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Sedoheptulose-1,7-bisphosphatase (SBPase) is a crucial enzyme for photosynthetic carbon assimilation in the Calvin-Benson cycle. Previous studies have shown that overexpression of SBPase is advantageous to chilling tolerance in plants; however, the mechanisms of SBPase acting in the improvement of chilling tolerance remain largely unknown. In the present study, we aimed to uncover the essential role of SBPase in the response of tomato plants to oxidative stress induced by low temperature. To fulfill that, we performed an array of comparative studies between slsbpase mutant plants that we previously generated using CRISPR/Cas9 genome editing system and their wild-type counterparts under chilling stress. It was observed that following a 24 h chilling treatment, slsbpase mutant plants accumulated higher levels of reactive oxygen species (ROS) than wild-type plants and consequently, more severe lipid peroxidation occurred in slsbpase plants. Activity assay of antioxidant enzymes showed that mutation in SlSBPASE significantly decreased activities of peroxidase (POD) and ascorbate peroxidase (APX), but surprisingly did not significantly alter activities of superoxide dismutase (SOD) and catalase (CAT) under the chilling condition. Notably, mutation in SlSBPASE reduced the contents of total ascorbate (AsA) and total glutathione (GSH) and suppressed the recycling of AsA and GSH in chilling-stressed tomato plants. In addition, activities of two GSH biosynthetic enzymes (gamma-glutamylcysteine synthetase and glutathione synthetase) and transcript abundance of their coding genes (GSH1 and GSH2) were markedly reduced in slsbpase mutant plants in comparison with those in wild-type plants under chilling stress. Furthermore, exogenous GSH remarkably mitigated chilling damage in slsbpase plants. Collectively, these results support that mutation in SlSBPASE aggravates chilling-induced oxidative stress by suppressing GSH biosynthesis and AsA-GSH recycling and suggest that SBPase is required for optimal response to chilling stress in tomato plants. The findings also shed light on the idea to mitigate chilling-induced damages by genetically manipulating a photosynthetic enzyme in plants.

Background Ferroptosis is the process of cell death triggered by lipid peroxides, and inhibition of glutathione (GSH) synthesis leads to ferroptosis. Liver cancer progression is closely linked to ferroptosis suppression. However, the mechanism by which inhibition of GSH synthesis suppresses potential ferroptosis of liver cancer cells and whether ferroptosis-related liver cancer biomarkers have a promising diagnostic value remain unknown. Methods Ribonucleotide reductase regulatory subunit M2 (RRM2) levels were measured using an enzyme linked immunosorbent assay (ELISA), quantitative RT-PCR (qPCR), immunoblotting (IB) and immunochemistry (IHC). Cell viability and cell death were measured by a CellTiter-Glo luminescent cell viability assay and staining with SYTOX Green followed by flow cytometry, respectively. Metabolites were measured using the indicated kits. The Interaction between glutathione synthetase (GSS) and RRM2 was measured using immunofluorescence (IF), co-immunoprecipitation (co-IP) and the proximal ligation assay (PLA). The diagnostic value was analyzed using the area under the receiver operating characteristic curve (AUC-ROC). Bioinformatics analysis was performed using the indicated database. Results RRM2 showed specifically elevated levels in liver cancer and inhibited ferroptosis by stimulating GSH synthesis via GSS. Mechanistically, phosphorylation of RRM2 at the Threonine 33 residue (T33) was maintained at normal levels to block the RRM2–GSS interaction and therefore protected RRM2 and GSS from further proteasome degradation. However, under ferroptotic stress, RRM2 was dephosphorylated at T33, thus the RRM2–GSS interaction was promoted. This resulted in the translocation of RRM2 and GSS to the proteasome for simultaneous degradation. Clinically, serum RRM2 was significantly associated with serum alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT), albumin (ALB) and total bilirubin. The AUC-ROC for the combination of RRM2 with AFP was 0.947, with a sensitivity of 88.7% and a specificity of 97.0%, which indicates better diagnostic performance compared to either RRM2 or AFP alone. Conclusion RRM2 exerts an anti-ferroptotic role in liver cancer cells by sustaining GSH synthesis. Serum RRM2 will be useful as a biomarker to evaluate the degree to which ferroptosis is suppressed and improve diagnostic efficiency for liver cancer.

