Postischemic injury in isolated rat hearts is not aggravated by prior depletion of myocardial glutathione

The aim of this study was to test the hypothesis that a decreased myocardial concentration of reduced glutathione (GSH) during ischemia renders the myocardium more susceptible to injury by reactive oxygen species generated during early reperfusion. To this end, rats were pretreated with L-buthionine-S,R-sulfoximine (2 mmol/kg), which depleted myocardial GSH by 55%. Isolated buffer-perfused hearts were subjected to 30 min of either hypothermic or normothermic no-flow ischemia followed by reperfusion. Prior depletion of myocardial GSH did not lead to oxidative stress during reperfusion, as myocardial concentration of glutathione disulfide (GSSG) was not increased after 5 and 30 min of reperfusion. In addition, prior depletion of GSH did not exacerbate myocardial enzyme release, nor did it impair the recoveries of tissue ATP, coronary flow rate and left ventricular developed pressure during reperfusion after either hypothermic or normothermic ischemia. Even administration of the prooxidant cumene hydroperoxide (20 M) to postischemic GSH-depleted hearts during the first 10 min of reperfusion did not aggravate postischemic injury, although this prooxidant load induced oxidative stress, as indicated by an increased myocardial concentration of GSSG. These results do not support the hypothesis that a reduced myocardial concentration of GSH during ischemia increases the susceptibility to injury mediated by reactive oxygen species generated during reperfusion. Apparently, myocardial tissue possesses a large excess of GSH compared to the quantity of reactive oxygen species generated upon reperfusion. (Mol Cell Biochem 156: 79-85, 1996)     

Alterations of the glutathione redox cycle status in fumonisin B1-treated pig kidney cells

Fumonisin B₁ (FB₁) causes equine leukoencephalomalacia, porcine pulmonary edema, and liver tumors and chronic nephritis in rats. To investigate mechanisms by which FB₁ induces toxicity, effects of FB₁ on cellular glutathione (GSH) redox status and GSH depletion on FB₁ toxicity in pig kidney (LLC-PK₁) cells were studied. Treatment of LLC-PK₁ cells with 50 μM FB₁ for 24 hours significantly decreased cellular GSH contents from 56 ± 3.2 to 42.7 ± 4.4 nmol/mg protein (p < 0.05) and increased the activities of glutathione reductase (GR) from 25.7 ± 2.4 to 35.7 ± 5.0 μmol NADPH/mg protein (p < 0.05). The activities of glutathione peroxidase (GSHpx), catalase, and Cu,Zn-superoxide dismutase (SOD) were not changed by this treatment. Treatment of LLC-PK₁ cells for 12 hours with 0.1. mM buthionine sulfoximine (BSO), a selective inhibitor of the enzyme γ-glutamylcysteine synthetase that catalyzes the rate-limiting reaction in de novo GSH synthesis, decreased cellular GSH levels to about 20% of that found in the control cells. The cells pretreated with 0.1 mM BSO for 12 hours were significantly sensitized to the FB₁ cytotoxicity as determined by a long-term survival assay (p < 0.05). The results demonstrate that FB₁ changes GSH redox cycle status in LLC-PK₁ cells, and GSH may play a role in cytoprotection against FB₁ toxicity. © 1997 John Wiley & Sons, Inc.     

Enhanced cadmium cytotoxicity in A549 cells with reduced glutathione levels is due to neither enhanced cadmium accumulation nor reduced metallothionein synthesis

