Effect of hypoxic cell radiosensitizers on glutathione level and related enzyme activities in isolated rat hepatocytes

A comparative study of the effect of misonidazole and novel radiosensitizers on glutathione (GSH) levels and related enzyme activities in isolated rat hepatocytes was performed. Incubation of hepatocytes with 5 mM radiosensitizers led to a decrease in the intracellular GSH level. The most pronounced decrease in cellular GSH was evoked by 2,4-dinitroimidazole-1-ethanol (DNIE); after incubation for only 15 min, GSH was hardly detected. DNIE-mediated GSH loss was dependent upon its concentration. DNIE reacted with GSH nonenzymatically as well as with diethylmaleate, while misonidazole and 1-methyl-2-methyl-sulfinyl-5-methoxycarbonylimidazole (KIH-3) did not. Addition of partially purified glutathione S-transferase (GST) did not enhance DNIE-mediated GSH loss in a cell-free system. DNIE inhibited glutathione peroxidase (GSH-Px), GST, and glutathione reductase (GSSG-R) activities in hepatocytes, while misonidazole and KIH-3 did not. GSH-Px activity assayed with H2O2 as substrate was the most inhibited. Inhibition of GSH-Px activity assayed with cumene hydroperoxide as substrate and GST was less than that of GSH-Px assayed with H2O2 as substrate. GSSG-R activity was decreased by DNIE, but not significantly. Incubation of purified GSH-Px with DNIE resulted in a little change in the activity when assayed with H2O2 as substrate.     

Glutathione-Mediated Regulation of ATP Sulfurylase Activity,SO42- Uptake,and Oxidative Stress Response in Intact Canola Roots

The dual role of glutathione as a transducer of S status (A.G. Lappartient and B. Touraine [1996] Plant Physiol 111: 147-157) and as an antioxidant was examined by comparing the effects of S deprivation, glutathione feeding, and H2O2 (oxidative stress) on SO42- uptake and ATP sulfurylase activity in roots of intact canola (Brassica napus L.). ATP sulfurylase activity increased and SO42- uptake rate severely decreased in roots exposed to 10 mM H2O2, whereas both increased in S-starved plants. In split-root experiments, an oxidative stress response was induced in roots remote from H2O2 exposure, as revealed by changes in the reduced glutathione (GSH) level and the GSH/oxidized glutathione (GSSG) ratio, but there was only a small decrease in SO42- uptake rate and no effect on ATP sulfurylase activity. Feeding plants with GSH increased GSH, but did not affect the GSH/GSSG ratio, and both ATP sulfurylase activity and SO42- uptake were inhibited. The responses of the H2O2-scavenging enzymes ascorbate peroxidase and glutathione reductase to S starvation, GSH treatment, and H2O2 treatment were not to glutathione-mediated S demand regulatory process. We conclude that the regulation of ATP sulfurylase activity and SO42- uptake by S demand is related to GSH rather than to the GSH/GSSG ratio, and is distinct from the oxidative stress response.     

Calcium homeostasis during thymocyte apoptosis. I. Increase in cytosolic Ca2+ concentration at the early stage of apoptosis induced by hydrogen peroxide

The concentration of free cytosolic Ca2+ ([Ca2+]i), 45Ca2+ entry and the level of reduced glutathione (GSH) after x-irradiation in a dose of 4.5 Gy or 0.1 mM H2O2-treatment were investigated in isolated rat thymocytes during the period preceding electrophoretically detected DNA intranucleosomal fragmentation. Using fura-2 it was shown that the level of [Ca2+]i in X-irradiated thymocytes was not changed as compared with the control, while the GSH content was increased. The gradual increase in [Ca2+]i along with GSH level falling was detected in the H2O2-treated cells. 45Ca2+ entry in the cells exposed to apoptogenic stimuli was not enhanced. After addition of H2O2 to the cells previously treated with thapsigargin further [Ca2+]i increase in both normal and nominally calcium-free medium was detected. Cyclosporine A inhibited Ca2+-mobilizing effect of H2O2, but did not prevent it completely. The role of intracellular calcium depots in calcium homeostasis disturbance during oxidative stress and apoptosis is discussed.     

