Glutathione-related Enzyme Activities Research Articles

Oxidative effects of the human antifungal drug clotrimazole on the eucaryotic model organism Saccharomyces cerevisiae.

Clotrimazole is a type of antifungal medication developed from azole compounds. It exhibits several biological actions linked to oxidative stress. This study focuses on the oxidative effects of clotrimazole on the eukaryotic model yeast, Saccharomyces cerevisiae. Our results showed that although initial nitric oxide levels were above control in clotrimazole exposed cells, they showed decreasing tendencies from the beginning of incubation and dropped below control at 125 µM from the 60th min. The highest superoxide anion and hydrogen peroxide levels were 1.95- and 2.85-folds of controls at 125 µM after 15 and 60min, respectively. Hydroxyl radical levels slightly increased throughout the incubation period in all concentrations and reached 1.3-fold of control, similarly at 110 and 125 µM in the 90th min. The highest level of reactive oxygen species was observed at 110 µM, 2.31-fold of control. Although NADH/NADPH oxidase activities showed similar tendencies for all conditions, the highest activities were found as 3.07- and 2.27-folds of control at 125 and 110 µM in the 15th and 30th min, respectively. The highest superoxide dismutase and catalase activities were 1.59- and 1.21-folds of controls at 110 µM clotrimazole in 30 and 90min, respectively. While the drug generally induced glutathione-related enzyme activities, the ratios of glutathione to oxidized glutathione were above the control only at low concentrations of the drug. The levels of lipid peroxidation in all treated cells were significantly higher than the controls. The findings crucially demonstrate that this medicine can generate serious oxidative stress in organisms.

Lemon Flavonoid Extract Eriomin Improves Pro/Antioxidant Status and Interferes with Cholesterol Metabolism without Affecting Serum Cholesterol Levels in Aged Rats.

This study aimed to assess the antioxidant capacity of lemon flavonoid extract Eriomin® (LE) and its impact on cholesterol metabolism in the context of healthy aging. We orally treated 24-month-old male Wistar rats with an LE (40 mg/kg) suspended in 0.3 mL of sunflower oil. At the same time, control groups received an equal volume of sunflower oil (CON) or remained untreated (ICON) daily for 4 weeks. We examined LE's effects on superoxide dismutase and catalase- and glutathione-related enzyme activities, the concentration of lipid peroxides and protein carbonyls, total oxidant status (TOS) and antioxidant status (TAS), and oxidative stress index (OSI) in the liver, jejunum, and ileum. We also measured total cholesterol, its biosynthetic precursors (lanosterol, lathosterol, desmosterol), its degradation products (bile acid precursors) in the serum, liver, jejunum, and ileum, and serum phytosterols (intestinal absorption markers). LE reduced TOS, TAS, and OSI (p < 0.05) compared with control values, indicating its consistent antioxidant action in all examined organs. LE lowered hepatic desmosterol (p < 0.05) while also reducing 7α- and 24-hydroxycholesterol levels in the liver and ileum (p < 0.01). Serum cholesterol, hepatic gene expression, and the immunostaining intensity of CYP7A1 were unchanged. In conclusion, LE exerted non-enzymatic antioxidant effects and reduced cholesterol degradation, reducing its biosynthesis products, thereby maintaining serum cholesterol levels.

Differences in Cholesterol Metabolism, Hepato-Intestinal Aging, and Hepatic Endocrine Milieu in Rats as Affected by the Sex and Age.

