Antioxidative stress proteins and their gene expression in brown trout (Salmo trutta) from three rivers with different heavy metal levels

Three populations of brown trout (Salmo trutta) exposed to different metal levels in their natural environments, were studied with respect to antioxidants metallothionein (MT), superoxide dismutase (SOD) and catalase (CAT) as well as for corresponding mRNA levels. In addition, mRNA levels were studied for glutathione peroxidase (GPx) and glutathione reductase (GR). The Cd/Zn-exposed trout (Naustebekken River) had higher accumulated levels of Cd, Cu and Zn in gills, and higher levels of MT (both protein and mRNA) in liver and kidney as well as in gills compared to the Cu-exposed trout (Rugla River) and trout from an uncontaminated reference river (Stribekken River). Less MT found in the Cu-exposed trout may increase susceptibility to oxidative stress, but no higher levels of antioxidant mRNAs were found in gills of these trouts. The data indicated that chronic exposures of brown trout to Cd, Zn and/or Cu did not involve maintenance of high activities of SOD and CAT enzymes in gills, although SOD mRNA levels were higher in the Cd/Zn-exposed trout. In livers, mRNA levels of SOD, CAT and GPx were higher in the metal-exposed trout, but in the case of GR this was only seen in kidneys of Cd/Zn-exposed trout. However, both metal-exposed groups had higher activities of SOD enzyme in liver compared to the unexposed reference trout, and CAT activity was found to be higher in kidneys of Cu-exposed trout. The Cu-exposed trout did not seem to rely on MT production to avoid Cu toxicity in gills, but rather by keeping the Cu uptake at a low level. A coordinated expression of different stress genes may also be important in chronic metal exposure. It may be concluded that the observed metal effects relies on acclimation rather than on genetic adaptation in the metal exposed populations.     

不同倍性虹鳟幼鱼对急性温度胁迫的抗氧化响应

虹鳟属于冷水性鱼类,其生存的最适温度为12~18 ℃,温度胁迫是虹鳟在夏秋季养殖过程中经常遇到的问题.枫叶鲑和硬头鳟均为品质优良的虹鳟选育种.为探讨急性温度胁迫对两种倍性虹鳟抗氧化响应的影响,本研究选取二倍体枫叶鲑和三倍体硬头鳟幼鱼,分别在13、17、21和25 ℃下进行热应激试验.在达到目标温度后的0、1、6和12 h取样,之后将受试鱼恢复至13 ℃的适宜温度下培养,并在恢复培养的1、12、24和48 h后取样,测定受试鱼肝脏中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPx)的活性,以及脂质过氧化物(LPO)、丙二醛(MDA)和热激蛋白70(HSP70)含量.结果表明: 枫叶鲑和硬头鳟的3种抗氧化酶活性在17 ℃组没有出现显著升高;21 ℃组枫叶鲑和硬头鳟的SOD活性在热应激期间出现显著升高,但枫叶鲑的SOD活性在恢复过程中恢复到正常水平;25 ℃组枫叶鲑和硬头鳟的SOD、CAT和GPx的活性均显著提高;但在恢复试验进行24 h后,CAT和GPx的活性恢复到正常水平.枫叶鲑在17、21和25 ℃组HSP70的产生量显著高于13 ℃组,而硬头鳟仅在21和25 ℃组HSP70的产生量显著高于13 ℃组.通过整合生物标志物响应指标值对多种抗氧化参数进行分析,发现枫叶鲑在17和21 ℃组急性温度胁迫下的抗氧化响应显著高于硬头鳟,但在25 ℃组硬头鳟的抗氧化响应高于枫叶鲑.这表明不同倍性虹鳟幼鱼在不同温度急性胁迫下的抗氧化响应有所差异.     

Activity of superoxide dismutase (SOD) and concentration of thiobarbituric acid reactive substances (TBARS) in liver and muscles of some fish

In this study we examined superoxide dismutase (SOD) activity and thiobarbituric acid reactive substances (TBARS) concentration in liver and muscles of four fish species: the carp, the brown trout, the white cod and the flounder. Higher SOD activity and higher TBARS concentration was revealed in the tissues of marine fish in comparison to freshwater fish. The highest SOD activity was observed in the cod while the highest TBARS concentration was in the flounder. The observed differences are probably an effect of the different living mode of the compared fish.     