Glutathione (GSH) depletion sensitizes human lung carcinoma (A549-727) cells to the cytotoxic effects of Cd⁺⁺. The effects of GSH depletion on Cd⁺⁺ accumulation and Cd⁺-induced metallothionein (MT) content were investigated to determine the possible role of these Cd⁺⁺ responses in the sensitization process. Cellular GSH was depleted to 20% to 25% of control levels with buthionine sulfoximine (BSO), or diethyl maleate (DEM), respectively. Neither treatment significantly affected Cd⁺⁺-induced accumulation of exogenous³⁵s-cysteine into intracellular MT in a dose-dependent fashion. The results indicate that neither enhanced Cd⁺⁺ accumulation nor reduced MT synthesis plays a primary role in affecting enhanced Cd⁺⁺ cytotoxicity in A549 cells with reduced GSH levels. Although BSO inhibition of GSH synthesis enhanced MT synthesis, it sensitized the cells to Cd⁺⁺, which suggests an additive effect of GSH and MT in cadmium cytoprotection. This observation also raises the possibility that intracellular cysteine levels limit Cd⁺⁺-induced MT accumulation rates.Abbreviations GSH glutathione - MT metallothionein - BSO DL-buthionine-[S,R]-sulfoximine - DMSO dimethyl sulfoximine - DEM diethyl maleate - NP-40 nonidet-P40 - PBS phosphate buffered saline - HBSS Hank's balanced salt solution - DTT dithiothreitol 3. This work was presented in part at the 72nd Annual Meeting of the Federation of American Societies for Experimental Biology, Las Vegas, Nevada, May 1–5, 1988.     

Effect of acute exercise on glutathione deficient heart

The role of glutathione (GSH) in myocardial antioxidant defense was investigated in Swiss-Webster mice either performing swim exercise to exhaustion or rested in both the GSH adequate (GSH-A) and GSH deficient (GSH-D) states. GSH deficiency was accomplished by injecting mice with L-buthionine [S,R]sulfoximine (BSO; 2 nmol/kg body wt, i.p.) and providing BSO (20 mM) in drinking water for 12 days. GSH and glutathione disulfide (GSSG) contents in the GSH-D hearts were decreased to 10 and 8%, respectively, of those in the GSH-A mice. This decrease was associated with a significant decline of the total glutathione level in the liver, skeletal muscle and plasma. Myocardial GSH peroxidase and GSH sulfur-transferase activities decreased significantly following GSH deficiency, whereas superoxide dismutase activity was significantly elevated. GSH deficiency did not affect exercise endurance performance. However, exhaustive exercise decreased GSH content in the myocardium of the GSH-A and GSH-D mice by 22 and 44% (p < 0.05), respectively. The GSH:GSSG ratio was not altered significantly following exercise because of a concomitant decrease in GSSG (p < 0.05). -Glutamyltranspeptidase activity was significantly increased after exercise, especially in the GSH-D hearts (72%; p < 0.05). GSH content after exercise correlated negatively with exercise time in both GSH-A and GSH-D mice (p < 0.05). These data indicate that GSH is actively used in the myocardium during prolonged exercise at moderate intensity and that GSH deficiency is tolerated by the heart, possibly compensated for by an increased GSH uptake from the plasma.     

Hematotoxicity and blood glutathione levels after cisplatin treatment of tumor-bearing mice

The involvement of glutathione, a major cellular antioxidant, in cisplatin-mediated development of various hematological changes in mice bearing ascites Dalton lymphoma tumor was investigated. With tumor growth, glutathione levels decreased in blood but increased in tumor cells. Cisplatin treatment of tumor-bearing mice caused a decrease in glutathione levels in blood, ascites supernatant, and tumor cells. Blood hemoglobin, erythrocytes, packed cell volume and leukocytes (eosinophils, basophils, and lymphocytes) were also decreased along with the development of various morphological abnormalities in erythrocytes (microcytes, macrocytes, echinocytes, acanthocytes, etc.) after cisplatin treatment. All these hematotoxic features were noted to be increased more when buthionine sulfoximine (a specific glutathione-depleting agent) was also given prior to cisplatin treatment. However, combination treatment of cysteine (precursor for glutathione synthesis) plus cisplatin resulted in an improvement in the glutathione levels and decrease in hematological toxicities. It is noted that the glutathione levels in blood and abnormalities in erythrocytes and other hematological parameters are inversely related in cisplatin-mediated cancer chemotherapy. It is suggested that blood glutathione may play an important role in the development of cisplatin-mediated hematological toxicity in the host. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of cyclosporin A treatment on renal calcium oxalate binding in experimental hyperoxaluria

