Differential expression of heat shock protein mRNAs under in vivo glutathione depletion in the mouse retina

Heat shock proteins (HSPs) are highly conserved proteins playing a protective role under deleterious conditions caused by a wide variety of pathophysiological, including environmental stresses. Glutathione (GSH) is known to play a critical role in the cellular defense against unregulated oxidative stress in mammalian cells including neurons. We previously demonstrated that GSH depletion induced cell death in the retina, but the mechanism(s) of cellular protection were not clear. Unregulated oxidative stress was induced by depletion of intracellular GSH by systematic administration of buthionine sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase. After 0, 1, 4 and 7 days of BSO administration, we examined expression of both large and small HSP mRNAs (hsp90alpha, hsp90beta, hsp70, hsp60 and hsp25) in oxidative-stressed mouse retina. Of large HSPs, only hsp70 expression was significantly decreased from 1 day after BSO injection, whereas expression of other large hsps was not changed on day 1. Expression of hsp60 decreased on 4 days, whereas expression of hsp90 decreased on 7 days after BSO administration. Different from large HSPs, a small HSP, hsp25 increased its expression to a great extent from 1 day after BSO administration. Taken together, our results show that unregulated oxidative stress could induce differential expression of HSPs, which, in turn, may play distinct roles in the cellular defense. Targeting HSPs, therefore, may provide novel tools for treatment of retinal degenerative diseases such as glaucoma, retinopathy or age-related macular degeneration.     

Anticonvulsant effects of four neuropeptides in the rat hippocampus during self-sustaining status epilepticus

The plaques in multiple sclerosis (MS) autopsy tissue contain tumor necrosis factor-alpha (TNF-alpha) at high concentrations. Moreover, microglia are able to convert L-tryptophan to quinolinic acid. Thus, TNF-alpha and quinolinic acid are endogenous compounds which may compromise oligodendrocytes during inflammatory demyelination. It is also known that cellular functions depend on adequate concentrations of glutathione (GSH). As some apoptotic oligodendrocytes have been observed in MS plaques, it was therefore logical to determine whether oligodendrocyte apoptosis would occur in response to TNF-alpha, quinolinic acid or GSH depletion. Oligodendrocytes were treated in vitro with TNF-alpha, quinolinic acid and the GSH-depleting agent, buthionine sulfoximine (BSO), respectively, and the numbers of intact and apoptotic cells were counted. TNF-alpha reduced the numbers of mature oligodendrocytes, but not immature oligodendrocytes, without producing apoptosis. Quinolinic acid and BSO each caused oligodendrocyte loss via apoptosis, and GSH ethyl ester partly protected the cells against BSO. The data suggest that oligodendrocytes undergo apoptosis under adverse conditions that result from an endogenous toxicant or depletion of GSH.     

TRPM2 channel protective properties of N-acetylcysteine on cytosolic glutathione depletion dependent oxidative stress and Ca2+ influx in rat dorsal root ganglion

N-acetylcysteine (NAC) is a thiol-containing (sulphydryl donor) antioxidant, which contributes to regeneration of glutathione (GSH) and also acts through a direct reaction with free radicals. Thiol depletion has been implicated in the neurobiology of sensory neurons and pain. We reported recently an activator role of intracellular GSH depletion on calcium influx through transient receptor potential melastatin-like 2 (TRPM2) channels in rat dorsal root ganglion (DRG). NAC may have a protective role on calcium influx through regulation of TRPM2 channels in the neurons. Therefore, we tested the effects of NAC on TRPM2 channel currents in cytosolic GSH depleted DRG in rats. DRG neurons were freshly isolated from rats and the neurons were incubated for 24 h with buthionine sulfoximine (BSO). In whole-cell patch clamp experiments, TRPM2 currents in the DRG incubated with BSO were gated by H(2)O(2). TRPM2 channels current densities, cytosolic free Ca(2+) content, and lipid peroxidation values in the neurons were higher in H(2)O(2) and BSO + H(2)O(2) group than in controls; however GSH and GSH peroxidase (GSH-Px) values were decreased. BSO + H(2)O(2)-induced TRPM2 channel gating was totally inhibited by extracellular NAC and partially inhibited by 2-aminoethyl diphenylborinate. GSH-Px activity, lipid peroxidation and GSH levels in the DRG neurons were also modulated by NAC. In conclusion, we observed a modulator role of NAC on Ca(2+) influx through a TRPM2 channel in intracellular GSH depleted DRG neurons. NAC incubation before BSO exposure appears to be more protective than NAC incubation after BSO exposure. Since cytosolic thiol group depletion is a common feature of neuropathic pain, our findings are relevant to the etiology and treatment of pain neuropathology in DRG neurons.     