Nitric oxide (NO) is a signal in the modulation of acclamatory responses to stress in plants. Here, the metabolic shift of Chlamydomonas reinhardtii to sub-lethal NO stress was approached by exposure to 0.1 mM S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, in the presence or the absence of the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (cPTIO). NO did not cause growth impairment but induced a decrease in glutathione (GSH) levels and redox state. NO upregulated the expression of glutathione-associated genes, glutathione synthetase (GSH1), and glutathione reductase (GSHR1) genes while it decreased that of the proteins associated with ER stress-induced unfolded protein response (UPR). Furthermore, the expression of NADPH oxidase isoform, respiratory burst oxygenase-like 2 (RBOL2), instead of RBOL1 increased under NO stress. NO-induced upregulation of GSH1 and GSHR1 upregulation and the downregulation of most UPR genes were not found in rbol2 mutant. The presence of cPTIO suppressed the NO-induced changes in GSH availability, UPR, and RBOL expression. Overall, NADPH oxidase (RBOL2)-dependent and -independent signaling pathways involve in the inhibition of UPR and the enhancement of GSH availability by NO.

Dichloroacetate (DCA) is an investigational drug that is used in the treatment of various congenital and acquired disorders of energy metabolism. Although DCA is generally well tolerated, some patients experience peripheral neuropathy, a side effect more common in adults than children. Repetitive DCA dosing causes downregulation of its metabolizing enzyme, glutathione transferase zeta 1 (GSTZ1), which is also critical in the detoxification of maleylacetoacetate and maleylacetone. GSTZ1 (-/-) knockout mice show upregulation of glutathione transferases (GSTs) and antioxidant enzymes as well as an increase in the ratio of oxidized glutathione (GSSG) to reduced glutathione (GSH), suggesting GSTZ1 deficiency causes oxidative stress. We hypothesized that DCA-mediated depletion of GSTZ1 causes oxidative stress and used the rat to examine induction of GSTs and antioxidant enzymes after repeated DCA exposure. We determined the expression of alpha, mu, pi, and omega class GSTs, NAD(P)H dehydrogenase [quinone] 1 (NQO1), gamma-glutamylcysteine ligase complex (GCLC), and glutathione synthetase (GSS). GSH and GSSG levels were measured by liquid chromatography-tandem mass spectrometry. Enzyme activity was measured in hepatic cytosol using 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, and 2,6-dichloroindophenol as substrates. In comparison with acetate-treated controls, DCA dosing increased the relative expression of GSTA1/A2 irrespective of rodent age, whereas only adults displayed higher levels of GSTM1 and GSTO1. NQO1 expression and activity were higher in juveniles after DCA dosing. GSH concentrations were increased by DCA in adults, but the GSH:GSSG ratio was not changed. Levels of GCLC and GSS were higher and lower, respectively, in adults treated with DCA. We conclude that DCA-mediated depletion of GSTZ1 causes oxidative stress and promotes the induction of antioxidant enzymes that may vary between age groups. SIGNIFICANCE STATEMENT: Treatment with the investigational drug, dichloroacetate (DCA), results in loss of glutathione transferase zeta 1 (GSTZ1) and subsequent increases in body burden of the electrophilic tyrosine metabolites, maleylacetoacetate and maleylacetone. Loss of GSTZ1 in genetically modified mice is associated with induction of glutathione transferases and alteration of the ratio of oxidized to reduced glutathione. Therefore, we determined whether pharmacological depletion of GSTZ1 through repeat administration of DCA produced similar changes in the liver, which could affect responses to other drugs and toxicants.

The cell free system has been paid more attention due to its potential of facilitating more efficient catalysis of multistep reactions. In this study, an efficient enzymatic cascade of GSH production was developed through the evolution of bifunctional glutathione synthetase (GshF), coupled with polyphosphate kinase (PPK). First, the stability and activity of GshF were enhanced by loop interchange and site-directed mutagenesis. As a result, the GshF half-value period increased 163.3-fold, and its activity raised 18 %. PPK from Jhaorihella thermophile (PPKJT) was characterized and used to regenerate ATP in the GSH synthesis, with hexametaphosphate (PolyP(6)) as the phosphate donor. After the process optimization, 99.9 mM GSH and 7.6 mM oxidized glutathione (GSSG) were produced within 2 h. The molar yield was 95.9 mol/mol based on the amino acid added, while the productivities of GSH achieved 49.95 mM/h, which was the highest yield and productivity ever reported about GSH synthesis.

