Methaemoglobin formation in the koala and the possum

Measurements were made of glutathione (GSH) levels, catalase activity and the oxidant sensitivity of the erythrocytes from the koala (Phascolarctos cinereus) and the common brushtail possum (Trichosurus vulpecula). The oxidant sensitivity was tested by treating the haemolysates with either 0.55 him H₂O₂ or 1.4mm NaNO₂. The erythrocytes of the koala had greater levels of GSH and catalase and yet were found to be more susceptible to oxidation induced by both these oxidants.     

The effect of eucalyptus oils on the erythrocytes of koalas

The oxidant effects of two fractions of eucalyptus oil; monoterpenes (C₁₀) and the larger sesquiterpenes (C₁₅), were investigated in the erythrocytes of the koala (Phascolarctos cinereus). The effects studied included the degree of haemolysis and changes in the intracellular levels of glutathione (GSH) and adenosine triphosphate (ATP) and in the membrane phospholipids. The results indicate that koala erythrocytes are susceptible to eucalyptus oil-induced oxidative damage. The two types of eucalyptus oils have different effects on the erythrocytes; monoterpenes appear to induce haemolysis through oxidative damage to the intracellular constituents, whereas sesquiterpenes may attack the red cell membrane.     

Erythrocyte metabolism in the Koala, the common brushtail possum and the whiptail wallaby

Erythrocyte metabolism was studied in the koala (Phascolarctos cinereus), the common brushtail possum (Trichosurus vulpecula) and the whiptail wallaby (Macropus parryi). Aspects of metabolism studied were the levels of ATP, 2,3-diphosphoglycerate (2,3-DPG) and reduced glutathione (GSH), activities of erythrocyte enzymes, glycolytic rate, the rate of glutathione S-conjugate transport (GCT), susceptibility of erythrocytes to an oxidising agent and the effect of eucalyptus oil on the red cells. Some of the major findings were that compared to common brushtail possum and the whiptail wallaby, the koala red cells (a) produced significantly greater amounts of lactate; (b) had significantly lower activity of glutathione S-transferase, (c) had significantly lower rate of GCT, (d) were more susceptible to oxidant stress induced by acetylphenylhydrazine (APH) and (e) exhibited a greater degree of haemolysis when incubated with eucalyptus oil.     

Role of Catalase in H2O2-induced oxidant stress in marsupial erythrocytes

We have examined the catalase activity and H₂O₂-induced oxidant stress on methaemoglobin formation and haemolysis in eight species of marsupials: the black striped wallaby (Macropus dorsalis), bridled nailtail wallaby (Onychogalea fraenata), proserpine rock wallaby (Petrogale persephone), red legged pademelon (Thylogale stigmatica), spectacled hare wallaby (Lagorchestes conspicillatus), whiptail wallaby (Macropus parryi), common brushtail possum (Trichosurus vulpecula), and the koala (Phascolarctos cinereus). The results indicate a significant relationship between the activity of catalase and methaemoglobin formation by H₂O₂.     

Erythrocyte metabolism in the gibbon, the elephant and the lion

Erythrocyte metabolism was studied in the white-handed gibbon (Hylobatus leucogenys), the Asian elephant (Elephas maximus) and the lion (Panthera leo). Aspects of metabolism studied were activities of the enzymes associated with glycolysis and the antioxidant defence systems, glycolytic rates using four different substrates and the susceptibility of haemoglobin to three different oxidising agents, acetylphenylhydrazine (APH), hydrogen peroxide (H₂0₂) and sodium nitrite (NaNO₂). The main findings were as follows. First, compared with humans, the erythrocytes from the gibbon were more resistant to H₂0₂-induced haemolysis and had very high activity of NADH-methaemoglobin reductase. Oxidation of haemoglobin by NaNO₂ was largely blocked by 0.1 mM GSH. Second, the erythrocytes from the lion had a very high activity of pyruvate kinase (PK). The rate of methaemoglobin formation by APH was also very high in these erythrocytes. Third, the erythrocytes of the elephant had very low levels of hexokinase and PK and were more resistant to oxidant stress than those of the lion and humans.     