Glutathione depletion induces differential apoptosis in cells of mouse retina, in vivo

Oxidative stress affects numerous intracellular macromolecules, and may result in cell death unless precisely regulated. Unregulated oxidative stress can be controlled by various cellular defense mechanisms such as glutathione (GSH) which can critically counteract the damaging effects of oxidative stress in mammalian cells. We determined the effects of unregulated oxidative stress induced by GSH depletion on cells in mouse retina. Mice were intraperitoneally injected with buthionine sulphoximine (BSO) at 1.5 g/kg. After 0, 1, 4, and 7 days of BSO administration, retinas were excised and sections were subjected to GSH assay and terminal uridine deoxynucleotidyl nick end labeling (TUNEL) analysis. After 4 days of BSO administration, the number of TUNEL positive cells was significantly increased. However, after 7 days, TUNEL positive cells returned to the basal level. The retinal region most affected by the BSO treatment appeared to be the outer nuclear layer where the photoreceptor cells reside. Different from cells in other regions, retinal cells in the inner nuclear layer increased in their apoptosis even after the first day of BSO injection, and the increase was further potentiated after 4 days. Taken together, our studies suggested that GSH depletion may cause unregulated oxidative stress to the cells in the retina and indeed increased cell death in the retina. The cells in the inner nuclear layer seemed to be affected earlier than the cells in other layers of the retina. The GSH level in the retina may be a crucial therapeutic target in preventing blindness.     

消耗还原型谷胱甘肽抑制血管紧张素Ⅱ激活巨噬细胞c-Jun/ATF-2及NF-κB

目的:探讨还原型谷胱甘肽(GSH)在血管紧张素Ⅱ(AngⅡ)激活巨噬细胞转录因子c-Jun/ATF-2及NF-κB中的作用。方法:用荧光分光光度法测定细胞内GSH含量;用丁基硫堇硫氧胺(BSO)消耗细胞内GSH;用免疫印迹法测定细胞c-Jun/ATF-2磷酸化表达及NF-κBp65表达;用凝胶滞留法测定细胞NF-κB活性;用细胞免疫组化观察细胞c-Jun/ATF-2磷酸化表达的时间模式。结果:AngⅡ(1μmol/L)刺激30-60min可降低RAW264.7细胞内GSH;而后GSH逐渐适应性恢复。同时,AngⅡ刺激60min,呈剂量依赖性降低RAW264.7细胞内GSH。GSH特异性消耗剂BSO(0.5mmol/L)与RAW264.7细胞共同孵育18h,即可完全消耗细胞内GSH。AngⅡ(1μmol/L)可诱导RAW264.7巨噬细胞c-Jun/ATF-2磷酸化表达;BSO预先完全消耗细胞内GSH,AngⅡ(1μmol/L)不能诱导巨噬细胞c-Jun/ATF-2磷酸化。同样,AngⅡ(1μmol/L)可激活RAW264.7巨噬细胞NF-κB;BSO预先完全消耗细胞内GSH,AngⅡ(1μmol/L)不能激活巨噬细胞NF-κB。结论:还原型谷胱甘肽可能参与调控巨噬细胞转录因子c-Jun/ATF-2及NF-κB的转录活性。     

Glutathione deficiency intensifies ischaemia-reperfusion induced cardiac dysfunction and oxidative stress.

