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.     

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.     

Effects of in vitro exposure to ozone and/or hyperoxia on superoxide dismutase, catalase, glutathione and lipid peroxidation in red blood cells and plasma of rainbow trout, Oncorhynchus mykiss (Walbaum)

In aquaculture, ozone is used as a disinfectant. In its production, extensive amounts of oxygen are formed resulting in hyperoxic conditions in culture units. Both ozone and hyperoxia have the potential to be toxic via pro‐oxidant mechanisms and to activate antioxidant defence systems in cultured species. To eliminate systemic effects, blood of rainbow trout, Oncorhynchus mykiss (Walbaum), was exposed in vitro for 5 min to ozone/hyperoxia or hyperoxia, and changes in antioxidant defences and lipid peroxidation were measured after exposure. Ozone exposure caused severe damage in red blood cells (rbc) detected as increased lipid peroxidation and oxidized glutathione (GSSG) levels in both plasma and rbc. Oxygen exposure alone increased intracellular lipid peroxidation and GSSG levels 10 min after exposure and was not evident in the plasma at any time. Ozone, but not oxygen exposure, decreased reduced glutathione (GSH) levels in plasma, and the changes were negatively correlated with increased lipid peroxidation in rbc, indicating that extracellular GSH has a dynamic role in the protection of rbc from direct oxidation by ozone. Both ozone and hyperoxic conditions increased superoxide dismutase (SOD) activity in rbc 3 and 6 h after exposure. In contrast, catalase activity was only increased 10 min after oxygen exposure, suggesting other catalase activation mechanisms rather than enzyme induction. The recovery of lipid peroxidation and GSSG levels in rbc after hyperoxia, but not ozone exposure, indicated a capacity to defend against hyperoxia‐produced oxidative damage, but an overwhelming of antioxidant defences by ozone in rainbow trout rbc in vitro.     

Oxidation of glutathione during hydroperoxide metabolism in isolated hepatocytes of rainbow trout (Salmo gairdneri)

Freshly isolated rainbow trout hepatocytes were exposed to tert-butyl hydroperoxide (BuOOH), a substrate for glutathione peroxidase. BuOOH at a concentration approximately equimolar (1 mM) with intracellular reduced glutathione (GSH) caused a reversible increase in intracellular glutathione disulphide (GSSG) but did not compromise cell viability or damage membrane lipids. BuOOH at 10 mM caused a large irreversible increase in intracellular GSSG followed by efflux into the medium. Considerable leakage of lactate dehydrogenase and loss of highly unsaturated fatty acids, particularly docosahexaenoic acid also occurred. Dependence of hydroperoxide removal on flux through the hexose monophosphate pathway was suggested by the increased release of ¹⁴CO₂ from [1-¹⁴C] glucose from hepatocytes incubated with BuOOH.     

多菌灵对蚯蚓体内3种解毒酶活性的影响

为明确多菌灵质量浓度与蚯蚓体内乙酰胆碱酯酶（AChE）、谷胱甘肽S-转移酶（GSTs）和羧酸酯酶（CarE）3种解毒酶活性之间的剂量效应关系,为多菌灵的安全使用及对相关动物的毒害研究提供参考,利用赤峰周边地区蔬菜大棚的蚯蚓,进行多菌灵0．75 g／L、1．00 g／L、1．25 g／L、1．50 g／L、1．75 g／L 和2．00 g／L 6个质量浓度对蚯蚓体内3种解毒酶活性的影响试验。结果表明：与对照组（蒸馏水）相比,多菌灵对蚯蚓体内的乙酰胆碱酯酶整体起抑制作用,在多菌灵浓度为1．75 g／L 时酶活性最低,为105．625μmol／L;对谷胱甘肽 S-转移酶整体起诱导促进作用,在多菌灵浓度为1．00 g／L 时酶活性最高,为17．343μmol／L;对羧酸酯酶活性,在多菌灵浓度小于1．25 g／L 时表现为诱导促进作用,大于1．25 g／L 时表现为抑制作用。     

磷脂氢谷胱甘肽过氧化物酶在精子发育中的作用研究进展

磷脂氢谷胱甘肽过氧化物酶(PHGPx)是广泛存在于哺乳动物细胞内唯一能够物接还原生物膜上脂类过氧化物的一类含硒酶.该酶在保护细胞膜完整、维持细胞的正常功能中发挥着重要作用;同时PHGPx还作为精子的结构物质,在精子的发育和成熟过程中发挥着重要作用;精液中PHGPx活性是评价精液品质的重要参数之一.对PHGPx的基因结构、体内分布以及与精子发育与成熟相关的生物学特性和功能等研究进展进行了总结.     

Reduced tillage in temperate organic farming: implications for crop management and forage production

To promote conservation tillage in organic farming systems, weed control and ley removal within arable-ley rotations need to be optimized. A long-term field trial was thus established in Frick, Switzerland in 2002 on a clayey soil and with a mean precipitation of 1000 mm/year. The tillage experiment distinguished between conventional tillage with mouldboard ploughing (CT, 15 cm depth) and reduced tillage (RT), including a chisel plough (15 cm) and a stubble cleaner (5 cm). Results of a 2-year grass-clover ley (2006/2007) and silage maize (2008) are presented. Due to dry conditions, mean grass-clover yields were 25% higher in RT than in CT, indicating better water retention of RT soils. Clover cover and mineral contents of the fodder mixture were also higher in RT. The ley was successfully removed in autumn 2007 in RT plots, and a winter pea catch crop was sown before maize. In CT, ploughing took place in spring 2008. Maize yields were 34% higher in RT than in CT, despite a two- to three-fold higher but still tolerable weed infestation. Maize in RT plots benefited from an additional 61.5 kg of easily decomposable organic N/ha incorporated into the soil via the pea mulch. Measurement of arbuscular mycorrhizal colonization of maize roots indicated a similar mechanical disturbance of the topsoil through the reduced ley removal system compared with ploughing. It is suggested that RT is applicable in organic farming, even in arable-ley rotations, but long-term effects need further assessment.     

AN IMPROVED TRANSFER SUBSTRUCTURE METHOD

The transfer substructure method is extended to application in substructures with different linking ways,thus the field of application of this method is expanded. Its relaibility and efficacy are proved by some examples.     

The Effects of the Growth Retardant Chlormequat (CCC) on Plant Height and Yield in GA Insensitive Wheats

Seven GA insensitive wheats differing in their final plant height were grown, over three years, in ran-domized field plots and analyzed for their response to CCC. Whereas the shorter lines Were virtually unaffected by the chemical, the ‘tall dwarfs’ responded with reduced straw height and higher yields as a consequence of higher number of grains per ear. Therefore, depending on the genetical background, spraying with growth retardants to achieve optimal plant height may be beneficial to grain yield.