Deficient metabolic utilization of hydrogen peroxide in Trypanosoma cruzi.

The glutathione peroxidase-glutathione reductase system, an alternative pathway for metabolic utilization of H2O2 [Chance, Sies & Boveris (1979) Physiol. Rev. 59, 527-605], was investigated in Trypanosoma cruzi, an organism lacking catalase and deficient in peroxidase [Boveris & Stoppani (1977) Experientia 33, 1306-1308]. The presence of glutathione (4.9 +/- 0.7 nmol of reduced glutathione/10(8) cells) and NADPH-dependent glutathione reductase (5.3 +/- 0.4 munit/10(8) cells) was demonstrated in the cytosolic fraction of the parasite, but with H2O2 as substrate glutathione peroxidase activity could not be demonstrated in the same extracts. With t-butyl hydroperoxide or cumene hydroperoxide as substrate, a very low NADPH-dependent glutathione peroxidase activity was detected (equivalent to 0.3-0.5 munit of peroxidase/10(8) cells, or about 10% of glutathione reductase activity). Blank reactions of the glutathione peroxidase assay (non-enzymic oxidation of glutathione by hydroperoxides and enzymic oxidation of NADPH) hampered accurate measurement of peroxidase activity. The presence of superoxide dismutase and ascorbate peroxidase activity in, as well as the absence of catalase from, epimastigote extracts was confirmed. Ascorbate peroxidase activity was cyanide-sensitive and heat-labile, but no activity could be demonstrated with diaminobenzidine, pyrogallol or guaiacol as electron donor. The summarized results support the view that T. cruzi epimastigotes lack an adequate enzyme defence against H2O2 and H2O2-related free radicals.     

Hepatocyte growth factor improves viability after H2O2-induced toxicity in bile duct epithelial cells

Intracellular defence mechanisms against oxidative stress may play an important role in the progression of liver diseases, including cholangiopathies. The multifunctional anti-apoptotic hepatocyte growth factor (HGF) has been suggested to have antioxidant functions. The effect of HGF upon cell viability, the generation of ROS, the expression of genes that play a role in ROS defence, and the activation of caspase-3 were measured in bile duct epithelial (BDE) cells in the presence or absence of H(2)O(2). HGF reduced H(2)O(2)-induced loss of viability, diminished H(2)O(2)-mediated ROS generation and abrogated H(2)O(2)-triggered changes in GSH/GSSG ratio. Furthermore, HGF increased the gene-expression of gamma-glutamylcysteine synthetase (GCLC) and glutathione reductase (GSR), while no effect was seen upon the gene-expression of superoxide dismutase 1 (SOD1), catalase (CAT), glutathione peroxidase (GPX1), and glutathione synthetase (GSR). Finally, HGF diminished the proteolytical activation of the key mediator of apoptosis (caspase-3) after H(2)O(2) exposure. Together, HGF may improve viability in bile duct epithelia cells after H(2)O(2) induced toxicity by proliferation, strengthening the intrinsic antioxidant defences, and/or by an attenuation of apoptosis. These in vitro results support the evaluation of HGF as antioxidative factor in hepatobiliary pathologies.     

Hydroperoxide metabolism in cyanobacteria

The enzymes involved in antioxidative activity and the cellular content of the antioxidants glutathione and ascorbate in the cyanobacteria Nostoc muscorum 7119 and Synechococcus 6311 have been examined for their roles in hydroperoxide removal. High activities of ascorbate peroxidase and catalase were found in vegetative cells of both species and in the heterocysts of N. muscorum. The affinity of ascorbate peroxidase for H2O2 was 15- to 25-fold higher than that of catalase. Increased activity of ascorbate peroxidase was observed in N. muscorum when H2O2 production was enhanced by photorespiration. Catalase activity was decreased in dilute cultures whereas ascorbate peroxidase activity increased. Ascorbate peroxidase activity also increased when the CO2 concentration was reduced. Ascorbate peroxidase appears to be a key enzyme in a cascade of reactions regenerating antioxidants. Dehydroascorbate reductase was found to regenerate ascorbate, and glutathione reductase recycled glutathione. In vegetative cells glutathione was present in high amounts (2-4 mM) whereas the ascorbate content was almost 100-fold lower (20-100 microM). Glutathione peroxidase was not detected in either cyanobacterium. It is concluded from the high activity of ascorbate peroxidase activity and the levels of antioxidants found that this enzyme can effectively remove low concentrations of peroxides. Catalase may remove H2O2 produced under photooxidative conditions where the peroxide concentration is higher.     