This study was aimed to investigate the effect of Cyclosporin A administration on renal calcium oxalate binding under hyperoxaluric condition.Cyclosporin A administration or ammonium oxalate treatment increased calcium oxalate binding, which was further increased in kidney treated with cyclosporin A and ammonium oxalate together. The increase of calcium oxalate binding was associated with lipid peroxidation as well as with a concomitant decrease in total thiol in both rat and human kdiney homogenate.Cyclosporin A administration to hyperoxaluric rats resulted with increased calcium oxalate binding protein. However there was no change with specific activity of the protein.In conclusion, Cyclosporin A administration either to normal or hyperoxaluric rats is resulted with increased concentration of calcium oxalate binding protein as well as enhanced activity due to membrane lipid peroxidation.     

Methionine sulfoximine supplementation enhances productivity in GS–CHOK1SV cell lines through glutathione biosynthesis

In Lonza Biologics' GS Gene Expression System?, recombinant protein‐producing GS–CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine‐free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER‐resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS–CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS–CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb‐producing GS–CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight‐fold and the concentration in harvest medium by two‐fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process‐driven method for increasing mAb productivity from GS–CHOK1SV cell lines. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:17–25, 2017     

Effect of cellular glutathione depletion on cadmium-induced cytotoxicity in human lung carcinoma cells

The effect of glutathione depletion on cellular toxicity of cadmium was investigated in a subpopulation (T27) of human lung carcinoma A549 cells with coordinately high glutathione levels and Cd⁺⁺-resistance. Cellular glutathione levels were depleted by exposing the cells to diethyl maleate or buthionine sulfoximine. Depletion was dose-dependent. Exposure of the cells to 0.5 mM diethyl maleate for 4 hours or to 10 mM buthionine sulfoximine for 8 hours eliminated the threshold for Cd⁺⁺ cytotoxic effect and deccreased the LD_50S. Cells that were pretreated with 0.5 mM diethyl maleate or 10 mM buthionine sulfoximine and then exposed to these same concentrations of diethyl maleate or buthionine sulfoximine during the subsequent assay for colony forming efficiency produced no colonies, reflecting an enhanced sensitivity to these agents at low cell density. Diethyl maleate was found to be more cytotoxic than buthionine sulfoximine. Synergistic cytotoxic effects were observed in the response of diethyl maleate pretreated cells exposed to Cd⁺⁺. Thus the results demostrated that depletion of most cellular glutathione in A549-T27 cells prior to Cd⁺⁺ exposure sensitizes them to the agent's cytotoxic effects. Glutathione thus may be involved in modulating the early cellular Cd⁺⁺ cytotoxic response. Comparison of reduced glutathione levels and of Cd⁺⁺ cytotoxic responses in buthionine sulfoximine-treated A549-T27 cells with those levels in other, untreated normal and tumor-derived cells suggests that the higher level of glutathione in A549-T27 is not the sole determinant of its higher level of Cd⁺⁺ resistance.Abbreviations BSO DL-buthionine-(R,S)-sulfoximine - DEM diethyl maleate - DMSO dimethyl sulfoxide - GSH reduced glutathione - MT metallothionein     

Differential effect of schisandrin B and dimethyl diphenyl bicarboxylate (DDB) on hepatic mitochondrial glutathione redox status in carbon tetrachloride intoxicated mice