The present study aimed at investigating the kinetic of inhibition of isoproturon to the GSH-associated enzymes [γ-glutamyl-cysteine synthetase (γ-GCS), glutathione synthetase (GS), glutathione reductase (GR), glutathione-S-transferase (GST) and glutathione peroxidase (GPX)] in wheat. Isoproturon, applied to 10-day-old seedlings for the following 12 days, provoked significant reductions in shoot fresh and dry weights, protein, thiols and glutathione (GSH); however, oxidized glutathione (GSSG) was elevated while GSH/GSSG ratio was declined with concomitant significant inhibitions in the activities of γ-GCS, GS, GR, GST and GPX; the effect was time dependent. IC50 and Ki values of isoproturon were lowest for GPX, highest for both GST and GR, and moderate for both γ-GCS and GS. The herbicide markedly decreased Vmax of γ-GCS, GS and GPX but unchanged that of GST and GR; however, Km of γ-GCS, GS, GST and GR increased but unchanged for GPX. The pattern of response of changing Vmax, Km, Vmax/Km, kcat and kcat/Km for in vivo and in vitro tests of each enzyme seemed most likely similar. These results indicate that a malfunction to defense system was induced in wheat by isoproturon resulting in inhibitions in GSH-associated enzymes, the magnitude of inhibition was most pronounced in GPX followed by γ-GCS, GS, GST, and GR. These findings could conclude that isoproturon competitively inhibited GST and GR; however, the inhibition was noncompetitive for GPX but mixed for both γ-GCS and GS.

Although Cd is threatening to the environment, animal, and human, the eco-friendly approach to mitigate the Cd-toxicity in alfalfa was barely studied. Therefore, this study aims at elucidating the role of S, a crucial macroelement, in alleviating Cd toxicity in alfalfa plants. The supplementation of S in Cd-stressed alfalfa reversed the detrimental effect on plant biomass, chlorophyll synthesis, and protein concentration. Interestingly, S surplus restored the photosynthetic kinetics, such as Fv/Fm, Pi_ABS, and Mo values in leaves of Cd-stressed alfalfa. Further, Cd-induced adverse effect on membrane stability, cell viability, and redox status was restored due to S under Cd stress. The exogenous S not only increased S status and the expression of sulfate transporters (MsSULRT1;2 and MsSULTR1;3), but also decreased the Cd concentration in the shoot by retaining elevated Cd in root tissue. Further analysis revealed the upregulation of MsGS (glutathione synthetase) and MsPCS1 (phytochelatin synthase) genes along with the increased concentration of glutathione and phytochelatin, predominantly in roots subjected to S surplus under Cd stress. The subcellular Cd analysis showed elevated Cd in the cell wall but not in the vacuole. It suggests that S-induced elevated glutathione enables the phytochelatin to bind with excess Cd leading to subcellular sequestration in the cell wall of roots. Also, S stimulates the S-metabolites and GR enzyme that coordinately counteracts Cd-induced oxidative damage. These findings can be utilized to popularize the application of S and to perform breeding/transgenic experiments to develop Cd-free forage crops.

Virulence factors of Helicobacter pylori (H. pylori) are diverse, so various biological responses happen in a host infected with H. pylori. The aim of this study is to conduct the metabolomics-based evaluation on H. pylori infection. AGS human gastric carcinoma cells were infected with H. pylori strain 26695, and then the altered metabolite pathways in the infected AGS cells were analyzed by metabolomics. Metabolites related to the glutathione (GSH) cycle were downregulated by H. pylori infection. Next, we evaluated the effects of H. pylori on the GSH-related pathway in AGS cells infected with H. pylori isolated from patients with atrophic gastritis (AG), duodenal ulcer (DU) and gastric cancer (GC). We found that the declined degree of GSH levels and oxidative stress were greater in AGS cells infected with GC strains than DU and AG-derived strains. There were no significant differences in almost mRNA expressions of GSH-related factors among different clinical strains, but the protein expression of glutathione synthetase was lower in AGS cells infected with GC-derived strains than DU and AG-derived strains. Our data demonstrates that GC-derived H. pylori-induced oxidative stress in a host is stronger and GC-derived strains may have suppressive influences on the host’s GSH-related defense systems.

Background Previous studies demonstrated an important role for connexin 43 (Cx43) in the regulation of apoptosis by influencing mitochondrial functions. This study aimed to investigate the relationship between Cx43 and lipopolysaccharide (LPS)-induced oxidative stress and apoptosis in human umbilical vein endothelial cells (HUVECs). Methods Western blot was performed to determine mitochondrial Cx43 (MtCx43) protein level and phosphorylation (p-MtCx43). Gap19, a selective Cx43 inhibitor, was used to examine the effects of Cx43 on LPS-induced oxidative stress and apoptosis in HUVECs. Expression of regulatory genes associated with oxidative stress was examined by quantitative polymerase chain reaction (qPCR) and Western blot. Apoptosis was assessed by flow cytometry. Results LPS stimulation resulted in increased levels of MtCx43 and p-MtCx43. Interestingly, Gap19 antagonized the upregulation of glutathione S-transferase Zeta 1 (GSTZ1) and cytochrome b alpha beta (CYBB), and the downregulation of antioxidant 1 (ATOX1), glutathione synthetase (GSS) and heme oxygenase 1 (HMOX1) induced by LPS or Cx43 overexpression. Moreover, the increased production of reactive oxygen species (ROS) and apoptosis elicited by LPS or Cx43 overexpression were reduced following treatment with Gap19. Conclusions Selective inhibition of Cx43 hemichannels protects HUVECs from LPS-induced apoptosis and this may be via a reduction in oxidative stress production.