A new strategy to enhance glutathione production by multiple H2O2 induced oxidative stresses in Candida utilis

Effect of H(2)O(2)-induced oxidative stress on glutathione (GSH) production in Candida utilis was investigated. Based on the results that H(2)O(2) can effectively stimulate GSH accumulation but inhibit cell growth simultaneously, a novel strategy of multiple H(2)O(2) stresses with different concentrations (1 mmol/L at 4h, 2 mmol/L at 8h, and 4 mmol/L at 12h) were developed to maximize GSH production. As a result, a maximal GSH yield of 218 mg/L was achieved and a corresponding intracellular GSH content was 2.15%, which were 54.6% and 58.1% higher than the control. By further applying this strategy to 7 L fermentor, GSH yield and intracellular GSH content were 328 mg/L and 2.30%. Moreover, increased activities of catalase (CAT) and GSH reductase (GR) indicated that GSH and CAT were directly involved in protecting cell against oxidative stress by H(2)O(2).     

Glutathione system adaptation to acute stress in the heart of rats during different regimes of hypoxia training

The intensity of lipid peroxidation (LPO), reduced and oxidized glutathione (GSH and GSSG) contents, glutathione reductase, glutathione peroxidase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase (G-6-PDH), and NADP-isocitrate dehydrogenase (NADP-IDH) activities were studied in the heart of male rats exposed to two modes of intermittent hypoxic training (IHT): I-breathing in normobaric chamber with 7% O2 gas mixture for 5 min with 15 min normoxic intervals 4 times daily during 3 weeks; II-breathing by 12% O2 gas mixture in the same manner). After adaptation to hypoxia, the rats were subjected to 6h-immobilization stress. It has been shown that stress action after IHT (regime I) caused the increase in LPO and the shift of GSH/GSSG to disulfides. A disbalance in antioxidative defense system was determined by the decrease in glutatione peroxidase, G-6-PDH activities, and GSH content. The support of glutathione reductase activity under stress in this group with simultaneous decrease of enzyme activity in the pentose phosphate pathway was realized through the participation of NADP-IDH. Hypoxic training in regime II induced LPO decrease in the heart tissue after stress. The increase in the heart GSH content, optimal balance of glutathione-related enzymes in this group evidences for the dependence of adaptation effects on the vigor of hypoxic exposition. Our results suggest the active participation of glutathione system in the formation of adaptation reactions under the extreme factor influences through the action on intracellular red/ox potential as well as effectiveness of antioxidant defense.     

Influence of estrogen on glutathione levels and glutathione-metabolizing enzymes in uteri and R3230AC mammary tumors of rats

The glutathione content and the activities of several enzymes in its metabolism, glutathione reductase, glutathione peroxidase and γ-glutamyl transpeptidase, were assayed in uteri obtained from estrogen-treated rats and in R3230AC mammary adenocarcinomas obtained from ovariectomized, intact and estrogen-treated hosts. Normal mammary glands, obtained 10–12 days post-partum, were also examined for these parameters.A daily pharmacological dose of 0.4 μg of estradiol-17β induced a maximal increase in uterine weight and in reduced glutathione (GSH); higher doses of estrogen did not significantly increase either of these parameters. Levels of oxidized glutathione (GSSG) were comparable in both estrogen-treated and untreated rats. The time course of the estrogen-induced uterotrophic response was associated with increases in glutathione reductase, glutathione peroxidase and γ-glutamyl transpeptidase activities with the increased GSH level preceding the increase in uterine weight. Compared to neoplasms from intact or ovariectomized animals, tumors from estrogen-treated hosts exhibited significant decreases in levels of GSSG and GSH, as well as in glutathione reductase and glutathione peroxidase activities, but demonstrated a significant elevation of γ-glutamyl transpeptidase activity. Normal glands from lactating rats had decreased GSH levels, lower activities of glutathione reductase and glutathione peroxidase, but elevated γ-glutamyl transpeptidase activity versus tumors from intact rats. Tumors from estrogen-treated rats more closely resembled mammary glands during lactation. The divergent growth responses elicited by estrogen in the uterus and mammary tumor are correlated with the observed changes in GSH levels and enzymes involved in glutathione metabolism.     

