Heat stress‐induced alterations of antioxidants in the freshwater fish Channa punctata Bloch

The effect of elevated temperature on the antioxidants in the freshwater fish Channa punctata was investigated. Fish stressed with an elevated temperature of 12° C, range ± 1° C over the ambient temperature for 3 h showed a significant ( P  < 0·05–0·01) reduction in the levels of antioxidants: reduced glutathione (GSH) and non‐protein thiols. Activity of glutathione reductase was also reduced in all the tissues (liver, kidney and gills) after 3 h of heat stress and 24 h recovery. Catalase (CAT) showed enhanced activity in liver in both the conditions while gills and kidney showed a decreased CAT activity. Glutathione S‐transferase (GST) activity in kidney and liver decreased significantly ( P  < 0·05–0·01) after 3 h of heat stress. At 24 h GST activity showed a tendency to normalize in all the tissues along with a concomitant increase in the GSH level in the kidney. Total and protein thiols in heat stressed fish when matched with controls, showed significant ( P  < 0·05) reduction in the kidney only with a transient increase in liver and gills. Heat shock also induced lipid peroxidation in 3 h heat‐treated and recovery groups when compared with controls. Elevated temperature therefore resulted in tissue specific and time‐dependent alterations of antioxidants in the fish. It also induced lipid peroxidation in various tissues.     

Antioxidant response and oxidative stress levels in Macrobrachium borellii (Crustacea: Palaemonidae) exposed to the water-soluble fraction of petroleum

The aim of the present work was to evaluate the effect of the water soluble fraction of hydrocarbons (WSF) on the antioxidant status of the freshwater prawn Macrobrachium borellii. First, seasonal variations were studied in a non-polluted area. Hepatopancreas and gills showed season-related fluctuations in catalase (CAT), glutathione-S-transferase (GST) activities and in lipid peroxidation levels (LPO), but not in superoxide dismutase (SOD). Then, adults were exposed semi-statically to sublethal doses for 7days. CAT, SOD, GST, and glutathione peroxidase (GPx) activities and LPO, reduced glutathione (GSH) and protein oxidation (PO) levels were determined. Exposed individuals showed significant increases in CAT, SOD, and GST activities in hepatopancreas and CAT activity in gills. GPx activity did not vary in either tissues. While LPO levels increased, GSH levels decreased significantly in hepatopancreas of exposed animals, but PO levels showed no variation. Induction of SOD was also assessed by Real-time PCR mRNA expression in hepatopancreas. The non-enzymatic antioxidant activity was also tested; ABTS 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) was higher in hemolymph of treated-prawns compared to controls, but ferric reducing activity of plasma assay (FRAP) values did not change. Taken together, the present results indicated that the antioxidant defenses of M. borellii, mainly in hepatopancreas, were significantly affected by aquatic hydrocarbon contamination, regardless of the season.     

Cytotoxic effects of 4-octylphenol on fish hepatocytes

The present study was conducted to determine cytotoxic effects of 4-octylphenol (4-OP) on primary cultured hepatocytes of pearl mullet (Alburnus tarichi). Lactate dehydrogenase (LDH) release, malondialdehyde (MDA) level, antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST)] and glutathione (GSH) content were measured after 24-h exposure to 4-OP. 4-OP caused dose- and time-dependent increases in LDH release. Significant induction of MDA level and decrease in GSH content were found. SOD and GPx activities were decreased while GST activity was increased. These findings suggest that 4-OP leads to cytotoxicity by depressing antioxidant defenses in fish hepatocytes.     

5-(Pentafluorobenzoylamino)fluorescein: A selective substrate for the determination of glutathione concentration and glutathione S-transferase activity