Nrf2 is a basic leucine zipper transcription factor that binds to the promoter region of the antioxidant response element (ARE), inducing the coordinated up-regulation of antioxidant and detoxification genes. We recently synthesized a set of new molecules by combining the functional moieties of curcumin and diallyl sulfide, both known to induce the expression of antioxidant phase II enzymes by activating Nrf2 pathway. The aim of the study is to investigate the ability of such compounds to activate Keap1/Nrf2/ARE cytoprotective pathway, in comparison with two reference Nrf2-activators: curcumin and dimethyl fumarate, a drug approved for the treatment of relapsing-remitting multiple sclerosis. Furthermore, since Nrf2 pathway is known to be regulated also by epigenetic modifications, including key modifications in microRNA (miRNA) expression, the effects of the hybrids on the expression levels of selected miRNAs, associated with Nrf2 signaling pathway have also been investigated. The results show that compounds exert antioxidant effect by activating Nrf2 signaling pathway and inducing the ARE-regulated expression of its downstream target genes, such as HO-1 and NQO1, with two hybrids to a higher extent than curcumin. In addition, some molecules induce changes in the expression levels of miR-125b-5p, even if to a lesser extent than curcumin. However, no changes have been observed in the expression levels of mRNA coding for glutathione synthetase, suggesting that the modulation of this mRNA is not strictly under the control of miR-125b-5p, which could be influenced by other miRNAs.

Phytoremediation is optimized when plants grow vigorously while accumulating the contaminant of interest. Here we show that sulphur supply alleviates aerial chlorosis and growth retardation caused by cesium stress without reducing cesium accumulation in Arabidopsis thaliana. This alleviation was not due to recovery of cesium-induced potassium decrease in plant tissues. Sulphur supply also alleviated sodium stress but not potassium deficiency stress. Cesium-induced root growth inhibition has previously been demonstrated as being mediated through jasmonate biosynthesis and signalling but it was found that sulphur supply did not decrease the levels of jasmonate accumulation or jasmonate-responsive transcripts. Instead, induction of a glutathione synthetase gene GSH2 and reduction of a phytochelatin synthase gene PCS1 as well as increased accumulation of glutathione and cysteine were observed in response to cesium. Exogenous application of glutathione or concomitant treatments of its biosynthetic intermediates indeed alleviated cesium stress. Interestingly, concomitant treatments of glutathione biosynthetic intermediates together with a glutathione biosynthesis inhibitor did not cancel the alleviatory effects against cesium suggesting the existence of a glutathione-independent pathway. Taken together, our findings demonstrate that plants exposed to cesium increase glutathione accumulation to alleviate the deleterious effects of cesium and that exogenous application of sulphur-containing compounds promotes this innate process.

Inflammation and oxidative stress are pivotal mechanisms for the pathogenesis of ischemia and reperfusion injury (IRI). Vagus nerve stimulation (VNS) may participate in maintaining oxidative homeostasis and response to external stimulus or injury. We investigated whether the in vivo VNS can protect the liver from IRI. In this study, hepatic IRI were induced by ligating the vessels supplying the left and middle lobes of the liver, which underwent 1 h occlusion followed with 24 h reperfusion. VNS was initiated 15 min after ischemia and continued 30 min. Hepatic function, histology, and apoptosis rates were evaluated after 24 h reperfusion. Compared with the IRI group, VNS significantly improved hepatic function. The protective effect was accompanied by a reduction in histological damage in the ischemic area, and the apoptosis rate of hepatocytes has considerable reduction. To find the underlying mechanism, proteomic analysis was performed and differential expression of glutathione synthetase (GSS) and glutathione S-transferase (GST) was observed. Subsequently, test results indicated that VNS upregulated the expression of mRNA and protein of GSS and GST. Meanwhile, VNS increased the plasma levels of glutathione and glutathione peroxidases. We found that VNS alleviated hepatic IRI by upregulating the antioxidant glutathione via the GSS/glutathione/GST signaling pathway.