Seasonal changes in antioxidant defence system of liver and gills of Salmo trutta caspius, Salmo trutta labrax and Salmo trutta macrostigma

Seasonal changes in antioxidant enzyme activities (superoxide dismutase, SOD, EC 1.15.1.1; catalase, CAT, EC 1.11.1.16; glutathione peroxidase, GPx, EC 1.11.1.9; glutathione reductase, GR, EC 1.6.4.2; glucose-6-phosphate dehydrogenase, G6PD, EC 1.1.1.49 and glutathione S -transferase, GST, EC 1.5.1.18) and lipid peroxidation (LPO) levels of livers and gills of female Caspian trout Salmo trutta caspius , Black Sea trout Salmo trutta labrax and mountain trout Salmo trutta macrostigma were investigated. SOD, CAT, GPx, G6PD and GST activities were higher in liver compared to gills of all sub-species; concomitantly, the GR activity was also higher in the livers of S. t. caspius and S. t. labrax , but the reverse was seen in S. t. macrostigma . LPO levels were higher in the gills compared to the liver of all sub-species. There was no general trend in the seasonal changes in the gill antioxidant enzyme (AE) activities or LPO levels. Higher AE activities, however, were found in the liver of each sub-species during autumn, and this coincided with an increase in the gonado-somatic index.     

Temporal genetic variation of brown trout <Emphasis Type="Italic">Salmo trutta</Emphasis> L. from Vorobiev creek (White sea) based on allozyme analysis

We performed an analysis of allozyme variation in brown trout from Vorobiev creek. Seventeen allozyme loci encoding glycerol-3-phosphate dehydrogenase (G3PDH), aspartate aminotransferase (AAT), malate dehydrogenase (MDH), lactate dehydrogenase (LDH), superoxide dismutase (SOD), and esterase D (EST-D) were studied. We found statistically significant differences in allele frequencies for the AAT-1,2*, G3PDH-2,3*, LDH-5*, and MDH-2* loci between brown trout samples collected in 1981–1982 and/or 1992–1995. We suggest that temporal changes of allele frequencies in brown trout from Vorobiev Creek are associated with gene drift.     

Metabolic responses to prolonged starvation, food restriction, and refeeding in the brown trout, Salmo trutta: oxidative stress and antioxidant defenses

The effects of long-term starvation and food restriction (49 days), followed by refeeding (21 days) have been studied with respect to antioxidant defense in the liver and gills (branchial tissues) of the brown trout, Salmo trutta. Malondialdehyde levels in both tissues increased in parallel with starvation and food restriction and these values did not return to normal after the refeeding period. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) in liver and gills increased during the 49 days of starvation, but glucose-6-phosphate dehydrogenase (G6PD) activities decreased. Glutathione S-transferase (GST) activity decreased in the liver at the 49th day of starvation, but increased in the branchial tissues. Some of the antioxidant enzyme activities (such as hepatic GST and branchial G6PD) returned to control values of fed fish after the refeeding period, but others (e.g. hepatic SOD and branchial GPx) did not return to normal values. In conclusion, our study indicates that total or partial food deprivation induces oxidative stress in brown trout.     

A test for endogenous interference in superoxide dismutase assays

Endogenous interfering substances can be detected by applying the techniques of parallel-line analysis of variance to the assay of superoxide dismutase (SOD) activities in crude tissue extracts. The technique also allows expression of specific activities in terms of units of activity as defined by commercially available standards as opposed to definition by the specific assay conditions utilized. This analysis may be broadly applied to many of the indirect SOD assays currently in use. In the current investigation, SOD was assayed by its ability to inhibit the rate of reduction of acetylated ferricytochrome c by superoxide anion ($\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$) generated via the xanthine-xanthine oxidase reaction. The parallel-line analysis was effective in detecting the presence of interference by exogenous ascorbate in the reaction system, and by unidentified endogenous reactants within extracts of whole blood and ocular choroidal tissues of the rainbow trout, Salmo gairdneri.     