5-(Pentafluorobenzoylamino)fluorescein (PFB-F), a new thiol-reactive molecule was synthesized to improve the detection limits and specificity of the assays for glutathione S-transferase (GST) activity and glutathione (GSH). A rapid assay method to measure GSH concentration or GST activity and the simultaneous analysis of multiple samples is possible because the glutathione adduct, GS-TFB-F, is separated from PFB-F by thin-layer chromatography (TLC) and can be quantitated by a fluorescence scanner. The detection limits for GSH and for GST activity using TLC were found to be as low as 10 pmol/microl and 1 ng/microl using equine liver GST, respectively. Determination of GSH concentration or GST activity in bovine pulmonary artery endothelial (BPAE) cell lysates gave a linear response for samples corresponding to 500-2500 cells. PFB-F could also measure GST activities of GST fusion proteins and prove to be a suitable substrate for determining the activities of human GST isozymes and other sources of mammalian GST. The selectivity of PFB-F with GSH was proven by comparing trace amount of the adducts that formed with cysteine and beta-galactosidase to that formed with GSH. The HPLC profile of a reaction mixture where cell lysate was used in place of purified GST, also shows only two main peaks, corresponding to GS-TFB-F and unreacted PFB-F. The selectivity of PFB-F for GSH was further confirmed by exposing BPAE cells to dl-buthionine-[S,R]-sulfoximine (BSO). Our results of GS-TFB-F determination indicate that 12-, 24-, or 36-h incubations with BSO caused 2-, 6-, or 7.6-fold reductions in GSH levels, respectively.     

The effects of selenium on oxidative stress biomarkers in the freshwater characid fish matrinxã, Brycon cephalus (Günther, 1869) exposed to organophosphate insecticide Folisuper 600 BR (methyl parathion)

Methyl parathion (MP), an organophosphate widely applied in agriculture and aquaculture, induces oxidative stress due to free radical generation and changes in the antioxidant defense system. The antioxidant roles of selenium (Se) were evaluated in Brycon cephalus exposed to 2 mg L(-1) of Folisuper 600 BR (MP commercial formulation - MPc, 600 g L(-1)) for 96 h. Catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione S-transferase (GST), reduced glutathione (GSH) and lipid peroxidation (LPO) levels in the gills, white muscle and liver were evaluated in fish fed on diets containing 0 or 1.5 mg Se kg(-1) for 8 weeks. In fish treated with a Se-free diet, the MPc exposure increased SOD and CAT activities in all tissues. However, the GPx activity decreased in white muscle and gills whereas no alterations were observed in the liver. MPc also increased GST activity in all tissues with a concurrent decrease in GSH levels. LPO values increased in white muscle and gills and did not change in liver after MPc exposure. A Se-supplemented diet reversed these findings, preventing increases in LPO levels and concurrent decreases in GPx activity in gills and white muscle. Similarly, GSH levels were maintained in all tissue after MPc exposure. These results suggest that dietary Se supplementation protects cells against MPc-induced oxidative stress.     

A Mu-class glutathione S-transferase from gills of the marine shrimp Litopenaeus vannamei: purification and characterization

Glutathione S-transferases (GSTs) are a family of detoxifying enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles, thereby increasing the solubility of GSH and aiding its excretion from the cell. In this study, a glutatione S-transferase from the gills of the marine shrimp Litopenaeus vannamei was purified by affinity chromatography using a glutathione-agarose affinity column. GST was purified to homogeneity as judged by reducing SDS-PAGE and zymograms. This enzyme is a homodimer composed of approximately 25-kDa subunits and identified as a Mu-class GST based on its activity against 1-chloro-2,4-dinitrobenzene (CDNB) and internal peptide sequence. The specific activity of purified GST was 440.12 micromol/(min mg), and the K(m) values for CDNB and GSH are very similar (390 and 335 microM, respectively). The intersecting pattern of the initial velocities of this enzyme in the Lineweaver-Burke plot is consistent with a sequential steady-state kinetic mechanism. The high specific activity of shrimp GST may be related to a highly effective detoxification mechanism necessary in gills since they are exposed to the external and frequently contaminated environment.     

Parameters related to oxygen free radicals in erythrocytes,plasma and epidermis of the hairless rat