夜间低温胁迫对番茄叶片活性氧代谢及AsA-GSH循环的影响

以番茄品种‘辽园多丽’为试材,利用人工气候室模拟设施生产中的夜间低温胁迫环境,研究9℃和6℃夜低温对番茄叶片活性氧代谢和AsA-GSH循环的影响。结果显示:9℃和6℃夜间低温胁迫3～9d可诱导番茄叶片中超氧阴离子(O2.-)产生速率、过氧化氢(H2O2)和丙二醛(MDA)含量上升;抑制过氧化物酶(POD)、过氧化氢酶(CAT)的活性,增加超氧化物歧化酶(SOD)和AsA-GSH循环中抗坏血酸过氧化物酶(APX)、脱氢抗坏血酸还原酶(DHAR)、谷胱甘肽还原酶(GR)的活性,并提高还原型抗坏血酸(AsA)、还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)的含量。研究表明,在夜间低温胁迫过程中,增加的番茄叶片中SOD活性和AsA-GSH循环清除活性氧的能力并未与氧还原的速率一致,从而导致番茄叶片中活性氧的累积,使细胞膜系统受到一定破坏,在6℃处理的植物中尤为明显。     

Tumoricidal activity of endothelial cells. Inhibition of endothelial nitric oxide production abrogates tumor cytotoxicity induced by hepatic sinusoidal endothelium in response to B16 melanoma adhesion in vitro

The mechanism of NO- and H(2)O(2)-induced tumor cytotoxicity was examined during B16 melanoma (B16M) adhesion to the hepatic sinusoidal endothelium (HSE) in vitro. We used endothelial nitric-oxide synthetase gene disruption and N(G)-nitro-l-arginine methyl ester-induced inhibition of nitric-oxide synthetase activity to study the effect of HSE-derived NO on B16M cell viability. Extracellular H(2)O(2) was removed by exogenous catalase. H(2)O(2) was not cytotoxic in the absence of NO. However, NO-induced tumor cytotoxicity was increased by H(2)O(2) due to the formation of potent oxidants, likely ( small middle dot)OH and (-)OONO radicals, via a trace metal-dependent process. B16M cells cultured to low density (LD cells), with high GSH content, were more resistant to NO and H(2)O(2) than B16M cells cultured to high density (HD cells; with approximately 25% of the GSH content found in LD cells). Resistance of LD cells decreased using buthionine sulfoximine, a specific GSH synthesis inhibitor, whereas resistance increased in HD cells using GSH ester, which delivers free intracellular GSH. Because NO and H(2)O(2) were particularly cytotoxic in HD cells, we investigated the enzyme activities that degrade H(2)O(2). NO and H(2)O(2) caused an approximately 75% (LD cells) and a 60% (HD cells) decrease in catalase activity without affecting the GSH peroxidase/GSH reductase system. Therefore, B16M resistance to the HSE-induced cytotoxicity appears highly dependent on GSH and GSH peroxidase, which are both required to eliminate H(2)O(2). In agreement with this fact, ebselen, a GSH peroxidase mimic, abrogated the increase in NO toxicity induced by H(2)O(2).     

Dissecting the superoxide dismutase-ascorbate-glutathione-pathway in chloroplasts by metabolic modeling. Computer simulations as a step towards flux analysis