Superoxide dismutase mimetics elevate superoxide dismutase activity in vivo but do not retard aging in the nematode Caenorhabditis elegans

According to the oxidative damage theory a primary cause of aging is the accrual of molecular damage from reactive oxygen species (ROS), particularly superoxide and its derivatives. This predicts that treatments that reduce ROS levels should retard aging. Using the nematode Caenorhabditis elegans, we tested the effects on stress resistance and life span of treatment with EUK-8 and EUK-134, synthetic mimetics of the antioxidant enzyme superoxide dismutase (SOD), which neutralises superoxide. Treatment with SOD mimetics elevated in vivo SOD activity levels, particularly in mitochondria, where up to 5-fold increases in SOD activity were recorded. Treatment with exogenous SOD mimetics did not affect endogenous protein SOD levels. Where life span was reduced by the superoxide generators paraquat and plumbagin, EUK-8 treatment increased life span in a dose-dependent fashion. Yet in the absence of a superoxide generator, treatment with EUK-8 or EUK-134 did not increase life span, even at doses that were optimal for protection against pro-oxidants. Thus, an elevation of SOD activity levels sufficient to increase life span when it is limited by superoxide generators does not retard aging in the absence of superoxide generators. This suggests that C. elegans life span is not normally limited by levels of superoxide and its derivatives.     

Transcriptional activation of the human Cu/Zn superoxide dismutase gene by 2,3,7,8-tetrachlorodibenzo-p-dioxin through the xenobiotic-responsive element

Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced during biological oxidations and environmental stress. Here we have investigated the effect of the most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the promoter of the Cu/Zn superoxide dismutase (SOD1) gene in HepG2 and HeLa cells using the chloramphenicol acetyltransferase gene as a reporter. The SOD1 promoter was activated 4- to 5-fold by TCDD treatment, in a concentration-dependent manner. In addition, the level of SOD1 mRNA and the enzymatic activity of the SOD1 protein were also enhanced on exposure of the cells to TCDD. Functional analysis of the regulatory region of the SOD1 gene by deletion and point mutation, and the use of a heterologous promoter system, showed that the SOD1 gene was transactivated by TCDD via the xenobiotic-responsive element (XRE). Gel mobility shift assays also confirmed the induction and the inducible binding of a receptor-ligand complex to XRE. Yeast cells that overexpress hSOD1 appeared to be more resistant to TCDD than the wild type. These results demonstrate that SOD1 is induced by TCDD via the XRE. The induced SOD1 may accelerate the neutralization of the superoxide anion and thus reduce the oxidative damage associated with dioxin toxicity.     

Hydrogen peroxide damages the zinc-binding site of zinc-deficient Cu,Zn superoxide dismutase

Mutations in Cu,Zn superoxide dismutase (Cu,Zn SOD) account for approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS), a late-onset neurodegenerative disease affecting motor neurons. These mutations decrease protein stability and lower zinc affinity. Zinc-deficient SOD (Cu,E SOD) has altered redox activities and is toxic to motor neurons in vitro. Using bovine SOD, we studied the effects of hydrogen peroxide (H(2)O(2)) on Cu,E SOD and Cu,Zn SOD. Hydrogen peroxide treatment of Cu,E SOD inactivated zinc binding activity six times faster than superoxide dismutase activity, whereas inactivation of dismutase activity occurred at the same rate for both Cu,Zn SOD and Cu,E SOD. Zinc binding by Cu,E SOD was also damaged by simultaneous generation of superoxide and hydrogen peroxide by xanthine oxidase plus xanthine. Although urate, xanthine, and ascorbate can protect superoxide dismutase activity of Cu,Zn SOD from inactivation, they were not effective at protecting Cu,E SOD. Hydrogen peroxide induced subtle changes in the tertiary structure but not the secondary structure of Cu,E SOD as detected by near and far UV circular dichroism. Our results suggest that low levels of hydrogen peroxide could potentially enhance the toxicity of zinc deficient SOD to motor neurons in ALS by rendering zinc loss from SOD irreversible.     

