Characterization of hepatic and extrahepatic glutathione S-transferases in rainbow trout (Oncorhynchus mykiss) and their induction by 3,3′,4,4′-tetrachlorobiphenyl

Liver, kidney, gill and olfactory epithelium cytosolic fractions of rainbow trout (Oncorhynchus mykiss) were examined for glutathione S-transferase (GST) contents. Proteins retained on a glutathione (GSH)-affinity matrix were separated as monomers by reversed-phase HPLC and characterized by immunoblotting, mass spectrometry and partial amino acid sequence. For each organ concerned, a specific pattern of these proteins was determined and appeared similar for liver and kidney on one hand, and for gill and olfactory epithelium on the other hand. It was confirmed that the prominent hepatic GST is a class enzyme, also constitutively expressed as a major isoform in the four organs studied. Moreover, a class variant and two new class GST subunits were characterized in minor fractions. An unknown protein, which was found major in gills and olfactory epithelium, exhibited some characteristics of class GSTs. Occurrence of possible GSH-adduct formation observed on two distinct monomers in specific experimental conditions is discussed. These results and methods were used to investigate the effect of 3,3,4,4-tetrachlorobiphenyl (TCB), a polychlorinated biphenyl (PCB), on GST expression in trout liver. From HPLC-profiling, significant co-induction of the major class and the two minor class GST subunits was observed in trout after waterborne exposure to TCB which was followed by a slight increase in 1-chloro-2,4-dinitrobenzene (CDNB) activity. The present work allows qualitative evaluation of the specific detoxification potential of rainbow trout. The use of HPLC-profiling of GSTs as a possible tool for the biomonitoring of polluted aquatic environment is suggested.     

饲料中添加谷胱甘肽对吉富罗非鱼抗氧化和抗亚硝基氮应激能力的影响

选择初始体重(3.27±0.04)g的吉富罗非鱼(GIFT Oreochromis niloticus),分别投喂基础饲料和添加80、160、240、320和400 mg/kg谷胱甘肽(GSH)的试验饲料,记作G0、G80、G160、G240、G320和G400。49 d后,G320血清和G160、G240肝脏谷胱甘肽S-转移酶,G160、G240血清和G320肝脏谷胱甘肽还原酶,G240肝脏超氧化物歧化酶,G320血清和G240肝脏过氧化氢酶活性,G240～G400血清和G240肝脏总抗氧化能力,G320血清抗超氧阴离子活性,与对照组相比分别显著升高。G80～G320肝脏丙二醛含量显著低于对照组。在亚硝基氮应激96 h内,G320累积死亡率显著降低。结果表明,饲料中添加一定量的GSH能显著提高罗非鱼的抗氧化性能和抗亚硝基氮应激能力。     

Sulphur accumulation after Verticillium dahliae infection of two pepper cultivars differing in degree of resistance

Elemental sulphur levels, sulphur localization in stems, and levels of sulphate, glutathione and cysteine were studied in pepper ( Capsicum annuum ) cvs Yolo Wonder (higher resistance) and Luesia (lower resistance) after inoculation with Verticillium dahliae , the cause of vascular wilt. Accumulation of elemental sulphur (S⁰) was first detected 10 days after inoculation in Yolo Wonder (mean S⁰ level 7·3  µ g g⁻¹ DW), and 15 days after inoculation in Luesia (mean S⁰ level 3·3  µ g g⁻¹ DW). The maximum level was reached 21 days after inoculation in Yolo Wonder (14·1  µ g g⁻¹ DW). In control plants, elemental sulphur was not detected. SEM-EDX (scanning electron microscopy-energy dispersive X-ray microanalysis) indicated that the sulphur was not restricted to a specific location, but was dispersed throughout the vascular tissue. Sulphate levels showed a decline at the end of the experiment in inoculated plants, possibly related to the increase in sulphur levels seen in the two cultivars. The differences in sulphate levels between the two cultivars may be due to faster sulphate breakdown in cv. Yolo Wonder.     

Lowered Antioxidant Status of Red Blood Cells in Post-Parturient Haemoglobinuria of Buffaloes

The antioxidant status of the red blood cells of buffaloes (n = 20) suffering from post-parturient haemoglobinuria (PPH) was assessed by comparing their tocopherol (vitamin E) and reduced glutathione contents with those of red blood cells from apparently healthy buffaloes (n = 20). The red cell tocopherol content of the diseased buffaloes (1.76±0.11 g/ml) was significantly (p<0.01) lower than that of healthy buffaloes (2.45±0.14 g/ml). This may be the first report comparing the concentration of tocopherol in the red blood cells of buffaloes suffering from PPH and apparently healthy buffaloes.There was a drastic reduction in the reduced glutathione content in the red cells of haemoglobinuric buffaloes (23.74±2.86 mg%) compared to the healthy control buffaloes (73.71±3.87 mg%). The diseased buffaloes also exhibited severe hypophosphataemia. These findings suggest that an impaired or insufficient antioxidant potential of the red blood cells in this disease in buffaloes is associated with the phosphorus deficiency.     