Age and sex influence serum cholesterol levels, but the underlying mechanisms remain unclear. To investigate further, we measured cholesterol, precursors (surrogate synthesis markers), degradation products (oxysterols and bile acid precursors) in serum, the liver, jejunum, and ileum, as well as serum plant sterols (intestinal absorption markers) in male and female Wistar rats (4 and 24 months old). The analysis of histomorphometric and oxidative stress parameters (superoxide dismutase, catalase, glutathione-related enzyme activities, lipid peroxide, and protein carbonyl concentrations) in the liver and jejunum offered further insights into the age- and sex-related differences. The hepatic gene expression analysis included AR, ERα, and sex-specific growth hormone-regulated (Cyp2c11 and Cyp2c12) and thyroid-responsive (Dio1, Tbg, and Spot 14) genes by qPCR. We observed age-related changes in both sexes, with greater prominence in females. Aged females had significantly higher serum cholesterol (p < 0.05), jejunum cholesterol (p < 0.05), and serum plant sterols (p < 0.05). They exhibited poorer hepato-intestinal health compared with males, which was characterized by mild liver dysfunction (hydropic degeneration, increased serum ALT, p < 0.05, and decreased activity of some antioxidant defense enzymes, p < 0.05), mononuclear inflammation in the jejunal lamina propria, and age-related decreases in jejunal catalase and glutathione peroxidase activity (p < 0.05). Aged females showed increased levels of 27-hydroxycholesterol (p < 0.05) and upregulated ERα gene expression (p < 0.05) in the liver. Our study suggests that the more significant age-related increase in serum cholesterol in females is associated with poorer hepato-intestinal health and increased jejunal cholesterol absorption. The local increase in 27-hydroxycholesterol during aging might reduce the hepatoprotective effects of endogenous estrogen in the female liver.

An Insight into the Impact of Serum Tellurium, Thallium, Osmium and Antimony on the Antioxidant/Redox Status of PCOS Patients: A Comprehensive Study.

Humans exploit heavy metals for various industrial and economic reasons. Although some heavy metals are essential for normal physiology, others such as Tellurium (Te), Thallium (TI), antimony (Sb), and Osmium (Os) are highly toxic and can lead to Polycystic Ovarian Syndrome (PCOS), a common female factor of infertility. The current study was undertaken to determine levels of the heavy metals TI, Te, Sb and Os in serum of PCOS females (n = 50) compared to healthy non-PCOS controls (n = 56), and to relate such levels with Total Antioxidant Capacity (TAC), activity of key antioxidant enzymes, oxidative stress marker levels and redox status. PCOS serum samples demonstrated significantly higher levels of TI, Te, Sb and Os and diminished TAC compared to control (p < 0.001). Furthermore, there was significant inhibition of SOD, CAT and several glutathione-related enzyme activities in sera of PCOS patients with concurrent elevations in superoxide anions, hydrogen and lipid peroxides, and protein carbonyls, along with disrupted glutathione homeostasis compared to those of controls (p < 0.001 for all parameters). Additionally, a significant negative correlation was found between the elevated levels of heavy metals and TAC, indicative of the role of metal-induced oxidative stress as a prominent phenomenon associated with the pathophysiology of the underlying PCOS. Data obtained in the study suggest toxic metals as risk factors causing PCOS, and thus protective measures should be considered to minimize exposure to prevent such reproductive anomalies.

Pretreatment of seeds with hydrogen peroxide improves deep-sowing tolerance of wheat seedlings

Drought is a prevalent natural factor limiting crop production in arid regions across the world. To overcome this limitation, seeds are sown much deeper to boost germination by soil moisture produced by underground water. Seed pretreatment can effectively induce deep-sowing tolerance in plants. In the present study, we evaluated whether H2O2 pretreatment of seeds can initiate metabolic changes and lead to improved deep-sowing tolerance in wheat. Pretreatment with 0.05 μM H2O2 promoted first internode elongation by 13% in the deep-sowing tolerant wheat cultivar “Tir” and by 32% in the sensitive cultivar “Kıraç-66” under deep-sowing conditions, whereas internode elongation was inhibited by diphenyleneiodonium chloride. In contrast to Tir seedlings, H2O2 levels in the first internode of Kıraç-66 seedlings increased under deep-sowing condition in the H2O2-treated group compared to controls. Moreover, these seedlings had significantly lower catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX) activities but higher NADPH oxidase (NOX) and superoxide dismutase (SOD) activities under the same conditions, which consequently induced greater H2O2 accumulation. Contrary to Tir, both total glutathione and glutathione S-transferase (GST) activity decreased in Kıraç-66 after deep-sowing at 10 cm. However, H2O2 treatment increased the total glutathione amounts and the activities of glutathione-related enzymes (except GST and GPX) in the first internode of Kıraç-66. Taken together, these data support that H2O2 acts as a signaling molecule in the activation of antioxidant enzymes (specifically NOX, SOD, and CAT), regulation of both glutathione-related enzymes and total glutathione content, and upregulation of the cell wall-loosening protein gene TaEXPB23.