Developmental Aspects of Detoxifying Enzymes in Fish (Salmo Iridaeus)

The activities of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione transferase and glyoxalase I have been studied during the embryologic development of rainbow trout (Salmo iridaeus) and in several other trout tissues to investigate the protective development metabolism.

A gradual increase of superoxide dismutase, catalase, glutathione reductase, glyoxalase I and glutathione transferase activities was noted throughout embryo development.

In all trout tissues investigated glutathione peroxidase was found to be extremely low compared to catalase activity. The highest activity of superoxide dismutase, glyoxalase I and glutathione reductase was found in liver followed by kidney.

No change in the number of GST subunits was noted with the transition from the embryonic to the adult stages of life according to the SDS/PAGE and HPLC analyses performed on the GSH-affinity purified fractions.     

Peroxisomal copper, zinc superoxide dismutase. Characterization of the isoenzyme from watermelon cotyledons.

The biochemical and immunochemical characterization of a superoxide dismutase (SOD, EC 1.15.1.1) from peroxisomal origin has been carried out. The enzyme is a Cu,Zn-containing SOD (CuZn-SOD) located in the matrix of peroxisomes from watermelon (Citrullus vulgaris Schrad.) cotyledons (L.M. Sandalio and L.A. del Río [1988] Plant Physiol 88: 1215-1218). The amino acid composition of the enzyme was determined. Analysis by reversed-phase high-performance liquid chromatography of the peroxisomal CuZn-SOD incubated with 6 M guanidine-HCl indicated that this enzyme contained a noncovalently bound chromophore group that was responsible for the absorbance peak of the native enzyme at 260 nm. The amino acid sequence of the peroxisomal CuZn-SOD was determined by Edman degradation. Comparison of its sequence with those reported for other plant SODs revealed homologies of about 70% with cytosolic CuZn-SODs and of 90% with chloroplastic CuZn-SODs. The peroxisomal SOD has a high thermal stability and resistance to inactivation by hydrogen peroxide. A polyclonal antibody was raised against peroxisomal CuZn-SOD, and by western blotting the antibody cross-reacted with plant CuZn-SODs but did not recognize either plant Mn-SOD or bacterial Fe-SOD. The antiSOD-immunoglobulin G showed a weak cross-reaction with bovine erythrocytes and liver CuZn-SODs, and also with cell-free extracts from trout liver. The possible function of this CuZn-SOD in the oxidative metabolism of peroxisomes is discussed.     

Nitric oxide produced by NOS2 copes with the cytotoxic effects of superoxide in macrophages

Nitric oxide (NO) reacts with superoxide to produce peroxynitrite, a potent oxidant and reportedly exerts cytotoxic action. Herein we validated the hypothesis that interaction of NO with superoxide exerts protection against superoxide toxicity using macrophages from mice with a knockout (KO) of inducible NO synthase (NOS2) and superoxide dismutase 1 (SOD1), either individually or both. While no difference was observed in viability between wild-type (WT) and NOS2KO macrophages, SOD1KO and SOD1-and NOS2-double knockout (DKO) macrophages were clearly vulnerable and cell death was observed within four days. A lipopolysaccharide (LPS) treatment induced the formation of NOS2, which resulted in NO production in WT and these levels were even higher in SOD1KO macrophages. The viability of the DKO macrophages but not SOD1KO macrophages were decreased by the LPS treatment. Supplementation of NOC18, a NO donor, improved the viability of SOD1KO and DKO macrophages both with and without the LPS treatment. The NOS2 inhibitor nitro-l-arginine methyl ester consistently decreased the viability of LPS-treated SOD1KO macrophages but not WT macrophages. Thus, in spite of the consequent production of peroxynitrite in LPS-stimulated macrophages, the coordinated elevation of NO appears to exert anti-oxidative affects by coping with superoxide cytotoxicity upon conditions of inflammatory stimuli.     