The glutathione synthetase system (GSS) is an important pathway of glutathione synthesis and plays a key role in heavy metal resistance. In this work, the response of Acidithiobacillus ferrooxidans to extracellular Cd2+ was investigated, and the interplay between Cd2+ resistance and the expression of GSS related-genes was analyzed by reverse-transcription quantitative PCR (RT-PCR). During growth in the presence of 5, 15 and 30 mM Cd2+, the transcript levels of eight GSS pathway genes were affected between 0.81- and 7.12-fold. Increased transcription was also reflected in increased enzyme activities: with those of glutathione reductase (GR) increased by 1.10-, 2.26- and 1.54-fold in the presence of 5, 15 and 30 mM Cd2+, respectively. In contrast, the activities of catalase (CAT) and superoxide dismutase (SOD) were decreased in the presence of Cd2+. At the metabolite level, intracellular methane dicarboxylic aldehyde (MDA) content was increased 1.97-, 3.31- and 1.92-fold in the presence of 5, 15 and 30 mM Cd2+, respectively. These results suggest that Cd2+ directly inhibits the activities of CAT and SOD, breaks the redox balance of the cells, which leads to the activation of the other antioxidant pathway of GSS. Resistance of A. ferrooxidans to Cd2+ may involve modulation of expression levels of glutathione S-transferase (GST), GR, and glutathione synthetase, which may protect against oxidative damage.

Bifunctional glutathione synthetase (GshF) has recently been reported to simultaneously catalyze the 2-step ATP-dependent biosynthesis of reduced glutathione (GSH). In this work, 19 putative gshF were mined from the complete sequenced genome of 20 representative Lactobacillus species. To functionally analyze these putative GshF, GshF from Lactobacillus plantarum and Lactobacillus casei were selected and successfully expressed in Escherichia coli. Compared with the control without expressing GshF, GSH titers were enhanced significantly in E. coli with overexpression of GshF, demonstrating that putative GshF from Lactobacillus have functional activities on GSH biosynthesis. Moreover, with the expression of GshF from L. plantarum in E. coli as a paradigm, GSH yield (286.5 μM) was strongly improved by 177.9% with optimized induced conditions and precursor concentration compared with the control under unoptimized conditions. Transcriptional analysis showed that key genes of endogenous GSH metabolism and precursor biosynthesis were remarkably suppressed by GshF expression, indicating that the increase of GSH titer was attributed to heterologous expression of GshF. Overall, our results suggested that gshF is enriched in Lactobacillus and that heterologous expression of GshF is an efficient strategy for improving GSH biosynthesis.

Glutathione is the most important part of a plant’s antioxidant defense system. Two enzymes are involved in cellular glutathione biosynthesis: glutamylcysteine ligase and glutathione synthetase, of which the latter catalyzes the transfer of glycine to the glutamylcysteine dipeptide. Transgenic plants with an elevated expression level of the glutathione synthetase genes are known to be tolerant to heavy metals. However, our knowledge on their tolerance to other types of the abiotic stress is insufficient. The goal of this study is to produce transgenic tobacco plants with a constitutive expression of the glutathione synthetase gene BnGSH from rapeseed and to estimate their growth parameters under the normal conditions, as well as under the effects of salt, drought, and cold stresses. We generate 17 lines of transgenic plants with the rapeseed BnGSH gene under the control of the 35S promoter by agrobacterium-mediated transformation. The transgene presence is confirmed by the PCR and histochemical analysis of the activity of the GUS reporter gene. Twelve lines with the highest expression level of the BnGSH gene are chosen based on the results of RT-PCR. The following morphological parameters are measured: stem height, leaf area, flower length, fresh and dry weights of shoots, and root length. Some transgenic plants demonstrated increased productivity both under normal conditions and under the effect of a high salinity stress. However, no change in the tolerance to drought and cold was observed in the transgenic plants.

