GSH Accumulation Research Articles

Intracellular Iron Deficiency and Abnormal Metabolism, Not Ferroptosis, Contributes to Homocysteine-Induced Vascular Endothelial Cell Death.

Background/Objectives: Homocysteine (Hcy) and iron are factors co-related with the progression of cardiovascular diseases. The vascular endothelium is an important barrier for physiological homeostasis, and its impairment initiates cardiovascular injury. However, the mechanism underlying Hcy-caused vascular endothelial cell injury and the participation of iron are not fully elucidated. This study aims to investigate the Hcy-induced vascular endothelial injury and iron metabolism dysfunction as well as the underlying molecular mechanism. Methods: Human umbilical vein endothelial cells (HUVECs) were employed as the experimental model to examine the Hcy-induced endothelial injury and its underlying mechanism via various biochemical assays. Results: Hcy suppressed the cell viability and proliferation and caused cell death in a concentration-dependent manner. Hcy induced cell cycle arrest, apoptosis, and autophagy as well as impairment of intracellular energy metabolism. Hcy disrupted the intracellular antioxidant system and mitochondrial function by increasing intracellular ROS, MDA and mitochondrial content, and decreasing the SOD activity and mitochondrial membrane potential. Hcy significantly reduced the GSH-Px activity along with the accumulation of intracellular GSH in a concentration-dependent manner. Ferroptosis inhibitors, Ferrostatin-1 (Fer-1), and Deferoxamine (DFO) significantly decreased the Hcy-caused cytotoxicity accompanied by a reduction in dysregulated mitochondria content, but only DFO ameliorated the elevation of intracellular ROS, and neither Fer-1 nor DFO affected the Hcy-caused reduction in intracellular ATP. In addition, Hcy decreased the intracellular concentration of iron, and supplementing Hcy with various concentrations of Fe3+ increased the cell viability and decreased the LDH release in a concentration-dependent manner. Hcy dramatically decreased the mRNA expression level of transferrin receptor while increasing the mRNA expression levels of transferrin, ferritin light chain, ferritin heavy chain, ferroportin, and SLC7A11. Moreover, Hcy suppressed the protein expression of phospho-Akt, phospho-mTOR, Beclin-1, LC3A/B, Nrf2, HO-1, phospho-MEK1/2, phospho-ERK1/2, and Caspase-3 in concentration- and time-dependent manners. Conclusions: Hcy-induced vascular endothelial injury is likely to be associated with apoptosis and autophagy, but not ferroptosis. The key underlying mechanisms are involved in the disruption of the intracellular antioxidant system and iron metabolism via regulation of PI3K/Akt/mTOR, MAPKs, Nrf2/HO-1, and iron metabolism.

Exogenous deferoxamine mesylate suppresses iron-dependent ferroptosis in postharvest fruit rot of blueberry caused by Alternaria alternata and Fusarium fujikuroi

Plant development relies heavily on regulated cell death, which is crucial for specific plant responses to biological stresses. Ferroptosis, an iron-dependent, oxidative, and non-apoptotic cell death form, has been recently discovered in animal cells. In this study, we investigated whether a ferroptosis-like process could be relevant to cell death in postharvest fruit rot of blueberry caused by Alternaria alternata and Fusarium fujikuroi. The results showed that deferoxamine mesylate and glutathione (GSH) treated fruit showed enhanced disease resistance with lower lesion areas, whereas an opposite result was observed in hydrogen peroxide (H2O2) treatment, indicating that intracellular ferroptosis might exist in the fungal infected blueberry fruit. Compared to control, deferoxamine mesylate or GSH treatment improved chlorophyll fluorescence parameters, which contributed to the stability of photosynthesis. In terms of ferroptosis pathway in the adjacent area of infected fruit, deferoxamine mesylate suppressed an iron-dependent cell death pathway that was characterized by depletion of H2O2 and MDA and accumulation of GSH, as well as the reduction of ferrous ions and total iron ions. Meanwhile, deferoxamine mesylate could effectively delay the decrease rate of unsaturation value and key phospholipids of fungi infected fruit during storage, which possibly maintained the integrity of cell membrane. Due to the serious rot of the lesion area, the peroxidation of membrane lipids was severe, accompanied by metabolic disorders. These combined results demonstrated that deferoxamine mesylate treatment could reduce Fenton reaction by chelating Fe2+ and avoid excessive accumulation of reactive oxygen species (ROS), suppress iron-dependent ferroptosis, thereby maintaining fruit quality and reducing postharvest diseases.