The present study introduces metabolic modeling as a new tool to analyze the network of redox reactions composing the superoxide dismutase-ascorbate (Asc)-glutathione (GSH) cycle. Based on previously determined concentrations of antioxidants and defense enzymes in chloroplasts, kinetic properties of antioxidative enzymes, and nonenzymatic rate constants of antioxidants with reactive oxygen, models were constructed to simulate oxidative stress and calculate changes in concentrations and fluxes of oxidants and antioxidants. Simulated oxidative stress in chloroplasts did not result in a significant accumulation of O2*- and H2O2 when the supply with reductant was sufficient. Model results suggest that the coupling between Asc- and GSH-related redox systems was weak because monodehydroascorbate radical reductase prevented dehydroascorbate (DHA) formation efficiently. DHA reductase activity was dispensable. Glutathione reductase was mainly required for the recycling of GSH oxidized in nonenzymatic reactions. In the absence of monodehydroascorbate radical reductase and DHA reductase, glutathione reductase and GSH were capable to maintain the Asc pool more than 99% reduced. This suggests that measured DHA/Asc ratios do not reflect a redox balance related to the Asc-GSH-cycle. Decreases in Asc peroxidase resulted in marked H2O2 accumulation without significant effects on the redox balance of Asc/DHA or GSH/GSSG. Simulated loss of SOD resulted in higher H2O2 production rates, thereby affecting all subsequent steps of the Asc-GSH-cycle. In conclusion, modeling approaches contribute to the theoretical understanding of the functioning of antioxidant systems by pointing out questions that need to be validated and provide additional information that is useful to develop breeding strategies for higher stress resistance in plants.     

Effects of Exogenous Spermidine on Antioxidant System Responses of Typha latifolia L. Under Cd^2＋ Stress

The effects of foliar spraying with spermidine (Spd), ranging in concentration from 0.25 to 0.50 mmol/L, on the antioxidant system under Cd^2 stress (range 0.1- 0.2 mmol/L Cd^2 ) in Typha latifolia L. grown hydroponically were investigated in order to offer a referenced evidence for an understanding of the mechanism by which polyamines (PAs) relieve the damage to plants by heavy metal and improve the phytoremediation efficiency of heavy metal-contaminated water. The results showed that Cd^2 stress induced oxidative injury, as evidenced by an increase in the generation of superoxide anion (O2), as well as the hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents in both leaves and caudices. With the exception of superoxide dismutase (SOD) activity in the leaves, an increase in the activities of catalase (CAT), guaiacol peroxidase (GPX), and glutathione reductase (GR) was observed in both leaves and caudices, SOD activity was increased in caudices, and ascorbate peroxidase (APX) activity was increased in leaves following Cd^2 treatment. The reduced glutathione (GSH) content in both leaves and caudices and the reductive ascorbate content in leaves was obviously increased, which were prompted by the application of exogenous Spd. Spraying with Spd increased the activity of GR and APX in both leaves and caudices, whereas the activity of SOD, CAT, and GPX was increased only in caudices following spraying with Spd. The generation of O2 and the H2O2 and MDA content in both leaves and caudices decreased after spraying with Spd. The decrease in MDA was more obvious following the application of 0.25 than 0.50 mmol/L Spd. It is supposed that exogenous Spd elevated the tolerance of T. latifolia under Cd^2 stress primarily by increasing GR activity and the GSH level.     

Hypoxia increases glutathione redox cycle and protects rat lungs against oxidants

Preexposure to hypoxia increased survival and lung reduced glutathione-to-oxidized glutathione ratios (GSH/GSSG) and decreased pleural effusions in rats subsequently exposed to continuous hyperoxia. In addition, lungs from hypoxia-preexposed rats developed less acute edematous injury (decreased lung weight gains and lung lavage albumin concentrations) than lungs from normoxia-preexposed rats when isolated and perfused with hydrogen peroxide (H2O2) generated by xanthine oxidase (XO) or glucose oxidase (GO). In contrast, when perfused with elastase or exposed to a hydrostatic left atrial pressure challenge, lungs isolated from hypoxia-preexposed rats developed the same acute edematous injury as lungs from normoxia-preexposed rats. The mechanism by which hypoxia preexposure conferred protection against H2O2 appeared to depend on hexose monophosphate shunt (HMPS)-dependent increases in lung glutathione redox cycle activity. First, before perfusion with GO, lungs from hypoxia-preexposed rats had increased glutathione peroxidase and glucose 6-phosphate dehydrogenase (but not catalase or glutathione reductase) activities compared with lungs from normoxia-preexposed rats. Second, after perfusion with GO, lungs from hypoxia-preexposed rats had increased H2O2 reducing equivalents, as reflected by increased GSH/GSSG and NADPH/NADPH+, compared with lungs from normoxia-preexposed rats. Third, pretreatment of rats with an HMPS inhibitor, (6-aminonicotinamide) or a glutathione reductase inhibitor, [1,3-bis(2-chloroethyl)-1-nitrosourea] prevented hypoxia-conferred protection against H2O2-mediated acute edematous injury in isolated lungs. These findings suggest that increased detoxification of H2O2 by glutathione redox cycle and HMPS-dependent mechanisms contributes to tolerance to hyperoxia and resistance to H2O2 of lungs from hypoxia-preexposed rats.     