Relationship between melanin content and superoxide dismutase (SOD) activity in the liver of various species of animals

The scavenger effect of melanin and of superoxide dismutase (SOD) activity on superoxide anion has been shown. In this work we show the relationship between melanin content and SOD activity in livers containing different quantities of melanin which were taken from various species of animals. The mitochondrial SOD activity disappears when the melanin content in the liver is very high; moreover it increases, in the liver of various species of animals examined, proportionally to the decrease of melanin content. No significant variation of the SOD activity localized in the soluble fraction has been detected when related to the melanin content. We think that in the pigmented liver the antioxidant activity of the melanin could mimic part of the function of SOD. The loss of Mn SOD activity could be mediated by a low intracellular level of superoxide anion due to the scavenger effect of melanin on superoxide anion; in fact, it is well known that the biosynthesis of Mn SOD is induced by intracellular levels of superoxide anion.     

Superoxide dismutase activity and expression in human venous and arterial bypass graft vessels.

Venous bypass grafts are more prone to accelerated atherosclerosis than arterial grafts, which is partly related to increased oxidative stress and diminished nitric oxide bioavailability. In veins superoxide production is dependent primarily on nox2 NAD(P)H oxidase expression, while in arteries nox4 appears to play an important role. This may in part explain differences in susceptibility to graft failure. Net levels of oxidative stress are however determined in parallel by the production as well as by degradation of free radicals (eg. by superoxide dismutases, catalases, thioredoxins etc). The differences in superoxide dismutase (SOD) expression and activity in human bypass conduit vessels remain unclear. Accordingly, we aimed to compare SOD activity and protein levels as well as its functional effects on superoxide production in segments of human internal mammary arteries (IMA) and saphenous veins (HSV) from patients undergoing bypass graft surgery (n=24). SOD activity was assessed by inhibition of pyrogallol autoxidation, Cu-Zn SOD and Mn SOD protein levels were studied by immunoblotting. Basal superoxide release was detected by lucigenin (5 microM) enhanced chemiluminescence. Total SOD activity did not differ significantly between HSV and IMA. Similarly, no difference was observed in SOD activity in the presence of KCN (Mn-SOD). Human bypass conduit vessels show amounts of Cu-Zn SOD or Mn-SOD protein levels. In both HSV and IMA segments superoxide production was more than doubled in the presence of SOD inhibitor-DETC. CONCLUSIONS: These studies suggest that the differences in oxidative stress between human arteries and veins are unlikely to be caused by SOD activity. However SOD plays and important role in amelioration of oxidative stress in both types of vessels.     

Anti-Oxidative Defences Are Modulated Differentially in Three Freshwater Teleosts in Response to Ammonia-Induced Oxidative Stress

Oxidative stress and the antioxidant response induced by high environmental ammonia (HEA) were investigated in the liver and gills of three freshwater teleosts differing in their sensitivities to ammonia. The highly ammonia-sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less ammonia sensitive cyprinid Cyprinus carpio (common carp) and the highly ammonia-resistant cyprinid Carassius auratus (goldfish) were exposed to 1 mM ammonia (as NH₄HCO₃) for 0 h (control), 3 h, 12 h, 24 h, 48 h, 84 h and 180 h. Results show that HEA exposure increased ammonia accumulation significantly in the liver of all the three fish species from 24 h–48 h onwards which was associated with an increment in oxidative stress, evidenced by elevation of xanthine oxidase activity and levels of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA). Unlike in trout, H₂O₂ and MDA accumulation in carp and goldfish liver was restored to control levels (84 h–180 h); which was accompanied by a concomitant increase in superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase activity and reduced ascorbate content. Many of these defence parameters remained unaffected in trout liver, while components of the glutathione redox cycle (reduced glutathione, glutathione peroxidase and glutathione reductase) enhanced to a greater extent. The present findings suggest that trout rely mainly on glutathione dependent defensive mechanism while carp utilize SOD, CAT and ascorbate as anti-oxidative sentinels. Hepatic cells of goldfish appear to utilize each of these protective systems, and showed more effective anti-oxidative compensatory responses towards HEA than carp, while trout were least effective. The present work also indicates that HEA exposure resulted in a relatively mild oxidative stress in the gills of all three species. This probably explains the almost complete lack of anti-oxidative responses in branchial tissue. This research suggests that oxidative stress, as well as the antioxidant potential clearly differ between salmonid and cyprinid species.     

Superoxide dismutase enhances the formation of hydroxyl radicals in the reaction of 3-hydroxyanthranilic acid with molecular oxygen.