The efficacy of glutathione (GSH) in protecting ischaemia-reperfusion (I-R) induced cardiac dysfunction and myocardial oxidative stress was studied in open-chest, stunned rat heart model. Female Sprague-Dawley rats were randomly divided into three experimental groups: (1) GSH-depletion, by injection of buthionine sulphoxamine (BSO, 4 mmol kg(-1), i.p.) 24 h prior to I-R, (2) BSO injection (4 mmol kg(-1), i.p.) in conjunction with acivicin (AT125, 0.05 mmol kg(-1), i.v.) infusion 1 h prior to I-R, and (3) control (C), receiving saline treatment. Each group was further divided into I-R, with surgical occlusion of the main left coronary artery (LCA) for 30 min followed by 20 min reperfusion, and sham. Myocardial GSH content and GSH : glutathione disulphide (GSSG) ratio were decreased by approximately 50% (P < 0.01) in both BSO and BSO + AT125 vs. C. Ischaemia-reperfusion suppressed GSH in both left and right ventricles of C (P < 0.01) and left ventricles of BSO and BSO + AT125 (P < 0.05). Contractility (+dP/dt and -dP/dt) in C heart decreased 55% (P < 0.01) after I and recovered 90% after I-R, whereas +/-dP/dt in BSO decreased 57% (P < 0.01) with ischaemia and recovered 76 and 84% (P < 0.05), respectively, after I-R. For BSO + AT125, +/-dP/dt were 64 and 76% (P < 0.01) lower after ischaemia, and recovered only 67 and 61% (P < 0.01) after I-R. Left ventricular systolic pressure in C, BSO and BSO + AT125 reached 95 (P > 0.05) 87 and 82% (P < 0.05) of their respective sham values after I-R. Rate-pressure double product was 11% (P > 0.05) and 25% (P < 0.05) lower in BSO and BSO + AT125, compared with Saline, respectively. BSO and BSO + AT125 rats demonstrated significantly lower liver GSH and heart Mn superoxide dismutase activity than C rats after I-R. These data indicate that GSH depletion by inhibition of its synthesis and transport can exacerbate cardiac dysfunction inflicted by in vivo I-R. Part of the aetiology may involve impaired myocardial antioxidant defenses and whole-body GSH homeostasis.     

Differential effects of L-buthionine sulfoximine and ethacrynic acid on glutathione levels and mitochondrial function in PC12 cells.

We investigated the effect of glutathione (GSH) depletion on mitochondrial function and generation of reactive oxygen intermediates (ROI) in PC12 cells in vitro. Direct depletion of cellular GSH using ethacrynic acid (EA, 500 mM) resulted in a concentration-dependent generation of ROI and cell death within 24 h. Treatment with 500 microM L-buthionine sulfoximine (BSO), which inhibits GSH synthesis, reduced cellular GSH but did not lead to generation of ROI. Furthermore, cells remained viable up to 72 h. Analysis of subcellular fractions revealed complete loss of cytosolic and mitochondrial GSH within 4 h of EA treatment. In contrast, BSO-treated cells still maintained 100% GSH in the mitochondrial fraction for 4 h and 6% for 48 h. Mitochondrial complex II/IIi and IV activities were not significantly decreased up to 48 h of BSO treatment while EA treatment resulted in a complete loss of complex II/III activity and a 70% reduction of complex IV activity within 4 h. These findings suggest that mitochondrial GSH is critical for the maintenance of mitochondrial function and cellular viability.     

Glutathione deficiency intensifies ischaemia‐reperfusion induced cardiac dysfunction and oxidative stress

The efficacy of glutathione (GSH) in protecting ischaemia‐reperfusion (I‐R) induced cardiac dysfunction and myocardial oxidative stress was studied in open‐chest, stunned rat heart model. Female Sprague–Dawley rats were randomly divided into three experimental groups: (1) GSH‐depletion, by injection of buthionine sulphoxamine (BSO, 4 mmol kg^–1, i.p.) 24 h prior to I‐R, (2) BSO injection (4 mmol kg^–1, i.p.) in conjunction with acivicin (AT125, 0.05 mmol kg^–1, i.v.) infusion 1 h prior to I‐R, and (3) control (C), receiving saline treatment. Each group was further divided into I‐R, with surgical occlusion of the main left coronary artery (LCA) for 30 min followed by 20 min reperfusion, and sham. Myocardial GSH content and GSH : glutathione disulphide (GSSG) ratio were decreased by ?50% (P < 0.01) in both BSO and BSO + AT125 vs. C. Ischaemia‐reperfusion suppressed GSH in both left and right ventricles of C (P < 0.01) and left ventricles of BSO and BSO + AT125 (P < 0.05). Contractility (+dP/dt and –dP/dt) in C heart decreased 55% (P < 0.01) after I and recovered 90% after I‐R, whereas ±dP/dt in BSO decreased 57% (P < 0.01) with ischaemia and recovered 76 and 84% (P < 0.05), respectively, after I‐R. For BSO + AT125, ±dP/dt were 64 and 76% (P < 0.01) lower after ischaemia, and recovered only 67 and 61% (P < 0.01) after I‐R. Left ventricular systolic pressure in C, BSO and BSO + AT125 reached 95 (P > 0.05) 87 and 82% (P < 0.05) of their respective sham values after I‐R. Rate‐pressure double product was 11% (P > 0.05) and 25% (P < 0.05) lower in BSO and BSO + AT125, compared with Saline, respectively. BSO and BSO + AT125 rats demonstrated significantly lower liver GSH and heart Mn superoxide dismutase activity than C rats after I‐R. These data indicate that GSH depletion by inhibition of its synthesis and transport can exacerbate cardiac dysfunction inflicted by in vivo I‐R. Part of the aetiology may involve impaired myocardial antioxidant defenses and whole‐body GSH homeostasis.     