The effects of dietary oxidized fat and selenium source on performance,glutathione peroxidase,and glutathione reductase activity in broiler chickens

Normal or elevated selenium status of broilers, which is influenced by dietary selenium sources, improves the bird’s ability to overcome the adverse effects of reactive oxygen metabolites. The objective of this study was to evaluate the effects of feeding graded levels of peroxidized poultry fat on blood and hepatic glutathione peroxidase (GSH-Px), and hepatic glutathione reductase activity in broiler chickens fed either inorganic sodium selenite (SEL) or organic selenium enriched in the organic selenium yeast product Sel-Plex (SP). Nine starter diets, varying in levels of oxidized fat (0, 3, and 6 mEq/kg) and dietary selenium sources, were fed to 360 male chicks from hatch to 21 d of age. Sel-Plex or SEL was added to the basal diet to provide either 0 or 0.2 ppm of supplemental selenium in the diets. Blood and hepatic samples were obtained for each treatment group at 21 d of age. Neither peroxidized fat nor selenium source significantly altered the activity of hepatic glutathione reductase (P ≤ 0.05). Blood GSH-Px was influenced significantly by both fat and selenium source (P ≤ 0.05), but the fat × selenium source interaction was not significant (P ≥ 0.3). A selenium source effect on the hepatic GSH-Px activity (P ≤ 0.05) was evidenced by higher GSH-Px activity, even in the basal diet with no added peroxidized fat. An increase in GSH-Px activity was seen in the erythrocyte and hepatic samples in both the SEL and SP treatments when peroxidized fat was given at 3 mEq/kg, but in the erythrocytes and in the hepatic tissues from SEL-supplemented birds, there was an apparent inhibition of GSH-Px activity. This inhibition was not seen in the hepatic tissue samples from SP-fed birds. Because elevated GSH-Px activity is indicative of oxidative stress, it was concluded that dietary SP supplementation resulted in better selenium and redox status in broilers than did SEL. These results indicate that the dietary selenium supplied in an organic form (selenium yeast as SP) improved the selenium and redox status in broilers, leading to greater resistance to oxidative stress than when the inorganic form of selenium (SEL) was fed.     

NADP-malic enzyme and glutathione reductase contribute to glutathione regeneration in Fragaria vesca fruit treated with protective high CO2 concentrations

Treatment of harvested strawberries (Fragaria vesca L. Mara des Bois) with high concentrations of CO₂ is an effective means of limiting fungal decay and avoiding disorders caused by low temperature storage. In the present study, we investigated the role of NADP-ME gene expression and activity in lowering malic acid contents and in the provision of NADPH required for the regeneration of the reduced form of glutathione (GSH). We also measured glutathione reductase (GR: EC 1.6.4.2) activity in strawberries treated with different high CO₂ concentrations (0, 20 and 40% for 3 days) during storage at 0 °C. A decrease in malic acid content in fruit exposed to 20% CO₂ was primarily mediated by the stimulation of NADP-ME activity, rather than associated with changes in the expression of cytosolic NADP-ME genes. Moreover, malic acid decarboxylation was associated with a marked increase in GR activity, which may account for the increased levels of glutathione in fruit following exposure to 20% CO₂. This chain of events was not observed in untreated fruit stored in air or in fruit treated with 40% CO₂, suggesting that the unique cellular redox status of 20% CO₂-treated fruit plays an important role in detoxification and protection from damage during storage. Based on these findings, we propose that NADP-ME activation in fruit exposed to 20% CO₂ provides NADPH for glutathione regeneration by GR, thereby conferring protection against the cellular damage caused by low temperatures or excessive high CO₂ levels.     