The effects of schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis, and dimethyl diphenyl bicarboxylate (DDB), a synthetic intermediate of schisandrin C (also a dibenzocyclooctadiene derivative), on hepatic mitochondrial glutathione redox status in control and carbon tetrachloride (CCl₄)-intoxicated mice were examined. Treating mice with Sch B or DDB at a daily oral dose of 1 mmol/kg for 3 d did not produce any significant alterations in plasma alanine aminotransferase (ALT) and sorbital dehydrogenase (SDH) activities. CCl₄ treatment caused drastic increases in both plasma ALT and SDH activities in mice. Pretreating mice with Sch B or DDB at the same dosage regimen significantly suppressed the CCl₄-induced increase in plasma ALT activity, with the inhibitory effect of Sch B being much more potent. Sch B, but not DDB, pretreatment could also decrease the plasma SDH activity in CCl₄-intoxicated mice. The lowering of plasma SDH activity, indicative of hepatoprotection against CCl₄ toxicity, by Sch B pretreatment was associated with an enhancement in hepatic mitochondrial glutathione redox status as well as an increase in mitochondrial glutathione reductase (mtGRD) activity in both non-CCl₄ and CCl₄-treated mice. DDB pretreatment, though enhancing both hepatic mitochondrial glutathione redox status and mtGRD activity in control animals, did not produce any beneficial effect in CCl₄-treated mice. The difference in hepatoprotective action against CCl₄ toxicity between Sch B and DDB may therefore be related to their ability to maintain hepatic mitochondrial glutathione redox status under oxidative stress condition.     

Changes in organ nonprotein sulfhydryl and glutathione concentrations during acute and chronic administration of inorganic lead to chicks

The effects of dietary and injected lead (Pb) on organ nonprotein sulfhydryl (NPSH) and glutathione (GSH) concentrations in the chick were studied. Lead acetate·3H₂O was administered either in the diet for 3 wk at 2000 ppm Pb or by intraperitoneal (ip) injection of 3-wkold chicks with 52 mg Pb/100 g body wt. In Exp. 1, NPSH concentrations in liver and kidney were increased by both dietary and injected Pb in comparison to chicks not receiving Pb. Thigh muscle NPSH was decreased by injected Pb, whereas dietary Pb had no effect. In Expt. 2, whole blood and plasma NPSH were measured at 0, 0.5, 1.0, 2.0, and 4.0 h following ip Pb injection. Both whole blood and plasma NPSH were increased by 30 min. Whole blood NPSH concentrations plateaued at 30 min, and plasma NPSH continued to rise for 2 h. In Expt. 3, injected Pb increased hepatic NPSH, but not GSH concentrations. The ratio of GSH/NPSH was therefore lowered. The incorporation of [1-¹⁴C]glycine into hepatic GSH was stimulated by injected Pb. Buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, reduced hepatic NPSH and [¹⁴C]glycine incorporation in Pb-treated chicks to below control (non-Pb injected) values. In Expt. 4, dietary Pb fed for 3 wk increased the hepatic concentrations of both NPSH and GSH such that the ratio of GSH/NPSH was unchanged in comparison to chicks not fed Pb. The data suggest that the initial response to acute Pb intoxication may involve a mobilization of nonprotein thiols via the interorgan translocation system for GSH. Such a response would help to maintain adequate levels of GSH in organs crucial to detoxification.     

Estimation and manipulation of glutathione levels in prepuberal mouse ovaries and ova:Relevance to sperm nucleus transformation in the fertilized egg

