Effect of thyroidectomy and adrenalectomy on changes in liver glutathione and malonaldehyde levels after acute ethanol injection

At low concentrations ethanol is metabolized largely by alcohol dehydrogenase to acetaldehyde, while at higher concentrations a microsomal ethanol oxidising system (MEOS) is involved, namely cytochrome P450 IIE1, which also probably generates free radical species. In hyperthyroidism hepatic glutathione stores are depleted and net superoxide anion production occurs. In contrast, in hypothyroidism hepatic glutathione may be increased and thus renders the liver less sensitive to alcohol generated free radical production. Steroid hormones inhibit lipid peroxidation. Sixty male Wistar rats either underwent thyroidectomy, adrenalectomy, or sham procedures. Twenty control animals were pair fed with thyroidectomized animals, whilst another twenty fed ad libitum. An intraperitoneal injection of alcohol (75 mmol/kg) was given 2.5 h prior to sacrifice to half the animals in each group, the remainder receiving saline. The total hepatic glutathione contents of the pair fed and the ad libitum groups were not different, but were significantly increased by thyroidectomy (p = <0.001). This effect was significantly reduced by alcohol (p < 0.01). The sham procedures and dietary restrictions had no effect. The ethanol alone reduced total hepatic glutathione, but this only reached statistical significance in the thyroidectomized and sham-adrenalectomized groups. Hepatic malonaldehyde (MDA) levels were significantly reduced in the thyroidectomy group but alcohol had no effect on them. We conclude that hypothyroidism increased hepatic glutathione status, presumably by reducing radical production by enzyme systems, which would otherwise consume this important scavenger. Long term exposure to ethanol with induction of MEOS is probably required for it to generate toxic levels of free radical species.     

Decreased catalase activity is the underlying mechanism of oxidant susceptibility in glucose-6-phosphate dehydrogenase-deficient erythrocytes

Historically, it has been theorized that the oxidant sensitivity of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes arises as a direct consequence of an inability to maintain cellular gluthione (GSH) levels. This study alternatively hypothesizes that decreased NADPH concentration leads to impaired to catalase activity which, in turn, underlies the observed oxidant susceptibility. To investigate this hypothesis, normal and G6PD-deficient erythrocytes and hemolysates were challenged with a H₂O₂-generating agent. The results of this study demonstrated that catalase activity was severely impaired upon H₂O₂ challenge in the G6PD-deficient cell whiel only decrease was observed in normal cells. Supplmentation of either normal or G6PD-deficient hemolysates with purified NADPH was found to significantly (P < 0.001) inhibit catalase inactivation upon oxidant challenge while addition of NADP⁺ had no effect. Analysis of these results demonstrated direct correlation between NADPH concentration and catalase activity (r = 0.881) and an inverse correlation between catalase activity and erythrocyte oxidant sensitivity (r = 0.906). In contrast, no correlation was found to exist between glutathione concentration (r = 0.170) and oxidant sensitivity. Analysis of NADPH/NADPt ration in acatalasemic mouse erythrocytes demonstrated that NADPH maintenance alone was not sufficient to explain oxidant resistance, and that catalase activity was required. This study supports the hypothesis that impaired catalase activity underlies the enhanced oxidant sensitivity of G6PD-deficient erythrocytes and elucidates the importance of NADPH in the maintenance of normal catalase activity.     