Effects of ascorbic acid on high GSH and normal dog erythrocytes (I)

Dog erythrocytes with high GSH and normal GSH concentrations were compared under effects of ascorbic acid. In the presence of glucose, these two types of erythrocytes showed a similar increase in intracellular ascorbic acid without change in GSH levels. Addition of iron (Fe³⁺) to the incubation medium enhanced ascorbic acid accumulation. In the absence of glucose, GSH fell markedly in both types of erythrocyte and less ascorbic acid accumulated in normal GSH cells than high GSH cells. With iron, normal cells showed GSH depletion and marked methaemoglobin formation. Lipid peroxidation with ascorbic acid and iron increased at a similar rate in both types of erythrocyte and was inhibited by catalase. Ferricyanide reduction in both erythrocytes loaded with ascorbic acid were similar and increased with glucose or catalase. There was a high correlation between intracellular ascorbic acid content and ferricyanide reduction. These results suggest that high GSH and normal GSH dog erythrocytes have a similar capacity for accumulating ascorbic acid and for reducing ferricyanide. However, normal GSH erythrocytes are more susceptible to the oxidant effect of ascorbic acid than high GSH cells; this is probably due to a smaller GSH reserve, which is exhausted more rapidly under oxidative stress.     

Role of Catalase in H2O2-induced oxidant stress in marsupial erythrocytes

We have examined the catalase activity and H₂O₂-induced oxidant stress on methaemoglobin formation and haemolysis in eight species of marsupials: the black striped wallaby (Macropus dorsalis), bridled nailtail wallaby (Onychogalea fraenata), proserpine rock wallaby (Petrogale persephone), red legged pademelon (Thylogale stigmatica), spectacled hare wallaby (Lagorchestes conspicillatus), whiptail wallaby (Macropus parryi), common brushtail possum (Trichosurus vulpecula), and the koala (Phascolarctos cinereus). The results indicate a significant relationship between the activity of catalase and methaemoglobin formation by H₂O₂.     

Redox imbalance and IL‐17 responses in memory CD4+ T cells from patients with psoriasis

All stages of the inflammatory process involved in T cell‐mediated chronic skin disorders like psoriasis are affected by redox imbalance. On the other hand, Th17 cells have a critical role in the pathogenesis of psoriasis. In this study, we evaluated redox status in memory CD4 + T cells and plasma of patients with psoriasis and its correlation with IL‐17 response. To this end, memory T cells were isolated from 10 patients with psoriasis and 10 controls. Intracellular Glutathione (GSH), reactive oxygen species (ROS) and superoxide as well as IL‐17 were measured using flow cytometry. Plasma total anti‐oxidant capacity (TAC) was quantified by ferric reducing ability of plasma (FRAP) assay. The expression of catalase (CAT), superoxide dismutase 1(SOD1), superoxide dismutase 2 (SOD2), nuclear factor, erythroid 2 like 2 (NFE2L2) and cytochrome b‐245 beta chain (CYBB) genes were analysed using real‐time PCR. Our results showed an increased intracellular ROS production in memory CD4 + T cells of patients compared to controls, (P = 0.04). Furthermore, a significant decrease in expression of catalase gene was found in patients, (P = 0.02). However, no significant differences were observed for intracellular GSH, IL‐17 and TAC levels between patients and controls. Also, no correlation was seen between the intracellular IL‐17 level and intracellular ROS, GSH and catalase gene expression levels. Collectively, we found an increased ROS production in stimulated memory T cells of patients that could be due to reduced expression of catalase gene. However, it seems that these redox abnormalities have no relationship with IL‐17 response in memory T cells.     

N-acetyl cysteine, L-cysteine, and beta-mercaptoethanol augment selenium-glutathione peroxidase activity in glucose-6-phosphate dehydrogenase-deficient human erythrocytes