Background Menopause is the most important sign of aging in women, and the ovary is the organ most sensitive to aging. Quercetin is a potential antioxidant and free radical scavenger that is widely found in fruits, vegetables, and leaves. However, the effect of quercetin on ovarian aging has not been elucidated, and the mechanism underlying its antioxidative effect remains unclear. The purpose of the current study was to investigate whether quercetin protects ovarian function by decreasing oxidative stress. Methods In an in vivo experiment, female menopausal rats (12 months old) were intragastrically administered quercetin at three doses (12.5 mg/kg, 25 mg/kg, and 50 mg/kg) for 90 days, and the estrous cycles were determined by vaginal smearing. In an in vitro experiment, rat primary ovarian granulosa cells were cultured and treated with H2O2 (400 μM) alone or H2O2 plus quercetin at 5 μM, 20 μM, or 50 μM. The levels of the hormones estradiol (E2), progesterone (P), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were detected by radioimmunoassay. The serum levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-PX) and glutathione S-transferase (GST) were examined. The expression levels of the oxidative stress-related genes SOD-1, catalase (CAT) and glutathione synthetase (GSS) in the ovaries and ovarian granulosa cells were detected by Western blot. Results The in vivo results demonstrated that quercetin had no effects on ovarian morphology, hormone secretion, or the estrous cycle in menopausal rats. Although no significant changes were detected in the serum levels of T-AOC, SOD, GSH, GSH-PX, and GST between the quercetin and control groups, the mRNA and protein expression levels of the oxidative stress-related genes SOD-1, CAT and GSS in menopausal rat ovaries were increased by low-dose quercetin. Moreover, the in vitro results demonstrated that quercetin significantly rescued the decrease in cell viability by H2О2-induced oxidative stress and enhanced the H2O2-induced decrease in expression of oxidative stress-related proteins. Conclusions Together, the results of this study indicated that quercetin increased the antioxidant capacity of the ovary by upregulating the expression of some oxidative stress-related genes both in vivo and in vitro.

Variations in embryo production between individual oocyte donors represent a serious problem in cattle production, when implementing ovum pick-up (OPU) and in vitro maturation (IVM) of oocytes. However, the precise cause of this problem is unknown. Here, we aimed to investigate the relationship between the glutathione (GSH) concentration in IVM oocytes and embryo development to explore a potential cause of the variations between individual donors. First, we found a high positive correlation (r = 0.80) between the GSH concentration in IVM oocytes and the blastocyst development rate of oocytes collected in the same OPU session for each donor (N = 11). Second, we selected two donors with significantly different blastocyst development rates. In samples from these donors, we examined the dynamics of oocytes GSH concentrations, and the gene expression of the glutathione synthetase (GSS) and glutathione peroxidase (GPX) genes in cumulus-oocyte complexes (COCs) during IVM. At 0 and 24 h after IVM, oocytes from the donor with the highest blastocyst development rate (high donor) exhibited significantly higher oocyte GSH concentrations than oocytes from the donor with the lowest blastocyst development rate (low donor, P

The purpose of this study was to investigate the short-term (48 hr) effects of feeding aflatoxin contaminated diet (170.3 μg/kg AFB1) in 49-week-old laying hens. Liver samples were taken at 12-hr intervals. Feed intake, body weight, absolute and relative liver weight were the same in groups. However, there was no feed intake during both dark periods (between 12nd to 24th and 36th to 48th hours of the experiment); therefore, aflatoxin intake was also negligible. Markers of initial phase of lipid peroxidation, conjugated dienes and trienes did not change as effect of aflatoxin, but terminal marker, malondialdehyde content was significantly higher at 12 hr as effect of aflatoxin. No significant difference was found in reduced glutathione concentration and glutathione peroxidase activity between the groups. Expression of glutathione peroxidase 4 gene (GPX4) was significantly reduced due to aflatoxin treatment at 12 and 24 hr, but induced later, while glutathione reductase gene (GSR) expression was significantly lower at 24 hr and glutathione synthetase gene (GSS) in aflatoxin-treated group at 12 hr. The results suggest that aflatoxin induced oxygen-free radical formation, but it did not reach critical level during this short period of time to cause activation of the expression of glutathione system.

Lipid peroxidation, glutathione content, glutathione peroxidase activity and gene expression of transcription factors, glutathione peroxidase, glutathione synthetase and glutathione reductase was investigated in common carp liver. Short term (24 h) exposure of aflatoxin B1 at different doses (100, 200 and 400 μg AFB1 kg−1 feed) was used. It was found that conjugated dienes and trienes elevated after 16 h, while thiobarbituric acid reactive substances content increased only at the lowest dose after 16 and 24 h of exposure. Glutathione content showed higher levels than control after 16 h of exposure and glutathione peroxidase (GPx4) activity was higher in all of the AFB1 treated than the control group after 8 h of exposure. Gene expression of transcription factors showed dual response. Expression of keap1 gene down-regulated after 8 h and 16 h and nrf2 gene after16 h, but up-regulated after 24 h of exposure in the lowest, and highest dose groups. Expression of gpx4a and gpx4b genes down-regulated after 8 h and induction was found after 16 and 24 h of exposure, irrespective of the dose. The results indicated that low dose of AFB1 provokes oxidative stress earlier than higher doses, which activated the Keap1-Nrf2 pathway. At higher doses this pathway activated later, but preformed GPx4 effectively prevented lipid peroxidation.