柑橘大实蝇精巢、精泵和精泵内骨骼的生长发育状况

【目的】柑橘大实蝇是一种严重为害柑橘类果实的经济害虫。研究其精巢、精泵以及精泵内骨骼生长发育状况,有助于提高柑橘大实蝇人工繁殖效率以及其田间防治效果,为柑橘大实蝇的预测预报及防治提供理论基础。【方法】基于光学显微镜测量恒温和室温条件下柑橘大实蝇雄虫的精巢、精泵以及精泵内骨骼的长度和宽度,并建立其长度、宽度和指数的函数模型,比较恒温和室温2种饲养条件下其雄虫器官发育状况差异。【结果】无论是在恒温还是室温条件下饲养,随着柑橘大实蝇雄成虫日龄的增加,其精巢、精泵以及精泵内骨骼的长度、宽度和指数变化趋势均符合幂函数增长模型。在恒温和室温条件下,其雄虫的精巢宽度、精泵的长度和宽度以及指数、精泵内骨骼宽度均无明显差异。在室温条件下饲养的雄虫的精巢长度[(4.00±0.14) mm]及指数(3.40±0.14)、精泵内骨骼长度[(1.65±0.03) mm]及指数(1.84±0.08)均分别显著高于在恒温条件下饲养的雄虫的精巢长度[(3.75±0.13) mm]及指数(3.19±0.14)、精泵内骨骼长度[(1.61±0.03) mm]及指数(1.77±0.08)。【结论】变温(室温)比恒温更有利于柑橘大实蝇雄虫的精巢和精泵内骨骼的发育,并推荐使用精泵长度推断其雄虫日龄的方法。     

Effect of metal ion catalyzed oxidation of rifamycin SV on cell viability and metabolic performance of isolated rat hepatocytes

The effect of rifamycin SV on metabolic performance and cell viability was studied using isolated hepatocytes from fed, starved and glutathione (GSH) depleted rats. The relationships between GSH depletion, nutritional status of the cells, glucose metabolism, lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) production in the presence of rifamycin SV and transition metal ions was investigated. Glucose metabolism was impaired in isolated hepatocytes from both fed and starved animals, the effect is dependent on the rifamycin SV concentration and is enhanced by copper (II). Oxygen consumption by isolated hepatocytes from starved rats was also increased by copper (II) and a partial inhibition due to catalase was observed. Cellular GSH levels which decrease with increasing the rifamycin SV concentration were almost depleted in the presence of copper (II). A correlation between GSH depletion and LDH leakage was observed in fed and starved cells. Catalase induced a slight inhibition of the impairment of gluconeogenesis, GSH depletion and LDH leakage in starved hepatocytes incubated with rifamycin SV, iron (II) and copper (II) salts. Lipid peroxidation measured as MDA production by isolated hepatocytes was also augmented by rifamycin SV and copper (II), especially in hepatic cells isolated from starved and GSH depleted rats. Higher cytotoxicity was observed in isolated hepatocytes from fasted animals when compared with fed or GSH depleted animals. It seems likely that in addition to GSH level, there are other factors which may have an influence on the susceptibility of hepatic cells towards xenobiotic induced cytotoxicity.     

CaATP inhibition of the MgATP-dependent proton pump (H+-ATPase) in bacterial photosynthetic membranes with a mechanism of alternative substrate inhibition

 The membrane-bound F1 sector of the H⁺–ATPase complex (F-type ATPase) in dark-adapted photosynthetic chromatophores is endowed with MgATP- and CaATP-dependent ATPase activities, both sensitive to inhibitors such as oligomycin and venturicidin. Because of contatamination of free Mg^{2 +} and Ca²⁺ ions in chromatophore preparations, kinetic characterization of the two hydrolitic reactions can be performed only in the presence of both substrates, using a model for two alternative substrates. The two activities are characterized by similar maximal rates and affinity constants [V_MgATP and V_CaATP: 13±1 and 10±1 nmol s^–1 ATP hydrolyzed (μmol BChl)^–1; K_MgATP and K_CaATP: 0.22±0.06 and 0.20±0.05 mm]. However, only the MgATP-dependent ATPase is coupled to Δ*_{H +} generation. In this process CaATP acts as an alternative substrate and a competitive inhibitor of the proton pump, with a K_I coincident with K_CaATP for the hydrolytic activity. This finding highlights the central role that the coordination chemistry of the ion-nucleotide complex plays in determining the proton gating mechanism at the catalytic site(s) of the enzyme complex. These results are discussed on the basis of the coordination properties of the ions and of the available information on the protein structure. Received: 5 December 1995 / Accepted: 7 March 1996     

In Situ Microdialysis for Monitoring of Extracellular Glutathione Levels in Normal, Ischemic and Post-Ischemic Skeletal Muscle