Glutathione and oxidized nicotinamide adenine dinucleotide (NAD+) redox status in plasma and placental tissue of Saudi patients with intrauterine growth restriction.

Objectives:To assess the oxidation state and gene expression profiles of relevant enzymes in intrauterine growth restriction (IUGR) patients in Saudi Arabia.Methods:Current case-control study involved plasma and placental tissue samples from 25 IUGR patients and 25 healthy pregnant (HP) women attending the Obstetrics and Gynecology Clinic, King Khalid University Hospital, Riyadh, Saudi Arabia, between April and November 2017. We compared hydrogen peroxide, superoxide anions, malondialdehyde, and oxidative stress markers levels and the activities of glutathione-related enzymes (glutathione peroxidase [GPx], glutathione reductase [GR], glutathione S-transferase [GST], glutamate cysteine ligase [GCL], glutathione synthetase [GS], reduced glutathione [GSH], oxidized glutathione [GSSG], and oxidized nicotinamide adenine dinucleotide [NAD+], and reduced NAD [NADH]) between the 2 groups. We also compared differential expression levels of glutathione-related enzyme genes using reverse transcription-quantitative polymerase chain reaction.Results:Oxidative stress markers significantly differed in IUGR samples, while GSH levels and GPx, GR, GST, GCL, and GS activities and their placental mRNA transcriptional levels were significantly lower. Plasma and placental NAD+ levels were also significantly lower, while NADH levels were significantly higher, causing lowered NAD+-NADH ratios in the IUGR group compared to control.Conclusions:Intrauterine growth restriction patients show a metabolic shift in favor of oxidation compared to HP women.

Protective Effects of Antioxidant Chlorophyllin in Chemically Induced Breast Cancer Model In vivo.

Glutathione-related enzymes belong to the protection mechanism of the cells against harmful oxidative damage and chemicals. Glutathione S-transferase (GST) is frequently over-expressed in various cancer cells and is involved in drug resistance. Chlorophyllin is an antioxidant molecule interfering with the GST P1-1 activity. The purpose of this study is to evaluate the short- and long-term protective effects of chlorophyllin as an antioxidant molecule on DNA damage, antioxidant enzyme activities, trace elements, and minerals in chemically induced breast cancer model in vivo. In our study, N-methyl-N-nitrosourea (MNU) was used for inducing breast carcinogenesis in female Sprague-Dawley rats. A total of 36 rats were divided into groups as short term and long term. Each group was divided into four sub-groups as control group received physiological saline solution (n= 3), Chl group (n= 5) received chlorophyllin, MNU group (n= 5) was administered MNU, and Chl + MNU group (n= 5) was treated with both chlorophyllin and MNU. Results illustrated that chlorophyllin had a significant anti-genotoxic effect in the short term, and glutathione-related enzyme activities were protected by chlorophyllin treatment in MNU-induced breast cancer model. Additionally, MNU administration impaired mineral and trace element levels including Na, Mg, K, Fe, Zn, and Co in the liver, kidney, spleen, heart, and tumor tissues; however, adverse effects of MNU were recovered upon chlorophyllin treatment in the indicated tissues of the rats. In conclusion, chlorophyllin can be used as an antioxidant molecule to ameliorate adverse effects of MNU by enhancing antioxidant enzyme activities and regulating trace element and mineral balance in several organs and tumor tissue in the breast cancer model.

Effect of acute ammonia exposure on the glutathione redox system in FFRC strain common carp (Cyprinus carpio L.)