Human immunodeficiency virus (HIV) infection induces oxidative stress and alcohol use accelerates disease progression, subsequently causing immune dysfunction. However, HIV and alcohol impact on lipid rafts-mediated immune dysfunction remains unknown. In this study, we investigate the modulation by which oxidative stress induces reactive oxygen species (ROS) affecting redox expression, lipid rafts caveiloin-1, ATP-binding cassette (ABC) transporters, and transcriptional sterol regulatory element-binding protein (SREBP) gene and protein modification and how these mechanisms are associated with arachidonic acid (AA) metabolites in HIV positive alcohol users, and how they escalate immune dysfunction.In both alcohol using HIV-positive human subjects and in vitro studies of alcohol with HIV-1 gp120 protein in peripheral blood mononuclear cells, increased ROS production significantly affected redox expression in glutathione synthetase (GSS), super oxide dismutase (SOD), and glutathione peroxidase (GPx), and subsequently impacted lipid rafts Cav-1, ABC transporters ABCA1, ABCG1, ABCB1, and ABCG4, and SREBP transcription. The increased level of rate-limiting enzyme 3-hydroxy-3-methylglutaryl HMG-CoA reductase (HMGCR), subsequently, inhibited 7-dehydrocholesterol reductase (DHCR-7). Moreover, the expression of cyclooxygenase-2 (COX-2) and lipoxygenase-5 (5-LOX) mRNA and protein modification tentatively increased the levels of prostaglandin E2 synthases (PGEThis article suggests for the first time that the redox inhibition affects lipid rafts, ABC-transporter, and SREBP transcription and modulates AA metabolites, serving as an important intermediate signaling network during immune cell dysfunction in HIV-positive alcohol users.These findings indicate that HIV infection induces oxidative stress and redox inhibition, affecting lipid rafts and ABC transports, subsequently upregulating AA metabolites and leading to immune toxicity, and further exacerbation with alcohol use. Antioxid. Redox Signal. 28, 324-337.

Consumption of rice grains contaminated with high concentrations of cadmium (Cd) can cause serious long-term health problems. Moreover, even low Cd concentrations present in the soil can result in the abatement of plant performance, leading to lower grain yield. Studies examining the molecular basis of plant defense against Cd-induced oxidative stress could pave the way in creating superior rice varieties that display an optimal antioxidative defense system to cope with Cd toxicity. In this study, we showed that after one day of Cd exposure, hydroponically grown rice plants exhibited adverse shoot biomass and leaf growth effects. Cadmium accumulates especially in the roots and the leaf meristematic region, leading to a disturbance of manganese homeostasis in both the roots and leaves. The leaf growth zone showed an increased amount of lipid peroxidation indicating that Cd exposure disturbed the oxidative balance. We propose that an increased expression of genes related to the glutathione metabolism such as glutathione synthetase 2, glutathione reductase and phytochelatin synthase 2, rather than genes encoding for antioxidant enzymes, is important in combating early Cd toxicity within the leaves of rice plants. Furthermore, the upregulation of two RESPIRATORY BURST OXIDASE HOMOLOG genes together with a Cd concentration-dependent increase of abscisic acid might cause stomatal closure or cell wall modification, potentially leading to the observed leaf growth reduction. Whereas abscisic acid was also elevated at long term exposure, a decrease of the growth hormone auxin might further contribute to growth inhibition and concomitantly, an increase in salicylic acid might stimulate the activity of antioxidative enzymes after a longer period of Cd exposure. In conclusion, a clear interplay between phytohormones and the oxidative challenge affect plant growth and acclimation during exposure to Cd stress.

The major aim of this study was to investigate the effect of one-time ozone elicitation on glutathione metabolism in selected berry fruit. Raspberry, highbush blueberry, strawberry, blackberry and blackcurrant fruit was ozonated with an ozone concentration of 15 ppm for 30 min. Research showed that ozonation process causes clear and positive changes in the metabolism of glutathione in berries. After ozonation, the fruit was characterized by increased level of glutathione (GSH), which resulted from higher activity of glutathione synthetase. Moreover, ozonation improved the metabolism activity of mitochondria leading to an increase in biosynthesis of ATP, which is the source of energy necessary for the GSH production. The higher activity of the enzymes involved in glutathione metabolism i.e. glutathione peroxidase and glutathione reductase and increased level of glutathione contributed to the higher ability of the fruit to scavenging the hydrogen peroxide.