血管紧张素II对单核细胞趋化因子及粘附分子表达的影响

为了观察血管紧张素II (AII)对人单核细胞株THP 1分泌单核细胞趋化因子 (MCP 1)、人类脐静脉内皮细胞(HUVEC )分泌细胞间粘附分子 (ICAM 1)的影响及AII对HUVEC与THP 1的粘附功能的影响 ,我们利用ELISA法检测AII作用后THP 1分泌MCP 1及HUVEC分泌ICAM 1的表达 ,利用计数法观察经AII作用的HUVEC与THP 1的粘附率。结果发现四种不同浓度的AII(10 6、 10 7、 10 8、 10 9mol/L )刺激THP 1及HUVEC 2 4h后 ,MCP 1及ICAM 1的分泌明显增加 ,AII(10 6mol/L )作用HUVEC 6h后 ,其对THP 1细胞粘附率明显增加 ,表明AII具有调节THP 1分泌MCP 1及HUVEC分泌ICAM 1的作用 ,AII可通过致炎症作用参与动脉粥样硬化的发病过程。     

Effects of buthionine sulfoximine on the development of ozone-induced pulmonary fibrosis

The capacity of reduced glutathione (GSH) to protect lung tissue against ozone-induced pulmonary fibrosis was investigated. Male B6C3F1 mice were exposed to 0, 0.2, 0.5, and 1.0 ppm ozone for 23 hr/day for 14 days. During exposures and/or for a period of 90 days after exposures, subgroups of mice at each exposure level were given drinking water containing 30 mM L-buthionine-S,R-sulfoximine (BSO) to lower in vivo levels of GSH. These BSO treatments reduced blood glutamylcysteine synthetase (GCS) activity (regulatory enzyme for GSH biosynthesis) and lung nonprotein sulfhydryl (NPSH) levels in nonexposed animals by approximately half. In contrast, ozone exposures increased blood GCS activity and lung NPSH levels in a concentration-dependent manner, with smaller increases in the BSO-treated mice. Immediately after exposures, an ozone-related inflammatory response was seen in lungs, but no histopathological signs of developing fibrosis were evident. Ninety days later, mice exposed to 1 ppm ozone and not treated with BSO had modest evidence of pulmonary fibrosis. Mice exposed to 1 ppm ozone and treated with BSO during this post-exposure period (regardless of BSO treatment during exposures) showed histopathological evidence of exacerbated pulmonary fibrosis, compared to similarly exposed mice not treated with BSO postexposure. These results indicated that interference with the body's normal defense mechanisms against oxidant damage, including suppression of GSH biosynthesis, exacerbates the subsequent development of pulmonary fibrosis.     