Ammonia is one of the most common aquatic pollutants. To analyze the effect of ammonia exposure on the glutathione redox system, we investigated the levels of hydrogen peroxide (H2O2) and glutathione, and transcription and activities of glutathione-related enzymes in liver and gills of FFRC strain common carp (Cyprinus carpio L.) exposed to 0, 10, 20, and 30 mg/L of ammonia. The results showed that H2O2 content reached a maximum level at 48 h of exposure in the liver of fish. In gills, H2O2 increased rapidly at 6 h and reached to maximum levels at 24 h of exposure, indicating that gills experienced oxidative stress earlier than the liver of fish exposed to ammonia. Reduced glutathione (GSH) content and reduced glutathione/oxidized glutathione (GSH/GSSG) ratio increased significantly within 24 h of exposure. Meanwhile, the transcription and activities of glutathione S-transferase (GST) and glutathione reductase (GR) increased significantly in the liver, and glutathione peroxidase (GSH-Px) and GST increased in the gills of fish exposed to ammonia. Malondialdehyde (MDA) content kept at a low level after exposure to low concentration of ammonia, but increased significantly after exposure to 30 mg/L ammonia for 48 h along with a decrease in GSH content and GSH/GSSG ratio. These data showed that the glutathione redox system played an important role in protection against ammonia-induced oxidative stress in the liver and gills of FFRC strain common carp, though the defense capacity was not able to completely prevent oxidative damage occurring after exposure to higher concentration of ammonia. This research systematically studied the response of the glutathione redox system to ammonia stress and would provide novel information for a better understanding of the adaptive mechanisms of fish to environmental stress.

Glutathione levels and activities of glutathione metabolism enzymes in patients with schizophrenia: A systematic review and meta-analysis.

Glutathione is among the important antioxidants to prevent oxidative stress. However, the relationships between abnormality in the glutathione system and pathophysiology of schizophrenia remain uncertain due to inconsistent findings on glutathione levels and/or glutathione-related enzyme activities in patients with schizophrenia. A systematic literature search was conducted using Embase, Medline, PsycINFO, and PubMed. Original studies, in which three metabolite levels (glutathione, glutathione disulfide, and total glutathione (glutathione+glutathione disulfide)) and five enzyme activities (glutathione peroxidase, glutathione reductase, glutamate-cysteine ligase, glutathione synthetase, and glutathione S-transferase) were measured with any techniques in both patients with schizophrenia and healthy controls, were included. Standardized mean differences were calculated to determine the group differences in the glutathione levels with a random-effects model. We identified 41, 9, 15, 38, and seven studies which examined glutathione, glutathione disulfide, total glutathione, glutathione peroxidase, and glutathione reductase, respectively. Patients with schizophrenia had lower levels of both glutathione and total glutathione and decreased activity of glutathione peroxidase compared to controls. Glutathione levels were lower in unmedicated patients with schizophrenia than those in controls while glutathione levels did not differ between patients with first-episode psychosis and controls. Our findings suggested that there may be glutathione deficits and abnormalities in the glutathione redox cycle in patients with schizophrenia. However, given the small number of studies examined the entire glutathione system, further studies are needed to elucidate a better understanding of disrupted glutathione function in schizophrenia, which may pave the way for the development of novel therapeutic strategies in this disorder.

Protective role of l-glutamic acid and l-cysteine in mitigation the chlorpyrifos-induced oxidative stress in rats

Exposure to the organophosphorus insecticide chlorpyrifos (CPF) can lead to oxidative stress. The aim of this work was to investigate and compare the protective effects of amino acids (l-glutamic acid (l-Glu) and l-cysteine (L-Cys) alone or in combination) for the purpose of suppression and mitigation of CPF-induced oxidative stress in rats. Rats were divided into five groups: CPF, CPF/L-Glu, CPF/L-Glu and l-Cys, CPF/L-Cys, control. The level of GSH and the activities of glutathione-related enzymes were determined. The content of lipid peroxidation products was also monitored. The obtained results suggest that level of GSH and activity of GSH-related enzymes was significantly inhibited by CPF. l-Glu and l-Cys were able to prevent CPF-induced oxidative stress. In rats treated with amino acids, we observed less significant or no changes in studied parameters. It was established that the above-mentioned amino acids, administered alone and in their combination, can mitigate and suppress CPF-induced oxidative stress. The most significant mitigation effect was found in rats treated with l-Glu only.