Abstract.   In glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes, failure to maintain normal levels of reduced glutathione (GSH) due to decreased NADPH regeneration in the hexose monophosphate pathway results in acute hemolytic anemia following exposure to oxidative insults, such as ingestion of Vicia fava beans or use of certain drugs. GSH is a source of protection against oxidative attack, used by the selenium-dependent glutathione peroxidase (Se-GSH-Px)/reductase (GR) system to detoxify hydrogen peroxide and organic peroxides, provided that sufficient GSH is made available. In this study, Se-GSH-Px activity was analyzed in G6PD-deficient patients in the presence of reducing agents such as N-Acetyl cysteine, L-cysteine, and β-mercaptoethanol. Se-GSH-Px activity was decreased in G6PD-deficient red blood cells (RBCs). N-Acetyl cysteine, L-cysteine, and β-mercaptoethanol increased Se-GSH-Px activity in G6PD-deficient human erythrocytes, indicating that other reducing agents can be utilized to complement Se-GSH-Px activity in G6PD deficiency. Based on the increased susceptibility of G6PD-deficient patients to oxidative stress, the reported increase in Se- GSH-Px activity can facilitate the detoxification of reactive oxygen species.     

Effects of ascorbic acid on high-GSH and normal-GSH dog erythrocytes (II)

Mechanisms of ascorbic acid uptake and oxidative effects were studied in dog erythrocytes with high GSH and normal GSH concentrations. It was noted that ascorbic acid enters dog erythrocytes very slowly in the reduced form compared with the oxidised form (DHA) and by a glucose-independent mechanism. In the absence of glucose, accumulation of ascorbic acid, GSH depletion and increased methaemoglobin formation occurred successively. Compared with erythrocytes with normal GSH levels, those with high GSH levels showed a greater accumulation and prolonged presence of ascorbic acid, considerable delay in GSH depletion and less methaemoglobin formation. This is due to there being a larger reserve of GSH in high-GSH cells, which is exhausted more slowly. GSH-depleted erythrocytes were no longer able to accumulate ascorbic acid. Hydrogen peroxide (H₂O₂) was found to be the sole reactive oxygen species responsible for oxidative effects of ascorbic acid, while superoxide (O ₂ ^– ), hydroxyl radical (OH) and metal ions had relatively minor roles. Overall, these results clearly imply that GSH is essential in accumulating ascorbic acid and resisting oxidative effects in dog erythrocytes and that cells with high GSH levels containing larger reserve of GSH have an advantage with regard to the accumulation of ascorbic acid and its function.     

Exogenous reactive oxygen and nitric oxide alter intracellular oxidant status of skeletal muscle fibres

To test whether exogenous oxidants alter intracellular oxidant levels in skeletal muscle fibres, we exposed rat diaphragm to donors of nitric oxide (NO_x), reactive oxygen species (ROS) or hyperoxia, and monitored intracellular oxidant levels using a fluorescent probe. Fibre bundles were dissected from the diaphragm and loaded with 2’,7’-dichlorodihydrofluorescein (DCFH); emissions were monitored using a fluorescence microscope. DCFH-loaded muscles were exposed to either a NO_x donor (1 m M S-nitroso-N-acetyl penicillamine, SNAP; 1 m M sodium nitroprusside, SNP; 400 μM 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazen, NOC-7), an ROS donor (100 μM hydrogen peroxide, H₂O₂; 100 μM tert-butyl hydroperoxide; 1 m M hypoxanthine plus 0.01 U mL^–1 xanthine oxidase, HXXO) or a range of PO ₂s (25, 60 or 95% O₂ oxygenating Krebs–Ringer solution) for 40 min; time-matched control bundles remained in Krebs–Ringer solution. Control muscles oxidized DCFH at a rate of 0.32 ± 0.1 greyscale units min^–1. SNAP (766%), SNP (1244%), NOC-7 (851%), H₂O₂ (543%), and HXXO (541%) increased DCFH oxidation from control levels. The increase in emissions caused by NOC-7 and SNP were blunted by the NO_x scavenger haemoglobin (1 μM ). DCFH oxidation by HXXO was unaffected by 1000 U mL^–1 superoxide dismutase but was significantly decreased by 1000 U mL^–1 catalase and 1 m M salicylate. PO ₂ had no effect on intracellular oxidant levels. Therefore, extracellular NO_x and ROS can alter intracellular oxidant status in skeletal muscle fibres. These observations suggest that intrafibre oxidant levels could be the result of both intracellular and extracellular oxidant production.     