The influence of glutathione on redox regulation by antioxidant proteins and apoptosis in macrophages exposed to 2-hydroxyethyl methacrylate (HEMA)

Resin monomers like 2-hydroxyethyl methacrylate (HEMA) disturb cell functions including responses of the innate immune system, mineralization and differentiation, or induce cell death via apoptosis. These phenomena are associated with oxidative stress and a reduction in the concentration of the antioxidant glutathione (GSH), resulting in imbalanced redox homeostasis. Thus far, the precise mechanism of how resin monomers interfere with cellular redox regulation is unknown. The present study provides insight into the induction of apoptosis and the differential expression of antioxidant enzymes depending on the availability of GSH. Buthionine sulfoximine (BSO) was used to inhibit GSH synthesis, while 2-oxothiazolidine-4-carboxylate (OTC), and N-acetylcysteine (NAC) as prodrugs supported GSH synthesis in RAW264.7 mouse macrophages exposed to HEMA (0-8 mm) for 24 h. The level of GSH was significantly decreased after cells were preincubated with BSO, and the formation of reactive oxygen species (ROS) increased in cultures subsequently exposed to HEMA. Apoptosis was drastically increased by BSO in HEMA-exposed cell cultures as well, but OTC and NAC retracted HEMA-induced cell death. These results show that dental monomer-induced apoptosis is causally related to the availability of GSH. The hydrogen peroxide decomposing enzymes glutathione peroxidase (GPx1/2) and catalase were differentially regulated in HEMA-exposed cultures. Expression of GPx1/2 was inhibited by HEMA and further reduced in the presence of BSO. SOD1 (superoxide dismutase) expression was inhibited in the presence of HEMA, and was decreased to an even greater extent by BSO, possibly due to H(2)O(2)-feedback inhibition. The expression of catalase was considerably up-regulated in HEMA-exposed cultures, implying that H(2)O(2) is the type of ROS that is significantly increased in monomer-exposed cells. OTC and NAC counteracted the effect of HEMA on GPx1/2, SOD1, and catalase expression. HO-1 (heme oxygenase) expression was strongly enhanced by HEMA, suggesting the need for further antioxidants like bilirubin to support enzyme activities that directly regulate H(2)O(2) equilibrium. Expression of the oxidoreductase thioredoxin (TRX1), the second major thiol-dependent antioxidant system in eukaryotic cells, was slightly reduced, while the oxygen-sensing protein HIF-1α was downregulated in HEMA-exposed cell cultures. These results indicate that cells and tissues actively respond to monomer-induced oxidative stress by the differential expression of enzymatic antioxidants.     

Oxidized laminin‐1 induces increased monocyte attachment and expression of ICAM‐1 in endothelial cells

Atherosclerosis has been associated with increased oxidative stress and monocyte recruitment by endothelial cells. Sub‐endothelial basement membrane proteins, such as laminins that play a central role in cell adhesion, are exposed to reactive oxygen species. In the present study monocyte attachment on human umbilical cord vein endothelial cells (HUVEC) that were preattached to oxidized or native laminin, was investigated. Intracellular cell adhesion molecule‐1 (ICAM‐1) expression by HUVEC was estimated by an enzyme‐linked immunosorbent assay. HUVEC attachment to oxidized or native laminin‐1 was examined using the Hemacolor kit. Anti‐alphaL, anti‐alphaM, anti‐alpha2 and anti‐beta2 integrin subunit antibodies were used in order to further investigate the above phenomena. HUVEC that were preattached to oxidized laminin expressed higher levels of ICAM‐1 and monocytes attached at a higher degree to these cells as compared to HUVEC that were preattached to native laminin. Incubation of monocytes with monoclonal antibodies against the alphaM and beta2 integrin subunits equalized the above mentioned differences. Moreover, HUVEC attached to oxidized laminin at a higher degree as compared to native laminin. This difference was equalized after incubation with the antibody against the alpha2 integrin subunit. These results indicate a modified interaction between HUVEC and the basement membranes in cases where laminin is oxidatively modified. This modified interaction results in increased ICAM‐1 expression by endothelial cells and consequently increased monocyte recruitment capacity.     