Acute exposure to copper induces variable intensity of oxidative stress in goldfish tissues

Copper is an essential element, but at high concentrations, it is toxic for living organisms. The present study investigated the responses of goldfish, Carassius auratus, to 96h exposure to 30, 300, or 700μgL-1 of copper II chloride (Cu2+). The content of protein carbonyls was higher in kidney (by 158%) after exposure to 700mgL-1 copper, whereas in gills, liver, and brain, we observed lower content of protein carbonyls after exposure to copper compared with control values. Exposure to copper resulted in increased levels of lipid peroxides in gills (76%) and liver (95-110%) after exposure to 300 and 700μgL-1 Cu2+. Low molecular mass thiols were depleted by 23-40% in liver and by 29-67% in kidney in response to copper treatment and can be used as biomarkers toxicity of copper. The activities of primary antioxidant enzymes, superoxide dismutase and catalase, were increased in liver as a result of Cu2+ exposure, whereas in kidney catalase activity was decreased. The activities of glutathione-related enzymes, glutathione peroxidase, glutathione-S-transferase, and glutathione reductase were decreased as a result of copper exposure, but glutathione reductase activity increased by 25-40% in liver. Taken together, these data show that exposure of fish to Cu2+ ions results in the development of low/high intensity oxidative stress reflected in enhanced activities of antioxidant and associated enzymes in different goldfish tissues.

Cytochrome c peroxidase regulates intracellular reactive oxygen species and methylglyoxal via enzyme activities of erythroascorbate peroxidase and glutathione-related enzymes in Candida albicans.

D-erythroascorbate peroxidase (EAPX1) deficiency causes glutathione deprivation, leading to the accumulation of methylglyoxal and reactive oxygen species (ROS), and especially, induction of cytochrome c peroxidase (Ccp1) in Candida albicans. Nevertheless, reciprocal effects between changes in Ccp1 activity and the antioxidative D-erythroascorbic acid- and glutathione-dependent redox status, which reflects methylglyoxal biosynthesis altering pathophysiology are unclear in eukaryotes. To elucidate the effect of CCP1 expression on EAPX1 and glutathione reductase (Glr1) activity-mediated D-erythroascorbic acid biosynthesis and redox homeostasis, the CCP1 gene was disrupted and overexpressed. First, we demonstrated both glutathione-independent and-dependent metabolite contents and their corresponding gene transcripts and enzyme activities (i.e., Ccp1, catalase-peroxidase [KatG], superoxide dismutase [Sod], Eapx1, and Glr1) in CCP1 mutants. Second, methylglyoxal-oxidizing alcohol dehydrogenase (Adh1) and methylglyoxal-reducing oxidoreductase activity on glycolytic methylglyoxal and pyruvate production and NAD(P)H content were determined in these mutants. Contrary to our expectation, CCP1 disruption (42.19±3.22nmolO2h-1mgwetcell-1) failed to affect cell respiration compared to the wild-type strain (41.62±7.11nmolO2h-1mgwetcell-1) under cyanide treatment, and in contrast to hydrogen peroxide (H2O2) treatment (21.74±1.03nmol O2h-1mgwetcell-1). Additionally, Ccp1 predominantly detoxified H2O2 rather than negligible scavenging activities towards methylglyoxal and other oxidants. CCP1 deficiency stimulated Sod and Adh1 activity but downregulated Glr1, Eapx1, catalase, and peroxidase activity while enhancing KatG, EAPX1, and GLR1 transcription by decreasing glutathione and D-erythroascorbic acid and increasing pyruvate. Noticeably, the ROS-accumulating CCP1-deficient mutant maintained steady-state levels of methylglyoxal, which was revealed to be regulated by methylglyoxal-oxidizing and -reducing activity with drastic changes in NAD(P)H. We confirmed and clarified our results by showing that CCP1/EAPX1 double disruptants underwent severe growth defects due to the D-erythroascorbic acid and glutathione depletion because of pyruvate overaccumulation. These observations were made in both budding and hyphal-growing CCP1 mutants. The revealed metabolic network involving Ccp1 and other redox regulators affected ROS and methylglyoxal through D-erythroascorbic acid and glutathione-dependent metabolites, thereby influencing dimorphism. This is the first report of the Ccp1-mediated D-erythroascorbic acid and glutathione biosynthesis accompanying methylglyoxal scavengers for full fungal virulence.

Alterations in epididymal proteomics and antioxidant activity of mice exposed to fluoride.