Glutathione (GSH), the major low-molecular-weight thiol in mammalian cells, is believed to be a necessary factor for the transformation of the disulfide-stabilized sperm nucleus into the male pronucleus after fertilization. Its concentration in mouse ova, isolated from the ampulla of the oviduct after hormone-induced superovulation of 3–4-week-old mice, has been determined by an enzymic cycling microassay. The level found was 1.80 pmol per ovum. Mean ovum diameter was estimated as 71–72 μm, indicating a GSH concentration of 9–10 mM in the mouse egg. Administration of L-buthionine S, R-sulfoximine, an inhibitor of GSH biosynthesis, during the 2 days preceding ovulation, reduced ovum GSH content below 0.20 pmol (<1.0 mM). The mean GSH concentration of the hormone-stimulated ovaries was reduced from 3.2 mM to 0.2 mM under these conditions. It has also been demonstrated that measurement and manipulation of ovum and ovarian levels of GSH can aid in studying its function in ovaries, ova, and early embryos. Hormone-induced superovulation was achieved in BSO-treated prepuberal C57B1/6 X SJL mice whose ovaries contained less than 10% of control levels of GSH. Over 50% of the isolated ova were fertilized in vitro. However, abnormal one-cell embryos resulted in which the maternally derived pronucleus coexisted with an unchanged sperm nucleus, thus confirming that adequate levels of GSH are necessary for initiating transformation of the fertilizing sperm nucleus.     

Role of mitochondrial calcium transport in the control of substrate oxidation

This paper reviews the model of the control of mitochondrial substrate oxidation by Ca²⁺ ions. The mechanism is the activation by Ca²⁺ of four mitochondrial dehydrogenases, viz: glycerol 3-phosphate dehydrogenase, the pyruvate dehydrogenase multienzyme complex (PDH), NAD-linked isocitrate dehydrogenase (NAD-IDH) and 2-oxoglutarate dehydrogenase (OGDH). This results in the increase, or near-maintenance, of mitochondrial NADH/NAD ratios in the activated state, depending upon the tissue and the degree of "downstream" activation by Ca²⁺, likely at the level of the F₁F₀ ATP-ase. Higher values of the redox span of the respiratory chain allow for greatly increased fluxes through oxidative phosphorylation with a minimal drop in protonmotive force and phosphorylation potential. As PDH, NAD-IDH and OGDH are all located within the inner mitochondrial membrane, it is changes in matrix free Ca²⁺ ( [Ca²⁺]m ) which act as a signal to these activities. In this article, we review recent work in which ([Ca²⁺]m) is measured in cells and tissues, using different techniques, with special emphasis on the question of the degree of damping of ([Ca²⁺]m) relative to changes in cytosol free Ca²⁺ in cells with rapid transients in cytosol Ca²⁺, e.g. cardiac myocytes. Further, we put forward the point of view that the failure of mitochondrial energy transduction to keep pace with cellular energy needs in some forms of heart failure may involve a failure of ([Ca²⁺]m) to be raised adequately to allow the activation of the dehydrogenases. We present new data to show that this is so in cardiac myocytes isolated from animals suffering from chronic, atreptozocin-induced diabetes. This raises the possibility of therapy based upon partial inhibition of mitochondrial Ca²⁺ efflux pathways, thereby raising ([Ca²⁺]m) at a given, time-average value of cytosol free Ca²⁺.     

Role of Biomolecules from human renal stone matrix on COM crystal growth

Human renal calculi surgically removed from kidney stone patients were obtained and chemically analysed. Stones with CaOx (calcium oxalate) as the major component were washed in 0.15 M NaCl with gentle stirring for 48 h and then pulverised to a fine powder. The powder was extracted with 0.05 M EGTA, 1 mM PMSF and 1% - mercaptoethanol for 4 days at 4°C, the suspensions and the supernatants obtained were filtered through an Amicon Model 200 apparatus (mol. wt. cut off of 10,000 daltons) under nitrogen at 40 p.s.i. and concentrated to a known volume. The method of Nakagawa et al. [7] was employed to study the ability of > 10 kDa fractions to influence COM growth using metastable solution of CaCl₂ and Na₂C₂O₂ containing traces of ¹⁴C-oxalic acid. Potent biomolecules having the ability to influence CaOx precipitation were subjected to isolation, purification and characterization. Standard biochemical procedures, e.g. ultracentrifugation, ion-exchange chromatography, molecular sieve chromatography and SDS-PAGE, etc., were employed. Results revealed that human renal calculi extract contains biomolecules that can inhibit as well as stimulate the growth of preformed COM (calcium oxalate monohydrate) crystals. Most potent stimulator of CaOx growth was found to have a molecular weight of 66 kDa.     