Methaemoglobin Formation in Euros and Bettong

Oxidant sensitivity was investigated in erythrocytes from Barrow Island euro (Macropus robustus isabellinus), mainland euro (Macropus robustus erubescens) and brushtailed-bettong (Bettongia pericillata). Oxidant sensitivity was tested by measuring methaemoglobin formation in haemolysates treated with H₂O₂ or NaNO₂. Barrow Island euro produced the highest methaemoglobin formation with H₂O₂. There was a significant negative correlation between catalase activity and H₂O₂-induced methaemoglobin formation. Our results provide further evidence supporting previous findings on eight species of marsupials.     

N-Acetyl cysteine, L-cysteine, and ?-mercaptoethanol augment seleniumglutathione peroxidase activity in glucose-6-phosphate dehydrogenasedeficient human erythrocytes

Abstract. In glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes, failure to maintain normal levels of reduced glutathione (GSH) due to decreased NADPH regeneration in the hexose monophosphate pathway results in acute hemolytic anemia following exposure to oxidative insults, such as ingestion of Vicia fava beans or use of certain drugs. GSH is a source of protection against oxidative attack, used by the selenium-dependent glutathione peroxidase (Se-GSH-Px)/reductase (GR) system to detoxify hydrogen peroxide and organic peroxides, provided that sufficient GSH is made available. In this study, Se-GSH-Px activity was analyzed in G6PD-deficient patients in the presence of reducing agents such as N-Acetyl cysteine, L-cysteine, and -mercaptoethanol. Se-GSH-Px activity was decreased in G6PD-deficient red blood cells (RBCs). N-Acetyl cysteine, L-cysteine, and -mercaptoethanol increased Se-GSH-Px activity in G6PD-deficient human erythrocytes, indicating that other reducing agents can be utilized to complement Se-GSH-Px activity in G6PD deficiency. Based on the increased susceptibility of G6PD-deficient patients to oxidative stress, the reported increase in Se- GSH-Px activity can facilitate the detoxification of reactive oxygen species.     

吩嗪甲酸硫酯对葡萄糖-6-磷酸脱氢酶缺乏的人红细胞某些生化指标的影响

为探讨葡萄糖－6－磷酸脱氢酶（C6PD）缺乏的人红细胞在在作用下的变化情况。通过测定在作用前后G6PD缺乏人红细胞变形能力，高铁血红蛋白生成，过氧化氢酶活力，还原型谷胱甘肽（GSH），还原型三磷酸吡啶核苷（NADPH）的浓度，并与正常红细胞比较。结果显示G6PD缺乏红细胞在氧自由基作用下变形能力，过氧化氢酶活力，GSH及NADPH水平与正常红细胞相比显著下降（P＜0．01），而高铁血红蛋白生成显著增加（P＜0．01），从而得出G6PD缺乏红细胞氧化易感性增强导致细胞损伤，变形能力下降是其易发溶血的主要原因的结论。     

ATP levels and glutathione regeneration in canine erythrocytes

The effect of ATP levels on GSH regeneration was examined in canine erythrocytes. The main findings were: (1) The GSH regeneration was dependent on glucose and ATP; (2) cytochalasin B and polymyxin B, both glucose transport inhibitors, reduced ATP synthesis and GSH regeneration; (3) inosine, a substrate of the salvage pathway, was not effective for ATP synthesis and GSH regeneration. These results indicate that glucose transport and its metabolism play an important role in oxidant defence systems in general and GSH regeneration in particular in canine erythrocytes.     

Comparative erythrocyte metabolism in three species of marsupials from Western Australia

A comparative study was carried out to investigate various aspects of erythrocyte metabolism in three species of marsupials located in the western part of Australia. The burrowing bettong (Bettongia lesueur) and the Barrow Island euro (Macropus robustus isabellinus) were captured on Barrow Island whereas the mainland euro (Macropus robustus erubescens) was captured from the Pilbara region of northwest Western Australia. Aspects of erythrocyte metabolism studied included: levels of glycolytic intermediates, enzyme activities, sensitivity of erythrocytes to oxidants and methaemoglobin reducing capacity. Some important findings were: (1) no relationship was observed between haemoglobin and diphosphoglycerate levels; (2) both species from Barrow Island had lower levels of catalase than the mainland species; and (3) methaemoglobin reductive capacity was greater in the erythrocytes of the burrowing bettong compared to the two species of euro.     