The following parameters related to oxygen free radicals (OFR) were determined in erythrocytes and the epidermis of hairless rats: catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), reduced (GSH) and oxidized (GSSG) glutathione, glutathione S-transferase (GST), superoxide dismutase (SOD) and thiobarbituric acid reactive substances (TBARS). GSH, GSSG and TBARS were also analyzed in plasma. In erythrocytes, the Pearson correlation coefficients (r) were significant (p < 0.001) between glutathione and other parameters as follows: GSH correlated negatively with GSSG (r = -0.665) and TBARS (r = -0.669); GSSG correlated positively with SOD (r = 0.709) and TBARS (r = 0.752). Plasma GSSG correlated negatively with erythrocytic thermostable GST activity (r = -0.608; p=0.001) and with erythrocytic total GST activity (r = -0.677; p < 0.001). In epidermis (p < 0.001 in all cases), GSH content correlated with GSSG (r = 0.682) and with GPx (r = 0.663); GSSG correlated with GPx (r = 0.731) and with GR (r = 0.794). By multiple linear regression analysis some predictor variables (R(2)) were found: in erythrocytes, thermostable GST was predicted by total GST activity and GSSG, GSSG content was predicted by GSH and by the GSH/GSSG ratio and GPx activity was predicted by GST, CAT and SOD activities; in epidermis, GSSG was predicted by GR and SOD activities and GR was predicted by GSSG, TBARS and GPx. It is concluded that the hairless rat is a good model for studying OFR-related parameters simultaneously in blood and skin, and that it may provide valuable information about other animals under oxidative stress.     

Metal-ion-mediated oxidative stress in the gill homogenate of rainbow trout (Oncorhynchus mykiss)

Human activities play a major role in toxic and carcinogenic metal pollution of the environment. This study was undertaken to evaluate the effects of copper and mercury at the 400-to 1000-μM concentration range on some biochemical markers of oxidative stress, such as lipid peroxidation (LPO), glutathione-S-transferase (GST) activity, and reduced glutathione (GSH) content in the rainbow trout gill homogenates with or without supplementation of manganese, selenium, and bovine serum albumin (BSA). The integrity of DNA was also measured to assess metal ion toxicity. The results showed that the LPO and specific activity of GST were elevated. This indicated that cell-protecting antioxidant mechanisms were overtaxed and could not prevent membrane peroxidation. Following the addition of metals, the GSH content was also significantly reduced in a concentration-dependent manner. Mercury was found to be more effective than copper. The application of antioxidants proved beneficial in inhibiting LPO, reducing GST activity, and elevating the GSH levels in the gill samples. Manganese was more effective than selenium and BSA. Surprisingly, when BSA (1.0%) was added to the gill homogenates treated with a 1000-μM concentration of metal ions, instead of alleviating malondialdehyde (MDA) generation, a drastic elevation in the MDA levels, alleviation in GST activity, and a further decrease in glutathione (GSH) levels were observed, which were most likely the result of pro-oxidant activity of BSA. The results also indicated that mercury and copper functioned as genotoxic pollutants, which altered the DNA integrity by inducing the single and double-stranded DNA breaks in the gill cell nuclei. Collectively, toxicity of metal ions is related to the depletion of GSH content and inhibition of antioxidant enzyme GST, resulting in the propagation of LPO and DNA damage.     

铜对梨形环棱螺抗氧化酶活性和金属硫蛋白含量的影响

本实验采用暴露重金属的方法,研究了不同浓度硫酸铜（Cu2+ 分别为0、0.005、0.01、0.02、0.05 mg/L）在不同暴露时间（0—14d）下对梨形环棱螺（Bellamya purificata）过氧化氢酶(CAT)、超氧化物歧化酶（SOD）、谷胱甘肽硫转移酶（GST）的活性、还原性谷胱甘肽（GSH）和金属硫蛋白（MT）含量的影响,以探讨Cu2+ 对梨形环棱螺的氧化损伤及其防御作用的机理,并为水环境Cu2+ 污染的早期诊断及生态风险评价提供科学的依据。结果表明：Cu2+对梨形环棱螺肝脏和鳃中CAT、SOD、GST、GSH 和MT 均有明显影响,表现出时间剂量效应。SOD在前4天、CAT在前3天酶活性总体上表现出诱导趋势, GST在前4天酶活性处于诱导状态,随着暴露时间的延长,酶活性下降,到第5天时表现出抑制趋势;随着时间的进一步增长,至14d时, 0.005 mg/L剂量组酶活性维持在正常值附近波动, 0.01 mg/L剂量组酶活性被诱导, 0.02 mg/L剂量组酶活性在肝脏中表现为诱导而在鳃中则被抑制,0.05 mg/L剂量组酶活性被抑制。肝脏和鳃GSH含量的变化与GST相似,在短时间内表现出诱导效应,肝脏GSH在暴露的前5天、鳃GSH在暴露的前4天均处于诱导状态,随着暴露时间的延长,0.005 mg/L剂量组表现出诱导,0.05 mg/L剂量组则受到抑制。MT在整个实验期间均处于诱导状态,各剂量组在0.5d被极显著诱导,随后MT含量出现起伏波动,有上升和下降,至第14天时达到一稳定水平。其中,0.01 mg/L剂量组肝脏的MT在整个实验期间均被极显著地诱导（P <0.01）,0.01 mg/L 剂量组的鳃组织MT除第10天外也被极显著诱导（P <0.01）。在暴露14d时,除0.05 mg/L剂量组的肝脏MT外,其余处于极显著诱导状态（P <0.01）。     