Fraxetin prevents rotenone-induced apoptosis by induction of endogenous glutathione in human neuroblastoma cells

Fraxetin belongs to an extensive group of natural phenolic anti-oxidants. In the present study, using a human neuroblastoma SH-SY5Y cells, we have investigated the protective effects of this compound on modifications in endogenous reduced glutathione (GSH), intracellular oxygen species (ROS) and apoptotic death on rotenone-mediated cytoxicity. Incubation of cells with the fraxetin led to a significant elevation dose-dependent of cellular GSH and this was accompanied by a marked protection against rotenone-mediated toxicity, which was also significantly reversed in the cells with buthionine sulfoximine (BSO) co-treatment. Taken together, this study suggested that intracellular GSH appeared to be an important factor in fraxetin-mediated cytoprotection against rotenone-toxicity in SH-SY5Y cells. Fraxetin at 10-100 muM inhibited the formation of ROS, cytochrome c release, activation of caspase-3 and 9, and suppressed the up-regulation of Bax, whereas no significant change occurred in Bcl-2 levels. Our results indicated that the anti-oxidative and anti-apoptotic properties render this natural compound potentially protective against rotenone-induced cytotoxicity.     

Antiapoptotic response to induced GSH depletion: involvement of heat shock proteins and NF-kappaB activation

Alteration of glutathione (GSH) homeostasis represents one of the earliest events during the commitment of stress-induced apoptosis. Extrusion of GSH into the extracellular milieu, in response to several oxidative stimuli, has been suggested as a molecular switch triggering apoptosis. However, chemical depletion of GSH does not induce cell death even though cytochrome c release from mitochondria has been observed. Here we report that U937 cells treated with buthionine sulfoximine (BSO) are able to survive and to inhibit the apoptotic program downstream of cytochrome c release. BSO treatment induces a highly significant decrease of GSH in both the cytosolic and mitochondrial fractions. The concomitant release of cytochrome c into the cytosol was associated with nuclear translocation of apoptosis-inducing factor. GSH depletion also resulted in reactive oxygen species production and in a specific increase of mitochondrial protein carbonyls. However, all these events were transiently present inside cells and efficiently counteracted by cell-repairing systems. We observed an increase in the proteasome activity and in the expression levels of heat shock protein 27 (Hsp27) and Hsp70. Moreover, nuclear factor-kappaB (NF-kappaB) was activated in our system as a survival cell response against the oxidative injury. Overall results suggest that activation of NF-kappaB and Hsp could allow cell adaptation and survival under exhaustive GSH depletion.     

Subcellular compartmentalization of glutathione: correlations with parameters of oxidative stress related to genotoxicity

Glutathione (GSH) is a major component of the antioxidant defence system of mammalian cells and is found in subcellular pools within the cytoplasm, nucleus and mitochondria. To evaluate the relationships between these pools and parameters of oxidative stress related to genotoxicity, wild type (WT) and 8-oxo-2'-deoxyguanosine glycosylase 1 (OGG1)-null (mOGG1(-/-)) mouse embryonic fibroblasts (MEF) were treated with buthionine sulphoximine (BSO; 0-1000 microM, 24 h), an inhibitor of GSH biosynthesis. BSO treatment resulted in a concentration-dependent depletion of GSH from the cytoplasm, but depletion of mitochondrial and nuclear GSH occurred only at concentrations > or =100 microM. GSH levels were correlated with reactive oxygen species (ROS), lipid peroxidation (measured as the increase in the genotoxic end-product malondialdehyde (MDA)) and oxidative DNA modifications, measured as both frank DNA strand-breaks (FSB) and oxidized purine lesions (OxP) using the alkaline comet assay with formamidopyrimidine DNA glycosylase (FPG) modification; this system allowed for the identification of BSO-induced DNA modifications as primarily mutagenic 8-oxo-2'-deoxyguanosine lesions. A number of significant correlations were observed. First, negative linear correlations were observed between mitochondrial GSH and ROS (r = -0.985 and r = -0.961 for WT and mOGG1(-/-) MEF, respectively), and mitochondrial GSH and MDA (r = -0.967 and r = -0.963 for WT and mOGG1(-/-) MEF, respectively). Second, positive linear correlations were observed between ROS and MDA (r = 0.996 and r = 0.935 for WT and mOGG1(-/-) MEF, respectively), and ROS and OxP (r = 0.938 and r = 0.981 for WT and mOGG1(-/-) MEF, respectively). Finally, oxidative DNA modifications displayed a negative linear correlation with nuclear GSH (r = -0.963 and -0.951 between nuclear GSH and FSB and OxP, respectively, for WT MEF and r = -0.960 between nuclear GSH and OxP in mOGG1(-/-) MEF), thus, demonstrating the genotoxic potential of compounds that deplete GSH. The findings highlight the critical roles of the mitochondrial and nuclear GSH pools in protecting cellular components, particularly DNA, from oxidative modification.     