It is well known that high fluoride results in low fertility. Epididymis is the important place for spermatozoa maturation, which is essential for successful fertilization. In the previous studies, fluoride was reported to damage the epididymal structure of mouse and rabbit. However, the mechanism underlying sodium fluoride (NaF)-induced epididymal toxicity has not yet been well elucidated. The aim of this study is to explore the global protein alterations in epididymis of mice exposed to NaF using the iTRAQ technique. Results showed that 211 proteins were differentially expressed in both 25 and 100mg/L NaF groups. Some of them have been proved to be important for reproduction, such as low-density lipoprotein receptor-related protein 2 (Lrp2), cytochrome c, testis-specific (Cyct), sorbitol dehydrogenase (Sord), glutathione S-transferases (GSTs), acrosin, beta-defensin 126, cysteine-rich secretory protein (Crisp) 1, and Crisp2. Gene ontology (GO) analysis suggested cellular process, organelle and catalytic activity account for high percent and number of differentially expressed proteins. 171 pathways were found after the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, among which the representative maps, such as ribosome, focal adhesion, and phagosome, were involved. Different functional categories post-translational modification, protein turnover, chaperones; translation, ribosomal structure and biogenesis; cytoskeleton; energy production and conversion are implicated in the Cluster of Orthologous Groups (COG) of proteins analysis. Subsequently, the effect of NaF on the antioxidant activity in epididymis, especially glutathione and glutathione-related enzymes, was evaluated. Results exhibited high fluoride caused low total antioxidant capacity (T-AOC), high methane dicarboxylic aldehyde (MDA), decreased reduced glutathione (GSH), and the glutathione-related enzymes [GSH peroxidase (GPx), GSH reductase (GR), and GSH S-transferase (GST)] changes in activity, protein, and mRNA expressions. In summary, NaF decreased the antioxidant activity of epididymis, especially glutathione and glutathione-related enzymes, as well as iTRAQ results, providing new explanations for the low sperm quality induced by fluoride.

Endoplasmic reticulum stress regulates glutathione metabolism and activities of glutathione related enzymes in Arabidopsis.

Stress conditions generate an extra load on protein folding machinery in the endoplasmic reticulum (ER) and if the ER cannot overcome this load, unfolded proteins accumulate in the ER lumen, causing ER stress. ER lumen localised protein disulfide isomerase (PDI) catalyses the generation of disulfide bonds in conjugation with ER oxidoreductase1 (ERO1) during protein folding. Mismatched disulfide bonds are reduced by the conversion of GSH to GSSG. Under prolonged ER stress, GSH pool is oxidised and H2O2 is produced via increased activity of PDI-ERO1. However, it is not known how glutathione metabolism is regulated under ER stress in plants. So, in this study, ER stress was induced with tunicamycin (0.15, 0.3, 0.45μg mL-1 Tm) in Arabidopsis thaliana (L.) Heynh. Glutathione content was increased by ER stress, which was accompanied by induction of glutathione biosynthesis genes (GSH1, GSH2). Also, the apoplastic glutathione degradation pathway (GGT1) was induced. Further, the activities of glutathione reductase (GR), dehydroascorbate reductase (DHAR), glutathione peroxidase (GPX) and glutathione S-transferase (GST) were increased under ER stress. Results also showed that chloroplastic GPX genes were specifically downregulated with ER stress. This is the first report on regulation of glutathione metabolism and glutathione related enzymes in response to ER stress in plants.

GSR is not essential for the maintenance of antioxidant defenses in mouse cochlea: Possible role of the thioredoxin system as a functional backup for GSR.