Ontogenetic changes in hepatic glutathione system (synthesis, catabolism, export) of male Uje:WIST rats.

The age-courses of concentrations of reduced (GSH) and oxidized (GSSG) glutathione, of GSH synthesizing enzyme activities, of glutathione S-transferase (GST), of GSSG-reductase (GR) and of biliary GSH and GSSG export were measured in livers from male Uje:WIST rats. Additionally, the age-courses of plasma GSH and GSSG concentrations were investigated. The hepatic level of GSH showed a biphasic pattern with a first maximum immediately after birth and a small second peak at the 50th day of life. The GSSG level increased continuously up to day 60 of life. The cytosolic GSH synthesizing enzyme activities showed diverse developmental patterns indicating different regulation principles. The hepatic activity of GR was relatively constant in the different age groups after birth. The GST activity (with o-dinitrobenzene as substrate) was relatively low at birth (about 30% of the maximum measured at day 60 of life). The maximum of GSH plasma level was found at birth. With increasing age a significant decrease in this level was observed. The excretion rate of total GSH (GSH + 2 GSSG) in bile was found to increase about 9-fold between 15 and 105 days of age. The results indicate that changes of hepatic GSH concentration with age are dependent on numerous factors. The balance between synthesis, catabolism and export is important for the maintenance of this level.     

Use of Oxidative Stress Indices of Freshwater Bivalve <Emphasis Type="Italic">Dreissena polymorpha</Emphasis> (Pallas) as Biomarkers of Anthropogenic Pollution of Rybinsk Reservoir

This article analyzes the indices of oxidative stress activity in freshwater bivalve Dreissena polymorpha (Pallas) from areas of the Rybinsk Reservoir with different levels of anthropogenic load. The following indices are studied: activity of catalase (CAT), glutathione reductase (GST), glutathione S-transferase (GST) and content of reduced glutathione (GSH), malondialdehyde (MDA), and carbonyl groups of oxidized proteins (CG). During the study period (July), the indices of oxidative stress of zebra mussel D. Polymorpha did not differ between sexes. Mussels that were collected in the most polluted part of the reservoir, the Sheksna stretch near the industrial complex of the city of Cherepovets, had a higher activity of catalase, glutathione S-transferase, and glutathione reductase and a higher content of malondialdehyde than zebra mussels taken from the relatively clean Volga stretch.     

Tissue lipid peroxidation and glutathione‐dependent enzyme status in patients with oral squamous cell carcinoma

We examined the extent of lipid peroxidation and the status of reduced glutathione (GSH) and the GSH‐dependent enzymes—glutathione peroxidase (GPx) and glutathione‐S‐transferase (GST)—in oral tumour tissues from 33 adult oral cancer patients and an equal number of age‐ and sex‐matched normal subjects. Diminished lipid peroxidation in the oral tumour tissue was accompanied by a significant decrease in phospholipids and an increase in the cholesterol/phospholipid (C/P) ratio. The concentration of glutathione and the activities of GPx and GST were elevated in oral tumour tissues. These findings suggest that GSH‐ and GSH‐dependent enzymes play a crucial role in tobacco‐related tumourigenesis and may be considered as markers of carcinogen exposure. Copyright © 1999 John Wiley & Sons, Ltd.     