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     

Increased lipid peroxidation in the liver and kidney and their mitochondrial fractions following buthionine sulfoximine induced glutathione depletion in guinea pigs

Malondialdehyde (MDA) and diene conjugates (DC) and vitamin C levels and the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were determined in the liver and kidney and their mitochondrial fractions of guinea pigs 48 h after the injection of L-buthionine-(S,R)-sulfoximine (BSO), a glutathione (GSH) depleting agent. In BSO-induced GSH depletion, lipid peroxidation and SOD activities were found to be increased but GSH-Px activities did not change in the liver and kidney and their mitochondrial fractions. In addition, vitamin C levels remained unchanged in the liver and kidney homogenates. These results indicate that GSH depletion may influence oxidative stress in the liver and kidney and their mitochondrial fractions of guinea pigs.     

IL-4 primes human endothelial cells for secondary responses to histamine

Interleukin-4 (IL-4) is a multifunctional cytokine, which is involved in numerous disease states, including atopic asthma. IL-4 not only induces direct responses in cells but can also prime for secondary responses to stimuli. Little is known about the priming effects of IL-4 on endothelial cells; therefore, we chose to examine the ability of IL-4 to prime endothelial cells for platelet-activating factor (PAF) synthesis and prostaglandin E(2) (PGE(2)) release. IL-4 alone did not enhance PAF synthesis or PGE(2) release; however, pretreatment with IL-4 primed for PAF synthesis and PGE(2) release in response to subsequent stimulation with histamine. In contrast, tumor necrosis factor alpha (TNF-alpha), oncostatin M (OSM), and IL-1beta did not prime endothelial cells for PAF synthesis in response to histamine. The priming effects of IL-4 occurred without any detectable changes in the requirement for signaling pathways upstream of PGE(2) release. IL-4 treatment increased the expression of mRNA for histamine receptor 1 (HR1) and shifted the inhibition curve for pyrilamine, a specific HR1 antagonist. In addition, the dose-response curve for histamine-induced elevations in intracellular calcium was shifted following IL-4 stimulation. Together, these data indicate that HR1 is up-regulated in IL-4-stimulated human umbilical vein endothelial cells (HUVEC) and suggest that this up-regulation may contribute to the enhanced responsiveness of IL-4-stimulated HUVEC to histamine.     

Cigarette smoke extract induces expression of cell adhesion molecules in HUVEC via actin filament reorganization

Epidemiologic studies have shown a strong association between cigarette smoking and cardiovascular diseases. Various oxidative species and free radicals are produced during cigarette smoking and these lead to endothelial dysfunction and inflammation. Expression of adhesion molecules, such as intercellular adhesion molecule‐1 (ICAM‐1), E‐selectin, and vascular cell adhesion molecule‐1, and adhesion of leukocytes are present in atherosclerosis. We showed previously that a nonfractionated cigarette smoke extract (CSE) induces surface expression of ICAM‐1 and E‐selectin in human umbilical vein endothelial cells (HUVEC). We then investigated the role of the MAPKs (ERK1/2, JNK, and p38) and AP‐1 and the role of actin cytoskeleton reorganization in the CSE‐induced expression of ICAM‐1 and E‐selectin. Western blot analysis showed that CSE treatment rapidly and significantly caused phosphorylation of JNK and ERK1/2 but not of p38. Cytochalasin D (an actin filament disruptor) partially inhibited CSE‐induced ICAM‐1 and E‐selectin surface expression. However, inhibitors of ERK1/2 (PD98059) and JNK (SP600125) did not attenuate the CSE‐induced ICAM‐1 and E‐selectin surface expression. The results of electrophoretic mobility shift assay showed that CSE enhanced AP‐1 binding activity. Therefore, CSE activated AP‐1 and upregulated ICAM‐1 and E‐selectin surface expression in HUVEC seem to be via an MAPK‐independent pathway. Moreover, the dynamic reorganization of the actin cytoskeleton seems to be required for the CSE‐induced surface expression of ICAM‐1 and E‐selectin. Environ. Mol. Mutagen., 2009. © 2008 Wiley‐Liss, Inc.