Glutathione reductase (GSR), a key member of the glutathione antioxidant defense system, converts oxidized glutathione (GSSG) to reduced glutathione (GSH) and maintains the intracellular glutathione redox state to protect the cells from oxidative damage. Previous reports have shown that Gsr deficiency results in defects in host defense against bacterial infection, while diquat induces renal injury in Gsr hypomorphic mice. In flies, overexpression of GSR extended lifespan under hyperoxia. In the current study, we investigated the roles of GSR in cochlear antioxidant defense using Gsr homozygous knockout mice that were backcrossed onto the CBA/CaJ mouse strain, a normal-hearing strain that does not carry a specific Cdh23 mutation that causes progressive hair cell degeneration and early onset of hearing loss. Gsr-/- mice displayed a significant decrease in GSR activity and GSH/GSSG ratios in the cytosol of the inner ears. However, Gsr deficiency did not affect ABR (auditory brainstem response) hearing thresholds, wave I amplitudes or wave I latencies in young mice. No histological abnormalities were observed in the cochlea of Gsr-/- mice. Furthermore, there were no differences in the activities of cytosolic glutathione-related enzymes, including glutathione peroxidase and glutamate-cysteine ligase, or the levels of oxidative damage markers in the inner ears between WT and Gsr-/- mice. In contrast, Gsr deficiency resulted in increased activities of cytosolic thioredoxin and thioredoxin reductase in the inner ears. Therefore, under normal physiological conditions, GSR is not essential for the maintenance of antioxidant defenses in mouse cochlea. Given that the thioredoxin system is known to reduce GSSG to GSH in multiple species, our findings suggest that the thioredoxin system can support GSSG reduction in the mouse peripheral auditory system.

Up-regulation of glutathione-related genes, enzyme activities and transport proteins in human cervical cancer cells treated with doxorubicin.

Doxorubicin (DOX), one of the most effective anticancer drugs, acts in a variety of ways including DNA damage, enzyme inhibition and generation of reactive oxygen species. Glutathione (GSH) and glutathione-related enzymes including: glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferases (GST) may play a role in adaptive detoxification processes in response to the oxidative stress, thus contributing to drug resistance phenotype. In this study, we investigated effects of DOX treatment on expression and activity of GSH-related enzymes and multidrug resistance-associated proteins in cultured human cervical cancer cells displaying different resistance against this drug (HeLa and KB-V1). Determination of expression level of genes encoding GST isoforms and MRP proteins (GCS, GPX, GSR, GSTA1-3, GSTM1, GSTP1, ABCC1-3, MGST1-3) was performed using StellARray™ Technology. Enzymatic activities of GPX and GSR were measured using biochemical methods. Expression of MRP1 was examined by immunofluorescence microscopy. This study showed that native expression levels of GSTM1 and GSTA3 were markedly higher in KB-V1 cells (2000-fold and 200-fold) compared to HeLa cells. Resistant cells have also shown significantly elevated expression of GSTA1 and GSTA2 genes (200-fold and 50-fold) as a result of DOX treatment. In HeLa cells, exposure to DOX increased expression of all genes: GSTM1 (7-fold) and GSTA1-3 (550-fold, 150-fold and 300-fold). Exposure to DOX led to the slight increase of GCS expression as well as GPX activity in KB-V1 cells, while in HeLa cells it did not. Expression of ABCC1 (MRP1) was not increased in any of the tested cell lines. Our results indicate that expression of GSTM1 and GSTA1-3 genes is up-regulated by DOX treatment and suggest that activity of these genes may be associated with drug resistance of the tested cells. At the same time, involvement of MRP1 in DOX resistance in the given experimental conditions is unlikely.

Glutathione, glutathione-related enzymes, and oxidative stress in individuals with subacute occupational exposure to lead.

The aim of the study was to investigate the influence of subacute exposure to lead on the glutathione-related antioxidant defense and oxidative stress parameters in 36 males occupationally exposed to lead for 40±3.2days. Blood lead level in the examined population increased significantly by 359% due to lead exposure. Simultaneously, erythrocyte glutathione level decreased by 16%, whereas the activity of glutathione-6-phosphate dehydrogenase in erythrocytes and leukocytes decreased by 28% and 10%, respectively. Similarly, the activity of glutathione-S-transferase in erythrocytes decreased by 45%. However, the activity of glutathione reductase in erythrocytes and leukocytes increased by 26% and 6%, respectively, whereas the total oxidant status value in leukocytes increased by 37%. Subacute exposure to lead results in glutathione pool depletion and accumulation of lipid peroxidation products; however, it does not cause DNA damage. Besides, subacute exposure to lead modifies the activity of glutathione-related enzymes.