Oxidative stress biomarkers in the freshwater characid fish, Brycon cephalus, exposed to organophosphorus insecticide Folisuper 600 (methyl parathion)

Methyl parathion (MP) is an organophosphorus insecticide used worldwide in agriculture and aquaculture due to its high activity against a broad spectrum of insect pests. The effect of a single exposure to 2 mg L(- 1) of a commercial formulation of MP (MPc: Folisuper 600(R), MP 600 g L(- 1)) on catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione S-transferase (GST), reduced glutathione (GSH) and lipid peroxidation (LPO) of the liver, white muscle and gills of Brycon cephalus was evaluated after 96 h of treatment. MPc exposure resulted in a significant induction of SOD, CAT and GST activity in all tissues. However, the GPx activity decreased significantly in white muscle and gills, whereas no alterations were observed in hepatic GPx activity. MPc also induced a significant increase in LPO values in the white muscle and gills, while hepatic LPO levels did not show any significant alteration. The current data suggest that MPc has oxidative-stress-inducing potential in fish, and that gills and white muscle are the most sensitive organs of B. cephalus, with poor antioxidant potentials. The various parameters studied in this investigation can also be used as biomarkers of exposure to MPc.     

N‐nitrosodimethylamine changes the expression of glutathione S‐transferase in the liver of male mice: The role of antioxidants

The present study investigated the protective effect of gossypol, selenium, zinc, or glutathione (GSH) against dimethylnitrosamine (DMN)‐induced hepatotoxicity in the livers of male mice. The expression and the activity of glutathione S‐transferase (GST), levels of GSH, and free radicals (malondialdehyde (MDA)), as well as the activity of glutathione reductase were determined after the treatment of mice for seven consecutive days with low or high doses of gossypol, selenium, zinc, or GSH. In experimental groups, DMN was administered as a single dose for 2 h after the repeated dose treatments of mice for seven consecutive days with each antioxidant. DMN reduced the expression and inhibited the activity of GST. However, repeated treatments of mice with low‐dose gossypol or high dose of either selenium or GSH followed by a single dose of DMN induced the expression and the activity of GST. In contrast, low‐dose treatments of mice with zinc, selenium, or GSH followed by a single dose of DMN reduced the expression and the activity of GST compared to either control or DMN‐treated groups. In addition, high‐dose treatment with either gossypol or selenium markedly induced the levels of GSH compared to either control or DMN‐treated groups. Interestingly, pretreatment of mice with high dose of either gossypol or selenium for seven consecutive days followed by a single dose of DMN decreased the levels of MDA, whereas DMN induced such levels. It is concluded that high dose of either gossypol or selenium is a stronger protector than zinc and GSH in ameliorating the toxic effects of DMN. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:389–395, 2008; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20255     

Interactive effects of zinc and nickel on the glutathione system state in <Emphasis Type="Italic">Mimulus guttatus</Emphasis> plants

To determine whether the enhanced stress tolerance of ZnSO₄ with NiSO₄-treated Mimulus guttatus Fischer ex DC. plants was associated with the glutathione (GR-GSH) system, we investigated the changes in glutathione redox state (reduced (GSH), oxidized (GSSG) forms, total reduced (GSHt) glutathione, and GSH/GSSG ratio) and in the enzymatic activities of glutathione reductase (GR) and peroxidatic glutathione S-transferases (GST). The 6-week-old plants were grown in water culture during 4 weeks on a modified Rorison’s medium with ZnSO₄ (50, 100, and 200 μM) and NiSO₄ (20 and 80 μM) in a condition of separate or simultaneous supply of the components. Dry biomass accumulations of roots and shoots were not influenced by the examined treatments. The positive correlations between the total external concentrations of ZnSO₄ and NiSO₄ and the total Zn and Ni contents in roots and leaves were found. It was determined that the MDA content was higher in the ZnSO₄-treated plants than in the NiSO₄-treated ones. The supplementation of the ZnSO₄-treated plants with varied concentrations of NiSO₄ decreased the Zn-induced increase in the MDA levels. The inverse proportionality between the MDA and pigment levels in leaves was found. The Zn-Ni interactions were shown to induce the decreases in the GR activity, the total peroxidatic GST activity, and the GSH/GSSG ratio in roots. However, in leaves, the GR activity and the GSH/GSSG ratio were significantly increased and the total peroxidatic GST activity was decreased. The supplementation of the ZnSO₄-treated plants with varied concentrations of NiSO₄ restored the Zn-induced reduction in the GSHt levels in roots and decreased the Zn-induced increase in the GSSG levels in leaves, which resulted in more reduced state of the intracellular environment. It was likely to cause a decrease of the MDA level. Thus, our studies on the Zn?Ni interactions identified the antagonizing role of Ni in Zn toxicity by the GR-GSH system.     