线粒体钙单向转运体在心肌低氧/复氧损伤中的作用

目的:观察线粒体钙单向转运体在心肌低氧/复氧损伤中的作用并探讨其机制。方法:应用Langendorff大鼠心脏灌流模型,低氧/复氧(H/R)采用冠脉前降支结扎30 min、复灌120 min的方法。用生物信号采集系统记录左室发展压(LVDP)、左室压最大上升/下降速率(±dP/dtmax)、左室舒张末压(LVEDP);分光光度法分别检测冠脉流出液中乳酸脱氢酶(LDH)的含量和线粒体活性氧(ROS);TTC染色法检测心肌梗死面积。结果:与单纯低氧/复氧组相比,复氧起始给予线粒体钙单向转运体抑制剂钌红(5μmol/L)明显改善左心室各项功能指标,减小心肌梗死面积,降低线粒体ROS和冠脉流出液中LDH含量;而在复氧期起始给予线粒体钙单向转运体激动剂精胺(20μmol/L),显著升高了线粒体ROS活性,冠脉流出液中LDH含量在复氧5 min、20 min、30 min时显著增多,左心室各项功能指标与心肌梗死面积与单纯低氧/复氧组相比无显著差异。ROS清除剂MPG(1 mmol/L)与精胺联合应用则取消了精胺的作用。结论:抑制线粒体钙单向转运体可能通过减少线粒体ROS的生成减轻心脏低氧/复氧损伤。     

The Effect of Phosphinothricin (Glufosinate) on Glutathione Synthesis in Plants

The inhibitory effect of DL-phosphinothricin (glufosinate) on glutathione synthesis was studied in vivo and in vitro. The influence of phosphinothricin on γ-glutamylcysteine synthetase was compared with the already known effects of l -buthionine sulfoximine and l -methionine sulfoximine. The results showed that phosphinothricin and buthionine sulfoximine are inhibitors of γ-glutamylcysteine synthetase of plants. With both substances the enzyme was inhibited by 50 % at a concentration of 7 . 10^?4M (pI₅₀ = 3.15). Methionine sulfoximine reduced the enzyme activity by 50% at 5 . 10^?2 M (pI₅₀ = 1.30). It is discussed that the target enzyme of phosphinothricin is the glutamine synthetase whereas the γ-glutamylcysteine synthetase is only an accessory target.     

Role of mitochondrial quality control in exercise-induced health adaptation

Long-term endurance training or physical activity has been confirmed not only to improve physical performance, but to bring about an obvious beneficial effect on human health; however, the mechanism of this effect is not clear. The most studied health adaptations in skeletal muscle response to endurance exercise are increased muscle glycogen level and insulin sensitivity, fiber type transformation toward oxi- dative myofibers, and increased mitochondrial content/function. Mitochondria are dynamic organelles in eukaryotic cells critical in physical performance and disease occurrence. The mitochondrial life cycle spans biogenesis, maintenance, and clearance. Exercise training may promote each of these processes and confer positive impacts on skeletal muscle contractile and metabolic functions. This review focused on the regula- tion of these processes by endurance exercise and discussed its potential benefits in health and disease. We presented evidence suggesting that exercise training potentiates not only the biogenesis of mitochondria but also the removal of old and unhealthy mitochondria through mitochondrial quality control.     