Copper at low levels impairs memory of adult zebrafish (Danio rerio) and affects swimming performance of larvae

Metal contamination at low levels is an important issue because it usually produces health and environmental effects, either positive or deleterious. Contamination of surface waters with copper (Cu) is a worldwide event, usually originated by mining, agricultural, industrial, commercial, and residential activities. Water quality criteria for Cu are variable among countries but allowed limits are generally in the μg/L range, which can disrupt several functions in the early life-stages of fish species. Behavioral and biochemical alterations after Cu exposure have also been described at concentrations close to the allowed limits. Aiming to search for the effects of Cu in the range of the allowed limits, larvae and adult zebrafish (Danio rerio) were exposed to different concentrations of dissolved Cu (nominally: 0, 5, 9, 20 and 60μg/L; measured: 0.4, 5.7, 7.2 16.6 and 42.3μg/L, respectively) for 96h. Larvae swimming and body length, and adult behavior and biochemical biomarkers (activity of glutathione-related enzymes in gills, muscle, and brain) were assessed after Cu exposure. Several effects were observed in fish exposed to 9μg/L nominal Cu, including increased larvae swimming distance and velocity, abolishment of adult inhibitory avoidance memory, and decreased glutathione S-transferase (GST) activity in gills of adult fish. At the highest Cu concentration tested (nominally: 60μg/L), body length of larvae, spatial memory of adults, and gill GST activity were decreased. Social behavior (aggressiveness and conspecific interaction), and glutathione reductase (GR) activity were not affected in adult zebrafish. Exposure to Cu, at concentrations close to the water quality criteria for this metal in fresh water, was able to alter larvae swimming performance and to induce detrimental effects on the behavior of adult zebrafish, thus indicating the need for further studies to reevaluate the currently allowed limits for Cu in fresh water.

Acute restraint stress reduces hippocampal oxidative damage and behavior in rats: Effect of S-allyl cysteine.

This simple study was designed to investigate whether acute restraint stress can generate changes in behavioral tests and hippocampal endpoints of oxidative stress in rats, and if the antioxidant S-allyl cysteine (SAC) can prevent these alterations. We evaluated motor activity, forced swimming and anxiety behavior, as well as the hippocampal levels of lipid peroxidation and the activities of glutathione-related enzymes in animals submitted to mild immobilization. The effect of SAC (100 mg/kg, i.p.), given to rats every day 30 min before starting the immobilization session, was also investigated. Immobilization (restraint) stress was induced for a period of 6 h per day for five consecutive days. Our results indicate that, under the tested conditions, acute restraint stimulates compensatory behavioral tasks (motor activity, anxiety and forced swimming) to counteract the stressing conditions prevailing, and selectively increased the levels of lipid peroxidation and the enzyme activities of glutathione-S-transferase (GST) and glutathione peroxidase (GPx) in the hippocampus also as adaptive responses. SAC exhibited preventive effects in the stressed group as it improved behavior, reduced lipid peroxidation and prevented the increase of GST and GPx activities, suggesting that this antioxidant blunted primary pro-oxidative stimuli induced by restraint stress. Findings of this work also confirm that the use of antioxidants such as SAC can provide effective protection against the acute oxidative damage associated with anxiety produced by stressing conditions.

Neuroprotection induced by NMDA preconditioning as a strategy to understand brain tolerance mechanism.

Excitotoxicity refers to toxicity caused by abnormal concentrations of glutamate in the synaptic cleft that may lead to neuronal death. Since its description, the phenomenon of glutamatergic excitotoxicity has been implicated in the physiopathology of a wide range of neurological and psychiatric disorders, from acute brain damage such as traumatic brain injury, ischemia as well as chronic conditions like epilepsy, depression and neurodegenerative pathologies such as Huntington's, Parkinson's and Alzheimer's diseases. Excessive stimulation of glutamatergic receptors, mainly N-methyl-D-aspartate (NMDA) receptors (NMDAR), can have numerous adverse effects on the cell viability, including increased nitric oxide release (NO), activation of proteases, increased production of reactive oxygen (ROS) and nitrogen (RNS) species and massive influx of calcium ions (Ca2+), resulting in cell death. Thus, the use of strategies that modulate the excitotoxic cell damage represents a perspective for the treatment of diseases such as Parkinson's and Alzheimer's diseases, ischemia, traumatic brain injury (TBI) and seizures.