Subcellular localization and modification with ageing of glutathione, glutathione peroxidase and glutathione reductase activities in human fibroblasts

Differential centrifugation and isopycnic equilibration in density gradients were used to localize glutathione (GSH), glutathione peroxidase and glutathione reductase in the subcellular organelles of WI-38 fibroblasts. GSH was present in all the subcellular fractions, whereas the glutathione peroxidase and reductase activities were restrained to the cytoplasm and the mitochondrial fractions. After equilibration in density gradients, the results showed the presence of GSH, glutathione peroxidase and glutathione reductase in both the cytoplasm and mitochondria. GSH was also located in plasma membranes and probably in peroxisomes, endoplasmic reticulum and lysosomal membranes. Evolution of GSH in ageing fibroblasts showed a sudden increase of its concentration just before cell death. The glutathione peroxidase activity already decreases in the early passages, while the decrease of the glutathione reductase activity was constant and reached a drastic low level at the end of the culture. In conclusion, GSH is probably involved in the cell degeneration associated with ageing but because of its multiple functions and its ubiquitous localization, it is difficult to assert to which extent this metabolite is implicated in the ageing process.     

Effects of resveratrol and 4-hexylresorcinol on hydrogen peroxide-induced oxidative DNA damage in human lymphocytes

The protective effects of resveratrol and 4-hexylresorcinol against oxidative DNA damage in human lymphocytes induced by hydrogen peroxide were investigated. Resveratrol and 4-hexylresorcinol showed no cytotoxicity to human lymphocytes at the tested concentration (10-100 μM). In addition, DNA damage in human lymphocytes induced by H 2 O 2 was inhibited by resveratrol and 4-hexylresorcinol. Resveratrol and 4-hexylresorcinol at concentrations of 10-100 μM induced an increase in glutathione (GSH) levels in a concentration-dependent manner. Moreover, these two compounds also induced activity of glutathione peroxidase (GPX) and glutathione reductase (GR). The activity of glutathione-S-transferase (GST) in human lymphocytes was induced by resveratrol. Resveratrol and 4-hexylresorcinol inhibited the activity of catalase (CAT). These data indicate that the inhibition of resveratrol and 4-hexylresorcinol on oxidative DNA damage in human lymphocytes induced by H 2 O 2 might be attributed to increase levels of GSH and modulation of antioxidant enzymes (GPX, GR and GST).     

Effects of Exogenous Spermidine on Antioxidant System Responses of Typha latifolia L. Under Cd2+ Stress

The effects of foliar spraying with spermidine (Spd), ranging in concentration from 0.25 to 0.50mmol/L, on the antioxidant system under Cd2 stress (range 0.1- 0.2 mmol/L Cd2 ) in Typha latifolia L.grown hydroponically were investigated in order to offer a referenced evidence for an understanding of the mechanism by which polyamines (PAs) relieve the damage to plants by heavy metal and improve the phytoremediation efficiency of heavy metal-contaminated water. The results showed that Cd2 stress inhydrogen peroxide (H2O2) and malondialdehyde (MDA) contents in both leaves and caudices. With theexception of superoxide dismutase (SOD) activity in the leaves, an increase in the activities of catalase (CAT), guaiacol peroxidase (GPX), and glutathione reductase (GR) was observed in both leaves and caudices,SOD activity was increased in caudices, and ascorbate peroxidase (APX) activity was increased in leaves following Cd2 treatment. The reduced glutathione (GSH) content in both leaves and caudices and the reductive ascorbate content in leaves was obviously increased, which were prompted by the application of exogenous Spd. Spraying with Spd increased the activity of GR and APX in both leaves and caudices,whereas the activity of SOD, CAT, and GPX was increased only in caudices following spraying with Spd.with Spd. The decrease in MDA was more obvious following the application of 0.25 than 0.50 mmol/L Spd.It is supposed that exogenous Spd elevated the tolerance of T. latifolia under Cd2 stress primarily by increasing GR activity and the GSH level.     

