestine, stomach, nerve, heart and muscle. The mRNA expressions of three SODs of P. clarkii (ecCuZnSOD, mtMnSOD and cytosolic MnSOD (cytMnSOD)) were studied after Spiroplasma eriocheiris and Aeromonas hydrophila stimulations. After S. eriocheiris challenge, the mRNAs of three SODs in hemocytes and gill were up-regulated, and ecCuZnSOD also increased in the hepatopancreas. However, the mtMnSOD and cytosolic MnSOD in the hepatopancreas were down-regulated. After A. hydrophila challenge, the mRNA expressions of three SODs in hepatopancreas and hemocytes were up-regulated and down-regulated in gill. The total SOD activity and CuZnSOD activity were increased compared to the control group with both S. eriocheiris and A. hydrophila challenges. The results may indicate that the SODs of P. clarkii have important roles in innate immune responses against S. eriocheiris and A. hydrophila.

A Novel Superoxide Dismutase from Cicer arietinum L. Seedlings: Isolation, Purification and Characterization

Superoxide dismutase is an important enzyme with various therapeutic applications. Search of a new source of superoxide dismutase with novel properties has significant importance. The current work reports purification of a novel superoxide dismutase enzyme with unique characteristics. A copper zinc superoxide dismutase (Cu-Zn SOD) was purified and characterized from Cicer arietinum L. seedlings germinated under aluminium (Al+3) stress. The specific activity of purified protein was 158 units/mg with 28 fold purification. The superoxide dismutase is a homodimeric protein with approx subunit molecular weight of 33.27 kDa. The enzyme is identified as Cu-Zn category of superoxide dismutase, reflected by H2O2 induced inhibition of in-gel activity and presence of quantifiable copper and zinc ions. The optimum pH range for purified Cu-Zn SOD activity was observed within 6.5-8.5 (highest at pH 8.0) and the pH stability was in the range of 6.0-8.5. The enzyme was more stable at low temperature (below 30°C) and the Km of purified Cu-Zn SOD for riboflavin as substrate was 10.16 ± 2.5 µM. The N-terminal amino acid sequence showed homology at conserved residues with other plant Cu-Zn SODs.

Changes in expression and activity of MnSOD and CuZnSOD in carotid body and adrenal medullary cells of rats exposed to chronic intermittent hypoxia.

Nitro‐oxidative stress is associated with hypertension and enhanced carotid body (CB) chemosensory responses induced by chronic intermittent hypoxia (CIH). Since, ROS levels are regulated by cytoplasmic (CuZnSOD) and mitochondrial (MnSOD) superoxide dismutases; it is plausible that changes in their expression or enzymatic activity due to tyrosine nitration may contribute to the chemosensory potentiation. We studied effects of CIH on 3‐nitrotyrosine (3‐NT), SODs levels and activities, and MnSOD nitration in CB and adrenal medullary cells from Sprague‐Dawley male rats exposed to CIH (5% O2, 12 times/h for 8 h/day) for 7 days. To determine the contribution of mitochondrial ROS to the chemosensory potentiation, we studied the effects of the mitochondrial targeted antioxidant MitoTEMPO on the CIH‐induced CB chemosensory potentiation. CIH increased the 3‐NT levels in the CB and adrenal cells, the MnSOD level and activity in the CB, but not in adrenal medullary cells. CuZnSOD levels did not increase in any tissue. MitoTEMPO treatment prevented the CIH‐induced CB chemosensory potentiation. These results indicate that mithochondrial ROS are crucial for the chemosensory potentiation, while the increased CB MnSOD level and activity may be the result of a compensatory mechanism to the increased oxidative stress.Supported by FONDECYT 1100405 &amp; AT 24110172

Oxidative damage associated with thermal stress in Pocillopora verrucosa from the Mexican Pacific

Coral communities are under increasing pressure from anomalous increases in sea surface temperature. These events have had negative effects on reef ecosystems and it is important to get a better understanding of the cellular mechanisms that allow organisms to survive the stress period before bleaching is evident. To gain insight into the effect of increased temperature on coral communities off the Pacific coast of central Mexico, we evaluated CuZnSOD (superoxide dismutase) activity in branches of Pocillopora verrucosa by simulating stress conditions (31 ºC) and non-stress conditions. There was an increase in SOD activity after 18 h of exposing the corals to 31 ºC, while no increase in activity was observed at 28 ºC. These results were used to quantify the maximum levels of CuZnSOD activity before Symbiodinium were expelled to prevent cell damage due to oxidative stress. We determined that CuZnSOD can be used as a molecular marker to quantify stress levels, prior to bleaching, in coral communities of the region.

In vitro interceptive and reparative effects of myo-inositol against copper-induced oxidative damage and antioxidant system disturbance in primary cultured fish enterocytes

Copper (Cu) is essential for normal cellular processes in most eukaryotic organisms but is toxic in excess. Our previous study reported that a nutrient antioxidant, myo-inositol (MI), can protect fish from Cu-induced oxidative injury; however, the mechanisms involved are not fully understood. Therefore, the present study aimed to analyze potential pathways. First, to investigate the hypothesis that MI protects enterocytes against Cu toxicity via the intercept pathway, enterocytes were treated with different concentrations of MI (0–75mg/L medium) in the presence of 6mg/L of Cu for 24h (Experiment 1). Next, we investigated the potential reparative role of MI after a Cu challenge (Experiment 2). The results of Experiment 1 indicated that cells exposed to Cu alone for 24h exhibited increases in lactate dehydrogenase release (LDH), malondialdehyde (MDA) formation and protein oxidation (P<0.05). Notably, a dose-dependent inhibitory effect on LDH release was observed with all doses of MI. Moreover, co-treatment with MI completely inhibited Cu-induced protein carbonyl (PC) formation. However, Cu-induced lipid peroxidation was not altered by MI co-treatment. Additionally, Cu exposure suppressed total-superoxide dismutase (T-SOD), CuZnSOD and catalase (CAT) activities, and these changes were completely blocked by co-treatment with sufficient MI concentrations. In contrast, cells exposed to Cu exhibited adaptive increases in reduced glutathione (GSH) content and the activities of anti-hydroxyl radical (AHR), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR). Interestingly, the Cu-stimulated increases in these antioxidants were blocked by co-treatment with sufficient MI concentrations. The results of Experiment 2 indicated that cell injury (LDH release), lipid peroxidation (MDA formation) and protein oxidation induced by Cu were reversed by subsequent MI treatment. Meanwhile, Cu-induced decreases in alkaline phosphatase (AKP), anti-superoxide anion (ASA), T-SOD and CuZnSOD activities were completely restored by subsequent MI treatment, while the reduced CAT activity was partially restored. However, MI rescues partially restored the adaptive increase in GPx activity induced by Cu, whereas the adaptive increase in reduced GSH content was completely reversed by 75mg/L of MI. However, subsequent MI treatments did not alter the induction of GST activity by Cu. In conclusion, we demonstrated for the first time that MI not only protected enterocytes from Cu-induced oxidative damage but also increased the repair activity in primary enterocytes after challenge with Cu. Moreover, MI-mediated increases in antioxidant enzyme activities contributed to lipid and protein oxidant repair.