Microdialysis probes were inserted into the tibialis anterior muscle and into the femoral vein of anaesthetised Sprague-Dawley rats for monitoring of reduced (GSH) and oxidized (GSSG) extracellular glutathione. The dialysates were analysed using HPLC. The levels of GSH and GSSG were high immediately after implantation in the skeletal muscle and declined to steady state levels after 90 minutes into the same range as that found in the venous dialysate. Total ischemia was induced two hours after implantation of the dialysis probe after steady state levels had been reached. The extracellular levels of GSH increased during total ischemia and had doubled at the end of the ischemic period compared to preischemic values. During the following initial 30 minutes of reperfusion the levels increased further to four-fold the preischemic levels. The levels of GSSG also increased (100%) during the initial 30 minutes of reperfusion. The extracellular GSH levels remained elevated for 1 hour of reperfusion, but the GSSG levels returned to preischemic levels. The results indicate that intermittent hypoxia or anoxia in muscle tissue through hypoperfusion or ischemia decreases intracellular GSH stores by leakage, reducing the intracellular antioxidative capacity and increasing the risk for oxidative reperfusion injury upon final normalization of tissue blood supply.     

A carbon monoxide irreducible form of cytochrome c oxidase and other unusual properties of the “monomeric” shark enzyme

Contrary to previous reports, the functional and spectral properties of “monomeric” shark cytochrome c oxidases are not entirely similar to those of the “dimeric” beef enzyme. Most significantly, unlike the behavior of beef oxidase, the fully oxidized shark enzyme is not reducible by carbon monoxide. Also, preparations of the shark enzyme, isolated at pH 7.8-8.0, lead to more than 60% of the sample always being obtained in a resting form, whereas similarly prepared beef oxidase is very often obtained, both by ourselves and others, exclusively in the pulsed form. Although the electronic absorption, magnetic circular dichroism and electron paramagnetic resonance (EPR) spectra of cytochrome c oxidase obtained from several shark species are similar to those of the beef enzyme, there are some significant differences. In particular, the Soret maximum is at 422 nm in the case of the fully oxidized resting shark oxidases at physiological pH and not 418 nm as commonly found for the beef enzyme. Moreover, the resting shark oxidases do not necessarily exhibit a “g = 12” signal in their EPR spectra. The turnover numbers of recent preparations of the shark enzyme are higher than previously reported and, interestingly, do not differ within experimental uncertainty from those documented for several beef isoenzymes assayed under comparable conditions.     

High Levels of Ascorbic Acid, Not Glutathione, in the CNS of Anoxia-Tolerant Reptiles Contrasted with Levels in Anoxia-Intolerant Species

Abstract: Ascorbic acid and glutathione (GSH) are antioxidants and free radical scavengers that provide the first line of defense against oxidative damage in the CNS. Using HPLC with electrochemical detection, we determined tissue contents of these antioxidants in brain and spinal cord in species with varying abilities to tolerate anoxia, including anoxia-tolerant pond and box turtles, moderately tolerant garter snakes, anoxia-intolerant clawed frogs (Xenopus laevis), and intolerant Long-Evans hooded rats. These data were compared with ascorbate and GSH levels in selected regions of guinea pig CNS, human cortex, and values from the literature. Ascorbate levels in turtles were typically 100% higher than those in rat. Cortex, olfactory bulb, and dorsal ventricular ridge had the highest content in turtle, 5–6 µmol g^?1 of tissue wet weight, which was twice that in rat cortex (2.82 ± 0.05 µmol g^?1) and threefold greater than in guinea pig cortex (1.71 ± 0.03 µmol g^?1). Regionally distinct levels (2–4 µmol g^?1) were found in turtle cerebellum, optic lobe, brainstem, and spinal cord, with a decreasing anterior-to-posterior gradient. Ascorbate was lowest in white matter (optic nerve) in each species. Snake cortex and brainstem had significantly higher ascorbate levels than in rat or guinea pig, although other regions had comparable or lower levels. Frog ascorbate was generally in an intermediate range between that in rat and guinea pig. In contrast to ascorbate, GSH levels in anoxia-tolerant turtles, 2–3 µmol g^?1 of tissue wet weight, were similar to those in mammalian or amphibian brain, with no consistent pattern associated with anoxia tolerance. GSH levels in pond turtle CNS were significantly higher (by 10–20%) than in rat for several regions but were generally lower than in guinea pig or frog. GSH in box turtle and snake CNS were the same or lower than in rat or guinea pig. The distribution GSH in the CNS also had a decreasing anterior-to-posterior gradient but with less variability than ascorbate; levels were similar in optic nerve, brainstem, and spinal cord. The paradoxically high levels of ascorbate in turtle brain, which has a lower rate of oxidative metabolism than mammalian, suggest that ascorbate is an essential cerebral antioxidant. High levels may have evolved to protect cells from oxidative damage when aerobic metabolism resumes after a hypoxic dive.     