Variation in glutathione status associated with induction and initiation of diapause in eggs of the bivoltine strain of the silkworm Bombyx mori

For the bivoltine (Dazao) strain of the silkworm Bombyx mori L., diapause expression in progeny is induced by exposure to conditions of 25 °C and continuous illumination (LL) during the maternal generation, whereas an environment of 15 °C and constant darkness (DD) results in nondiapause progeny. Initiation of diapause in progeny can be prevented by treatment of diapause‐programmed eggs with hydrochloric acid (HCl) at approximately 24 h post‐oviposition. To investigate whether glutathione is involved in the regulation of diapause induction and initiation in this species, measurements of total glutathione, reduced glutathione (GSH), oxidised glutathione (GSSG), GSH/GSSG ratio, glutathione S‐transferase (GST) and peroxiredoxins (Prdx) are compared in eggs incubated under LL and DD conditions, and between diapause eggs and those treated with HCl. Compared with DD, eggs incubated under LL have higher total glutathione (GSH + 2GSSG), lower GSH, higher GSSG, a lower GSH/GSSG ratio, lower GST activity and higher Prdx activity at stages 20–25 of maternal embryogenesis. The lower ratio of GSH/GSSG is indicative of pro‐oxidative conditions during diapause induction, which may result from the stronger oxidation of GSH. Compared with HCl‐treated eggs, diapause eggs have lower total glutathione, no difference in GSH, lower GSSG, a higher GSH/GSSG ratio, no difference in GST activity and lower Prdx between 36 and 72 h post‐oviposition. The higher ratio GSH/GSSG is indicative of reducing conditions during diapause initiation, which may a result of the weaker oxidation of GSH. Moreover, variations of Prdx and GST suggest that Prdx rather than GST plays an important role in the oxidation of GSH during the induction and initiation of diapause.     

Hepatic and branchial glutathione S-transferases of two fish species: Substrate specificity and biotransformation of microcystin-LR

Liver and gills of roach (Rutilus rutilus) and silver carp (Hypophthalmichthys molitrix) were examined for glutathione S-transferases (GSTs) contents and their substrate specificity and capacity to biotransform microcystin-LR (MC-LR). GSTs and other glutathione (GSH) affine proteins were purified using a GSH-agarose matrix and separated by anionic chromatography (AEC). Substrate specificities were determined photometrical for 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), 4-nitrobenzyl chloride (pNBC) and ethacrynic acid (ETHA). Biotransformation rate of MC-LR was determined by high performance liquid chromatography (HPLC). Roach exhibited different hepatic and branchial GST activities for used substrates (DNB, pNBC and DCNB) compared to silver carp but not for ethacrynic acid. It suggests that, both fish species have similar amount of pi and/or alpha class, which were the dominant GST classes in liver and gills. Gills of both fish species contained a higher number of GST isoenzymes, but with lower activities and ability of MC-LR biotransformation than livers. GST isoenzymes from roach had higher activity to biotransform MC-LR (conversion rate ranging up to 268 ng MC-LR min^? 1 mL^? 1 hepatic enzyme) than that isolated from silver carp. Without any prior contact to MC-LR or another GST inducer, roach seems to be better equipped for microcystin biotransformation than silver carp.     

State of the system of antioxidant defense in tissues of the black sea turbot during the spawning

The system of antioxidant (AO) defense and processes of lipid peroxidation (LP) of the Black Sea turbot Psetta (Scophtalmus) maxima maeotica (L., 1758) have been investigated during the spawning season. The activity of glutathione peroxidase (GP), glutathione reductase (GR), catalase and content of reduced glutathione (GSH) and TBA-reactive products have been determined in gonads, gills, liver, red and white muscles of males and females at different stages of gonad's maturity (V and VI stages). The peculiarities of AO complex and LP depended on tissue specificity and sexual distinctions of the turbot have been found. The turbot females at VI stage were found to have the most significant changes. In gonads and liver the level of TBA-reactive products decreased. In gonads the activities of GP and GR decreased, but the level of GSH increased. In gills of these females the activity of GP and the level of GSH increased, while in the red muscles the activity of catalase raised. In white muscles the activity of GR dropped. In the males' tissues of the turbot at VI stage the growth of the activity of GP in gills and GSH content in white muscles have been found. In all tissues of males the decrease of the TBA-reactive products content has been observed.     