Superoxide dismutase (SOD) enhanced the formation of hydroxyl radicals, which were detected by using the e.s.r. spin-trapping technique, in a reaction mixture containing 3-hydroxyanthranilic acid (or p-aminophenol), Fe3+ ions, EDTA and potassium phosphate buffer, pH 7.4. The hydroxyl-radical formation enhanced by SOD was inhibited by catalase and desferrioxamine, and stimulated by EDTA and diethylenetriaminepenta-acetic acid, suggesting that both hydrogen peroxide and iron ions participate in the reaction. The hydroxyl-radical formation enhanced by SOD may be considered to proceed via the following steps. First, 3-hydroxyanthranilic acid is spontaneously auto-oxidized in a process that requires molecular oxygen and yields superoxide anions and anthranilyl radicals. This reaction seems to be reversible. Secondly, the superoxide anions formed in the first step are dismuted by SOD to generate hydrogen peroxide and molecular oxygen, and hence the equilibrium in the first step is displaced in favour of the formation of superoxide anions. Thirdly, hydroxyl radicals are generated from hydrogen peroxide through the Fenton reaction. In this Fenton reaction Fe2+ ions are available since Fe3+ ions are readily reduced by 3-hydroxyanthranilic acid. The superoxide anions do not seem to participate in the reduction of Fe3+ ions, since superoxide anions are rapidly dismuted by SOD present in the reaction mixture.     

水分胁迫对木麻黄(Casuarina.Sp.)小枝活性氧伤害的定量分析

两种木麻黄在水分胁迫条件下水势、质膜相对透性、超氧离子、ＭＤＡ和ＳＯＤ之间都显著相关。但ＣＡＴ和ＰＯＤ与水势、质膜相对透性、超氧离子、ＭＤＡ和ＳＯＤ等指标之间的相关关系不一致,大部分不显著,水势对质膜相对透性、ＭＤＡ、超氧离子和ＳＯＤ的弹性系数均属于ＥＰ〈０,处于水势对这些生理指标的负效应阶段。ＳＯＤ对质膜相对透性、ＭＤＡ和超氧离子的弹性系数均小于１,属于０〈ＥＰ〈１,处于ＳＯＤ活性对质膜相对透性的效应的递减阶段。超氧离子对质膜相对透性和ＭＤＡ的弹性系数均小于１,属于０〈ＥＰ〈１,处于超氧离子对质膜相对透性的效应的递减阶段。抗旱能力不同的两种木麻黄弹性系数和相尖的边际量不同。     

Electron spin resonance study on the permeability of superoxide radicals in lipid bilayers and biological membranes.

The permeability of lipid bilayers and biological membranes to superoxide free radicals was examined by using superoxide dismutase (SOD)-loaded lipid vesicles and SOD-loaded erythrocyte ghosts. After exposing SOD lipid vesicles and SOD ghosts to enzymatically produced superoxide radicals and using spin-trapping and electron spin resonance (ESR) techniques, we found that SOD entrapped within erythrocyte ghosts effectively scavenges external O2.- while SOD inside the lipid bilayers has no effect. These results confirm that O2.- is able to cross through a biological plasma membrane but not across a pure lipid bilayer. The data provide instruction as to how and where anti-oxidant therapy is to be approached relative to the site of oxygen free radical production.     

Discovery of superoxide reductase: an historical perspective

For more than 30 years, the only enzymatic system known to catalyze the elimination of superoxide was superoxide dismutase, SOD. SOD has been found in almost all organisms living in the presence of oxygen, including some anaerobic bacteria, supporting the notion that superoxide is a key and general component of oxidative stress. Recently, a new concept in the field of the mechanisms of cellular defense against superoxide has emerged. It was discovered that elimination of superoxide in some anaerobic and microaerophilic bacteria could occur by reduction, a reaction catalyzed by a small metalloenzyme thus named superoxide reductase, SOR. Having played a major role in this discovery, we describe here how the concept of superoxide reduction emerged and how it was experimentally substantiated independently in our laboratory.Abbreviations Dfx desulfoferrodoxin - SOD superoxide dismutase - SOR superoxide reductase