Changes in GSH-antioxidant system induced by daunorubicin in human normal and diabetic fibroblasts

We investigated the effect of daunorubicin on glutathione content and activity of GSH-related enzymes in cultured normal and diabetic human fibroblasts. Cells were incubated with 4 microM daunorubicin (DNR) for 2 h followed by culture in drug-free medium for up to 72 h. Treatment of diabetic cells with the drug caused a time-dependent depletion of intracellular GSH and a decrease of the GSH to total glutathione ratio. GSH depletion was accompanied by apoptotic changes in morphology of the nucleus. Analysis of GSH-related enzymes showed a significant increase of the activities of Se-dependent and Se-independent peroxidases and glutathione S-transferase. In contrast, glutathione reductase activity was reduced by 50%. Significant differences between normal and diabetic cells exposed to DNR were observed in the level of GST and Se-dependent glutathione peroxidase activities. These findings indicated that daunorubicin efficiently affects the GSH antioxidant defense system both in normal and diabetic fibroblasts leading to disturbances in glutathione content as well as in the activity of GSH-related enzymes.     

Differential Implication of Glutathione, Glutathione-Metabolizing Enzymes and Ascorbate in Tomato Resistance to Pseudomonas syringae

We studied the response of glutathione‐ and ascorbate‐related antioxidant systems of the two tomato cultivars to Pseudomonas syringae pv. tomato infection. In the inoculated susceptible A 100 cultivar a substantial decrease in reduced glutathione (GSH) content, oxidised glutathione accumulation and GSH redox ratio decline as well as glutathione peroxidase activity increase were found. The enhanced glutathione reductase activity was insufficient to keep the glutathione pool reduced. A transiently increased dehydroascorbic acid (DHA) content and ascorbic acid (AA) redox ratio decrease together with ascorbate peroxidase activity suppression were observed. Adversely to the progressive reduction in GSH pool size, AA content tended to increase but the changes were more modest than those of GSH. By contrast, in interaction with the resistant Ontario cultivar the glutathione pool homeostasis was maintained throughout P. syringae attack and no significant effect on the ascorbate pool was observed. Moreover, in the resistant interaction there was a significantly higher constitutive and pathogen‐induced glutathione‐S‐transferase (GST) activity. The relationship between GST activity and DHA content found in this study indicates that this enzyme could also act as dehydroascorbate reductase. These results reflect the differential involvement of GSH and AA in tomato‐P. syringae interaction and, in favour of the former, they clearly indicate the role of GSH and GSH‐utilizing enzymes in resistance to P. syringae. The maintenance of glutathione pool homeostasis and GST induction appear to contribute to tissue inaccessibility to bacterial attack.     

Glutathione peroxidase, glutathione reductase, glutathione S-transferase, and gamma-glutamyltranspeptidase activities in the human early pregnancy placenta

GSH peroxidase, GSSG reductase, GSH S-transferase, and gamma-glutamyltranspeptidase activities were measured in the supernatant of 13 human early pregnancy placenta homogenates. From measurements of GSH peroxidase activity with both H2O2 and cumene hydroperoxide as second substrate it was deduced that immature placenta contains only the Se-dependent form. All the specimens investigated exhibited GSSG reductase and gamma-glutamyltranspeptidase activities. GSH S-transferase activity was noted only using 1-chloro-2,4-dinitrobenzene as electrophilic substrate, while no detectable activity was found with 1,2-dichloro-4-nitrobenzene, 1,2-epoxy-3-(p-nitrophenoxy) propane, and p-nitrobenzylchloride. It is concluded that human placenta is equipped, from early pregnancy, with the enzymatic systems which are involved in GSH-mediated cellular detoxication and in preserving the integrity of the sulfhydryl status of the cells.