Comparative erythrocyte metabolism in three species of marsupials from Western Australia

A comparative study was carried out to investigate various aspects of erythrocyte metabolism in three species of marsupials located in the western part of Australia. The burrowing bettong (Bettongia lesueur) and the Barrow Island euro (Macropus robustus isabellinus) were captured on Barrow Island whereas the mainland euro (Macropus robustus erubescens) was captured from the Pilbara region of northwest Western Australia. Aspects of erythrocyte metabolism studied included: levels of glycolytic intermediates, enzyme activities, sensitivity of erythrocytes to oxidants and methaemoglobin reducing capacity. Some important findings were: (1) no relationship was observed between haemoglobin and diphosphoglycerate levels; (2) both species from Barrow Island had lower levels of catalase than the mainland species; and (3) methaemoglobin reductive capacity was greater in the erythrocytes of the burrowing bettong compared to the two species of euro.     

Variables of the rubella hemagglutination test employing freeze-dried erythrocytes

Summary The variables which affect the interaction between freeze-dried one-day-old chick erythrocytes and rubella hemagglutinin prepared from rubella-infected porcine kidney cells were defined and evaluated. The sensitivity of the hemagglutination (HA) reaction is much greater at pH 6.0 to 6.2 than at pH 7.0 to 7.5. HEPES (N-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid) diluent with added Ca²⁺ or Mg²⁺ ion gave four- to eightfold higher HA titers than one without divalent cations.The development of agglutinated and non-agglutinated erythrocyte patterns depended much upon the concentrations of gelatin and albumin in the HEPES diluent. Gelatin especially was essential to obtain stable and clearly distinguishable patterns.Optimal conditions for the agglutination of freeze-dried erythrocytes by rubella hemagglutinin were provided when a HEPES-buffered saline at pH 6.2, containing 10^–3 m CaCl₂, 0.2 per cent bovine serum albumin, and 0.0025 per cent gelatin was employed throughout as a diluent for serum, hemagglutinin, and freeze-dried erythrocyte suspension. This diluent gave maximally sensitive and reproducible results in rubella HA and hemagglutination-inhibition (HI) tests employing freeze-dried erythrocytes.With 1 Figure     

N-acetyl-l-cysteine reduces the parasitism of BALB/c mice infected with Leishmania amazonensis

Leishmania amazonensis infection leads to progressive diseases in a majority of inbred strains of mice. Glutathione (GSH) participates in a large number of cellular phenomena and seems to be essential for several immune functions, including host defense during leishmaniasis. In this study, we evaluated the effects of N-acetyl-l-cysteine (NAC), as GSH supplement, on the course of L. amazonensis infection in susceptible BALB/c mice. The treatment with NAC (200 mg/kg daily) was effective in raising GSH levels in both lymph node and spleen cells. Although this treatment did not change the footpad swelling development in L. amazonensis-infected mice, it caused a significant decrease in the number of parasites recovered from the footpad lesion and draining popliteal lymph node. Our data suggest that intracellular Leishmania killing in vivo was improved by the augment of GSH levels through NAC administration.     

Inhibition of glutathione synthesis of Ascaris suum by buthionine sulfoximine

Abstrac t We investigated the effect of dl-buthionine-S,R-sulfoximine (BSO), a selective glutathione (GSH)-depleting agent, on the GSH synthesis of Ascaris suum. The GSH concentrations of the reproductive and muscle tissues of A. suum were determined to be 8.5±0.3 and 14.3±1.3 (n=3) nmol/mg protein, respectively. After treatment of the parasites with 10 μM BSO for 24 h, the GSH content of the reproductive tissue of A. suum was totally depleted as compared with that of untreated controls. However, the GSH levels of the muscle tissue were reduced to only 50% after treatment of the worms for 24 h with 10 μM BSO. Exogenous GSH had no significant effect on the GSH level of the parasites when the worms were incubated for 4 h in RPMI 1640 medium supplemented with 1 mM GSH. In the presence of exogenous GSH, BSO was less effective in depleting the GSH levels of the parasites, which may indicate that the parasites can replenish their GSH levels. GSH depletion, which has been discussed as being therapeutically effective when normal and tumor cells or parasites have markedly different requirements for GSH, may have applications in the development of drugs against nematode infections. Received: 7 September 1995 / Accepted: 15 November 1995