The low curative response to current treatment regimens for most soft tissue sarcomas indicates a strong need for alternative treatment strategies and predictive markers for treatment outcome. PCI (photochemical internalization) is a novel treatment strategy to translocate drugs into cytosol that otherwise would have been degraded in lysosomes. Two highly geno-and phenotypically different uterine and vulvar leiomyosarcoma cell lines, MES-SA and SK-LMS-1, were treated with bleomycin (BLM) activated by PCI (PCIBLM). The MES-SA cells were much more sensitive to PCIBLM than the SK-LMS-1 cells and the treatment induced a 7-8 fold higher increase in DNA double-strand breaks at the same dose of light as measure by γH2AX staining. A 3-fold higher induction of apoptosis and stronger activation of Bax and p21 was also measured in the P53WT MES-SA cells, compared to the P53mut SK-LMS-1 cells. The basal formation of reactive oxygen species (ROS) was 3-fold higher in SK-LMS-1 cells than in the MES-SA cells and SK-LMS-1 cells expressed glutathione peroxidase 1 (GPx1) and more superoxide dismutase 2 (SOD2) than the MES-SA cells. Glutathione depletion with the glutathione synthetase inhibitor buthionine sulfoximine increased the cytotoxic effect of the photochemical treatment (PDT) most strongly in the SK-LMS-1 cells, and reduced PCIBLM-induced H2AX activation in the MES-SA cells, but not in the SK-LMS-1 cells. The results indicate PCIBLM as a potential novel treatment strategy for soft tissue sarcomas, with antioxidant enzymes, in particular GPx1, and the P53 status as potential predictive markers for response to PCIBLM.

Buthionine sulfoximine (BSO) induces decreased GSH and trypanothione [T(SH)2] pools in trypanosomatids, presumably because only gamma-glutamylcysteine synthetase (γECS) is blocked. However, some BSO effects cannot be explained by exclusive γECS inhibition; therefore, its effect on the T(SH)2-metabolism pathway in Trypanosoma cruzi was re-examined. Parasites exposed to BSO did not synthesize T(SH)2 even when supplemented with cysteine or glutathione, suggesting trypanothione synthetase (TryS) inhibition by BSO. Indeed, recombinant γECS and TryS, but not glutathione synthetase, were inhibited by BSO and kinetics and docking analyses on a TcTryS 3D model suggested BSO binding at the glutathione site. Furthermore, parasites overexpressing γECS and TryS showed ~50% decreased activities after BSO treatment. These results indicated that BSO is also an inhibitor of TryS. This article is protected by copyright. All rights reserved.

Nitric oxide (NO) plays a key role in regulating a wide range of physiological and biochemical processes. This study investigated possible effects of NO, which was supplied by sodium nitroprusside (SNP), NO donor, on selenium (Se) concentration, Se species within roots, activities and gene transcription of selenite uptake, and metabolism-related transporters or enzymes using ICP-MS, HPLC-ICP/MS, qRT-PCR, fluorescence imaging, and biochemical analysis in the roots of rice (Oryza sativa L.) seedling. A suitable SNP concentration (10 µM) increased Se concentration in rice roots. NO enhanced the gene transcription of phosphate transporter OsPT2 and sulfate transporters OsSultr1;2 and OsSultr4;1 that contribute to selenite uptake. Furthermore, NO increased selenocysteine and methyl-selenocysteine concentrations in roots, stimulated glutathione biosynthesis by up-regulating the transcript levels of γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase (GS), and increased cysteine synthase (CS) activity. In conclusion, our results indicate that NO promotes Se concentration of rice seedling roots by enhancing selenite uptake, up-regulating gene transcription of selenite uptake-related transporters, and increasing Se metabolism through regulating glutathione biosynthesis.

CodY st is a global transcriptional regulator that modulates the metabolic network in Streptococcus thermophilus ST2017. In this study, experimental data showed that the cell survival of the codY st defective mutant obviously declined at the presence of 10 m M H 2 O 2 , suggesting CodY st was involved in response to the oxidative stress. To investigate this phenomenon, transcriptome analysis and real time-quantitative PCR were performed and the results indicated that the transcriptional level of a bifunctional glutathione synthetase gene ( gshF ) was downregulated by about 3-fold in the codY st defective mutant, along with a decrease by 20% of the glutathione yield compared with the wild-type in minimal chemical defined medium, whereas half of the viable cells remained after H 2 O 2 challenge. In vitro gel shift assays showed that the purified CodY st could bind to the promoter region of gshF , with a conserved CodY st box, confirming the regulation of CodY st on the gshF gene. To our knowledge, this is first report of CodY st in response to oxidative stress mediated by the regulation of gshF in S. thermophilus .