The effect of different concentrations of linseed oil or fish oil in the maternal diet on the fatty acid composition and oxidative status of sows and piglets

N‐3 polyunsaturated fatty acids (PUFA) are essential for foetal development. Hence, including n‐3 PUFA in the sow diet can be beneficial for reproduction. Both the amount and form (precursor fatty acids vs. long chain PUFA) of supplementation are important in this respect. Furthermore, including n‐3 PUFA in the diet can have negative effects, such as decreased arachidonic acid (ARA) concentration and increased oxidative stress. This study aimed to compare the efficacy to increase eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) concentrations in the piglet, when different concentrations of linseed oil (LO, source of precursor α‐linolenic acid) or fish oil (FO, source of EPA and DHA) were included in the maternal diet. Sows were fed a palm oil diet or a diet including 0.5% or 2% LO or FO from day 45 of gestation until weaning. Linoleic acid (LA) was kept constant in the diets to prevent a decrease in ARA, and all diets were supplemented with α‐tocopherol acetate (150 mg/kg) and organic selenium (0.4 mg/kg) to prevent oxidative stress. Feeding 0.5% LO or 0.5% FO to the sows resulted in comparable EPA concentrations in the 5‐day old piglet liver, but both diets resulted in lower EPA concentrations than when 2% LO was fed. The highest EPA concentration was obtained when 2% FO was fed. The DHA level in the piglet liver could only be increased when FO, but not LO, was fed to the sows. The 2% FO diet had no advantage over the 0.5% FO diet to increase DHA in the piglet. Despite the constant LA concentration in the sow diet, a decrease in ARA could not be avoided when LO or FO were included in the diet. Feeding 2% FO to the sows increased the malondialdehyde concentration (marker for lipid peroxidation) in sow plasma, but not in piglets.     

叶施谷胱甘肽对水稻镉和矿质元素含量的影响

为评估还原型谷胱甘肽（GSH）作为水稻降Cd叶面调理剂的可行性,本研究通过田间试验,检测开花期叶面喷施一次GSH对水稻各器官Cd和矿质元素含量的影响,并结合各元素在水稻不同器官中的分布情况,探究GSH抑制水稻体内Cd转运的作用机制。结果表明：开花期叶面喷施GSH可显著降低水稻籽粒中Cd含量,最高降幅可达76.5%,使稻米Cd含量由0.449 mg·kg^-1降至我国食品安全标准（0.2 mg·kg^-1）以内,并提高了稻米中K、Mg、Ca和Mn的含量。喷施GSH后水稻各营养器官的Cd含量均显著降低,降幅为68.8%~86.7%;同时,不同器官中K、Mg、Ca、Fe、Mn和Zn的含量也有不同程度的升高或降低。不同相邻器官之间的元素转移系数显示,叶面喷施GSH分别提高了Cd和Zn从第二节间到穗下节的转移系数,增幅可达46.2%和134.4%,降低了Cd和Zn从穗下节到旗叶的转移系数,降幅分别为44.5%和32.2%,并降低了Cd从穗下节继续向上到穗颈的转运系数,降幅可达31.4%。相关性分析发现,不同处理组水稻第二节及以上营养器官中,Zn含量与Cd含量相关系数最高,为0.809,为极强正相关（P<0.001）。由此可见,喷施GSH不但降低了水稻各器官中Cd含量,而且能够通过调控穗下节与其相连营养器官——穗颈、第二节间和旗叶之间的Cd转运,提高水稻自身主要Cd阻控器官——穗下节对Cd的拦截能力,进而抑制Cd向籽粒的转运和积累。同时,喷施GSH也提高了水稻穗下节对Zn的固定能力,改变了水稻不同营养器官中6种矿质元素的含量和分布,其中Zn与Cd的相关性最高,并最终提高了稻米中K、Mg、Ca和Mn的含量。     

Glutathione and its Related Enzymes in the Nile Fish

Glutathione (GSH) and related enzymes, glutathione transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) form an important phase 2 biotransformation enzymes system. The objective of this study was to compare this enzymes system in three fish species from the river Nile, Oreochromis niloticus, Claris lazera and Cyprinus carpio in order to establish the main differences and to purify and characterize GST from the liver of O. niloticus.The level of GSH and the activity of GST, GPx and GR in the liver, kidney and gills of the three fish species were examined. A simple reproducible procedure for the purification of GST from the liver of O. niloticus to homogeneity, which includes chromatography on DEAE- cellulose followed by affinity chromatography on GSH-sepharose was established. The molecular mass was found to be 25,460 Da by SDS-PAGE. The Michaelis-Meneten constants (K_m) of the enzyme for GSH and CDNB were 0.35 mM and 0.42 mM, respectively. The affinity purified enzyme exhibited maximum pH at pH 8.0 and increasing pH above 8.0 did not affect the observed maximum. The purified enzyme acts readily on CDNB, less readily on some standard transferase substrates (1,2-dichloro-4-nitrobenzene and p-nitrophenethyl bromide) and not at all on others (bromosulphophthalein and p-nitrobenzyl chloride). Bromosulfophthalein, cibacron blue and hematin inhibited CDNB-conjugating activity of the purified enzyme with IC₅₀ 0.079, 3.98 and 0.126 μM, respectively.