Involvement of plasma membrane potential in the tolerance mechanism of plant roots to aluminium toxicity

H⁺-ATPase activity of a plasma membrane-enriched fraction decreased after the treatment of barley (Hordeum vulgare) seedlings with Al for 5 days. A remarkably high level of Al was found in the membrane fraction of Al-treated roots. A long-term effect of Al was identified as the repression of the H⁺-ATPase of plasma membranes isolated from the roots of barley and wheat (Triticum aestivum) cultivars, Atlas 66 (Al-tolerant) and Scout 66 (Al-sensitive). To monitor short-term effects of Al, the electrical membrane potentials across plasma membranes of both wheat cultivars were compared indirectly by measuring the efflux of K⁺ for 40 min under various conditions. The rate of efflux of K⁺ in Scout was twice that in Atlas at low pH values such as 4.2. Vanadate, an inhibitor of the H⁺-ATPase of the plasma membrane, increased the efflux of K⁺. Al repressed this efflux at low pH, probably through an effect on K⁺ channels, and repression was more pronounced in Scout. Al strongly repressed the efflux of K⁺ irrespective of the presence of vanadate. Ca²⁺ also had a repressive effect on the efflux of K⁺ at low pH. The effect of Ca²⁺, greater in Scout, might be related to the regulation of the net influx of H⁺, since the effect was negated by vanadate. The results suggest that extracellular low pH may cause an increase in the influx of H⁺, which in turn is counteracted by the efflux of K⁺ and H⁺. These results suggest that the ability to maintain the integrity of the plasma membrane and the ability to recover the electrical balance at the plasma membrane through a net influx of H⁺ and the efflux of K⁺ seem to participate in the mechanism of tolerance to Al stress under acidic conditions.     

Glutathione S-transferase and S-crystallins of cephalopods: Evolution from active enzyme to lens-refractive proteins

Our previous studies have shown that the S-crystallins of cephalopod (Ommastrephes sloani pacificus) eye lenses comprise a family of at least ten members which are evolutionarily related to glutathione S-transferase (GST, EC 2.5.1.18). Here we show by cDNA cloning that there are at least 24 different S-crystallins that are 46–99% identical to each other by amino acid sequence in the squid Loligo opalescens. In each species, all but one S-crystallin (SL11 in O. pacificus and Lops4 in L. opalescens) examined has an inserted central peptide of variable length and sequence. cDNA expression studies conducted in Escherichia coli showed that squid GST (which is expressed little in the lens) has very high enzymatic activity using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate; by contrast, SL20-1 of O. pacificus and Lops 12 of L. opalescens (which are encoded by abundant lens mRNAs) have no GST activity. Interestingly, SL11 and Lops4 have some enzymatic activity with the CDNB substrate. Site-specific mutations at Y7 or W38, both residues essential for activity of vertebrate GSTs, or insertion of the central peptide present in the inactive SL20-1, reduced the specific activity of squid GST by 30- to 100-fold. These data indicate that the S-crystallins consist of a family of enzymatically inactive proteins (when using CDNB as a substrate) which is considerably larger than previously believed and that GST activity was lost by gradual drift in sequence as well as by insertion of an extra peptide by exon shuffling. The results are also consistent with the idea that SL11 and Lops4 are orthologous crystallins representing the first descendants of the ancestral GST gene in the pathway which gave rise to the extensive S-crystallin family of lens proteins. Correspondence to: S.I. Tomarev