Vitamin E, Ascorbate, Glutathione, Glutathicne Disulfide, and Enzymes of Glutathione Metabolism in Cultures of Chick Astrocytes and Neurons: Evidence that Astrocytes Play an Important Role in Antioxidative Processes in the Brain

Abstract: GSH, GSSG, vitamin E, and ascorbate were measured in 14-day cultures of chick astrocytes and neurons and compared with levels in the forebrains of chick embryos of comparable age. Activities of enzymes involved in GSH metabolism were also measured. These included -γ-glutamylcysteine synthetase, GSH synthetase, γ-glutamyl cyclotransferase, γ-glutamyltranspeptidase, glutathione transferase (GST), GSH peroxidase, and GSSG reductase. The concentration of lipid-soluble vitamin E in the cultured neurons was found to be comparable with that in the forebrain. On the other hand, the concentration of vitamin E in the astrocytes was significantly greater in the cultured astrocytes than in the neurons, suggesting that the astrocytes are able to accumulate exogenous vitamin E more extensively than neurons. The concentrations of major fatty acids were higher in the cell membranes of cultured neurons than those in the astrocytes. Ascorbate was not detected in cultured cells although the chick forebrains contained appreciable levels of this antioxidant. GSH, total glutathione (i.e., GSH and GSSG), and GST activity were much higher in cultured astrocytes than in neurons. γ-Glutamylcysteine synthetase activity was higher in the cultured astrocytes than in the cultured neurons. GSH reductase and GSH peroxidase activities were roughly comparable in cultured astrocytes and neurons. The high levels of GSH and GST in cultured astrocytes appears to reflect the situation in vivo. The data suggest that astrocytes are resistant to reactive oxygen species (and potentially toxic xenobiotics) and may play a protective role in the brain. Because enzymes of GSH metabolism are generally well represented in cultured astrocytes and neurons these cells may be ideally suited as probes for manipulating GSH levels in neural tissues in vitro. Cultured astrocytes and neurons should be amenable to the study of the effects of various metabolic insults on the GSH system. Such studies may provide insights into the design of therapeutic strategies to combat oxidative and xenobiotic stresses.     

Differential Implication of Glutathione, Glutathione-Metabolizing Enzymes and Ascorbate in Tomato Resistance to Pseudomonas syringae

We studied the response of glutathione‐ and ascorbate‐related antioxidant systems of the two tomato cultivars to Pseudomonas syringae pv. tomato infection. In the inoculated susceptible A 100 cultivar a substantial decrease in reduced glutathione (GSH) content, oxidised glutathione accumulation and GSH redox ratio decline as well as glutathione peroxidase activity increase were found. The enhanced glutathione reductase activity was insufficient to keep the glutathione pool reduced. A transiently increased dehydroascorbic acid (DHA) content and ascorbic acid (AA) redox ratio decrease together with ascorbate peroxidase activity suppression were observed. Adversely to the progressive reduction in GSH pool size, AA content tended to increase but the changes were more modest than those of GSH. By contrast, in interaction with the resistant Ontario cultivar the glutathione pool homeostasis was maintained throughout P. syringae attack and no significant effect on the ascorbate pool was observed. Moreover, in the resistant interaction there was a significantly higher constitutive and pathogen‐induced glutathione‐S‐transferase (GST) activity. The relationship between GST activity and DHA content found in this study indicates that this enzyme could also act as dehydroascorbate reductase. These results reflect the differential involvement of GSH and AA in tomato‐P. syringae interaction and, in favour of the former, they clearly indicate the role of GSH and GSH‐utilizing enzymes in resistance to P. syringae. The maintenance of glutathione pool homeostasis and GST induction appear to contribute to tissue inaccessibility to bacterial attack.