Intracellular adaptations of glutathione content in Cucurbita pepo L. induced by treatment with reduced glutathione and buthionine sulfoximine

The intracellular effects of GSH (reduced glutathione) and BSO (buthionine sulfoximine) treatment on glutathione content were investigated with immunogold labeling in individual cellular compartments of Cucurbita pepo L. seedlings. Generally, GSH treatment led to increased levels of glutathione in roots and leaves (up to 3.5-fold in nuclei), whereas BSO treatment significantly decreased glutathione content in all organs. Transmission electron microscopy revealed that glutathione levels in mitochondria, which showed the highest glutathione labeling density of all compartments, remained generally unaffected by both treatments. Since glutathione within mitochondria is involved in the regulation of cell death, these results indicate that high and stable levels of glutathione in mitochondria play an important role in cell survival strategies. BSO treatment significantly decreased glutathione levels (1) in roots by about 78% in plastids and 60.8% in the cytosol and (2) in cotyledons by about 55% in the cytosol and 38.6% in plastids. After a short recovery period, glutathione levels were significantly increased in plastids and the cytosol of root tip cells (up to 3.7-fold) and back to control values in cotyledons. These results indicate that plastids, either alone or together with the cytosol, are the main center of glutathione synthesis in leaves as well as in roots. After GSH treatment for 24 h, severe ultrastructural damage related to increased levels of glutathione was found in roots, in all organelles except mitochondria. Possible negative effects of GSH treatment leading to the observed ultrastructural damage are discussed.     

Role of calcium-induced mitochondrial hydroperoxide in induction of apoptosis of RBL2H3 cells with eicosapentaenoic acid treatment

Eicosapentaenoic acid (EPA) was previously shown to induce caspase-independent apoptosis in rat basophilic leukemia cells (RBL2H3 cells) by translocation of apoptosis-inducing factor (AIF) [Free Radic Res (2005) 39, 225-235]. Here, we attempted to investigate the mechanism of EPA-induced apoptosis. A rapid and sustained increase in calcium was observed in mitochondria at 2 h after the addition of EPA prior to apoptosis. Coincidently, hydroperoxide was generated in the mitochondria after exposure to EPA. Production of mitochondrial hydroperoxide was significantly reduced by ruthenium red, an inhibitor of mitochondrial calcium uniporter, and BAPTA-AM, a cytoplasmic calcium chelator, indicating that generation of hydroperoxide is triggered by an accumulation of calcium in the mitochondria. The production of mitochondrial hydroperoxide was markedly attenuated by overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in the mitochondria. Apoptosis was therefore, significantly prevented through inhibition of mitochondrial hydroperoxide generation with mitochondrial PHGPx, ruthenium red or BAPTA-AM. However, accumulation of calcium in the mitochondria was not prevented by mitochondrial PHGPx although apoptosis was blocked, indicating that elevated calcium does not directly induce apoptosis. Taken together, our results show that calcium-dependent hydroperoxide accumulation in the mitochondria is critical in EPA-induced apoptosis.     

Role in hemolysis of the interaction of tellurium compounds with glutathione

We have shown that tellurite and tellurate require the interaction with reduced glutathione (GSH) to hemolyze human erythrocytes. The study of the nature of this interaction is the main object of this paper. The degree of hemolysis was determined by the method of Angelone. The addition of extracellular 1 mM GSH or cysteine increased the rate of hemolysis. Concanavalin A (0.3 mg/mL) and/or 4 mg/mL adenosine did not affect the hemolysis by 0.1 mM tellurite. One tenth to 1 mM 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate (SITS) inhibited this hemolysis by 60–100%. Millimolar GSH released this inhibition. Incubation of 0.1 mM tellurite with 1 mM GSH for 90 min at 37°C, produced a hemolytic agent when prepared and tested under nitrogen, but one that was not active when prepared in air. The hemolysis byp-hydroxymercuribenzoate orp-hydroxymercuriphenylsulfonate did not involve GSH. Scanning electron micrographs showed a sphero-echinocyte transformation, in the pre-hemolytic stage, with all the agents tested. The rate of penetration of tellurite plays a role in determining the rate of hemolysis, as shown by the effect of SITS. The release by GSH of the inhibition by SITS poses questions concerning the site of action and cell membrane penetration of the hemolytic agent. Telluride or some intermediate in the interaction of GSH with tellurite is the actual hemolytic agent.