Selective PI3Kδ inhibitor TYM-3-98 suppresses AKT/mTOR/SREBP1-mediated lipogenesis and promotes ferroptosis in KRAS-mutant colorectal cancer

Colorectal cancer (CRC) is one of the most common tumors of the digestive system worldwide. KRAS mutations limit the use of anti-EGFR antibodies in combination with chemotherapy for the treatment of CRC. Therefore, novel targeted therapies are needed to overcome the KRAS-induced oncogenesis. Recent evidence suggests that inhibition of PI3K led to ferroptosis, a nonapoptotic cell death closely related to KRAS-mutant cells. Here, we showed that a selective PI3Kδ inhibitor TYM-3–98 can suppress the AKT/mTOR signaling and activate the ferroptosis pathway in KRAS-mutant CRC cells in a concentration-dependent manner. This was evidenced by the lipid peroxidation, iron accumulation, and depletion of GSH. Moreover, the overexpression of the sterol regulatory element-binding protein 1 (SREBP1), a downstream transcription factor regulating lipid metabolism, conferred CRC cells greater resistance to ferroptosis induced by TYM-3–98. In addition, the effect of TYM-3–98 was confirmed in a xenograft mouse model, which demonstrated significant tumor suppression without obvious hepatoxicity or renal toxicity. Taken together, our work demonstrated that the induction of ferroptosis contributed to the PI3Kδ inhibitor-induced cell death via the suppression of AKT/mTOR/SREBP1-mediated lipogenesis, thus displaying a promising therapeutic effect of TYM-3–98 in CRC treatment.

Effect of Plant Growth-Promoting Bacteria on Antioxidant Status, Acetolactate Synthase Activity, and Growth of Common Wheat and Canola Exposed to Metsulfuron-Methyl.

Metsulfuron-methyl, a widely used herbicide, could cause damage to the sensitive plants in crop-rotation systems at extremely low levels in the soil. The potential of plant growth-promoting bacteria (PGPB) for enhancing the resistance of plants against herbicide stress has been discovered recently. Therefore, it is poorly understood how physiological processes occur in plants, while PGPB reduce the phytotoxicity of herbicides for agricultural crops. In greenhouse studies, the effect of strains Pseudomonas protegens DA1.2 and Pseudomonas chlororaphis 4CH on oxidative damage, acetolactate synthase (ALS), enzymatic and non-enzymatic antioxidants in canola (Brassica napus L.), and wheat (Triticum aestivum L.) were investigated under two levels (0.05 and 0.25 mg∙kg-1) of metsulfuron-methyl using spectrophotometric assays. The inoculation of herbicide-exposed wheat with bacteria significantly increased the shoots fresh weight (24-28%), amount of glutathione GSH (60-73%), and flavonoids (5-14%), as well as activity of ascorbate peroxidase (129-140%), superoxide dismutase SOD (35-49%), and ALS (50-57%). Bacterial treatment stimulated the activity of SOD (37-94%), ALS (65-73%), glutathione reductase (19-20%), and the accumulation of GSH (61-261%), flavonoids (17-22%), and shoots weight (27-33%) in herbicide-exposed canola. Simultaneous inoculation prevented lipid peroxidation induced by metsulfuron-methyl in sensitive plants. Based on the findings, it is possible that the protective role of bacterial strains against metsulfuron-metil is linked to antioxidant system activation.

Quercetin attenuates cerebral ischemic injury by inhibiting ferroptosis via Nrf2/HO-1 signaling pathway

AimsIschemic stroke is a severe cerebrovascular disease in which neuronal death continually occurs through multiple forms, including apoptosis, autophagy, pyroptosis and ferroptosis. Quercetin (QRC), as a natural flavonoid compound, has been reported to have pharmacological effects on ischemic injury accompanied by unclear anti-ferroptotic mechanisms. This study is designed to investigate the therapeutic effects of QRC against ferroptosis in ischemic stroke. Materials and methodsIn vivo, the model of MCAO rats were used to assess the protective effect of QRC on cerebral ischemic. Additionally, we constructed oxidative stressed and ferroptotic cell models to explore the effects and mechanisms of QRC on ferroptosis. The related proteins were analysed by western blotting, immunohistochemical and immunofluorescence techniques. ResultsThe experiments demonstrated that QRC improves neurological deficits, infarct volume, and pathological features in MCAO rats, also increased the viability of HT-22 cells exposed to H2O2 and erastin. These results, including MDA, SOD, GSH, ROS levels and iron accumulation, indicated that QRC suppresses the generation of lipid peroxides and may involve in the regulatory of ferroptosis. Both in vitro and in vivo, QRC was found to inhibit ferroptosis by up-regulating GPX4 and FTH1, as well as down-regulating ACSL4. Furthermore, we observed that QRC enhances the nuclear translocation of Nrf2 and activates the downstream antioxidative proteins. Importantly, the effect of QRC on ferroptosis can be reversed by the Nrf2 inhibitor ML385. ConclusionsThis study provides evidence that QRC has a neuroprotective effect by inhibiting ferroptosis, demonstrating the therapeutic potential for cerebral ischemic stroke.

Reduced GSH Acts as a Metabolic Cue of OPDA Signaling in Coregulating Photosynthesis and Defense Activation under Stress.

12-oxo-phytodienoic acid (OPDA) is a primary precursor of jasmonates, able to trigger autonomous signaling cascades that activate and fine-tune plant defense responses, as well as growth and development. However, its mechanism of actions remains largely elusive. Here we describe a dual-function messenger of OPDA signaling, reduced glutathione (GSH), that cross-regulates photosynthesis machinery and stress protection/adaptation in concert, optimizing plant plasticity and survival potential. Under stress conditions, the rapid induction of OPDA production stimulates GSH accumulation in the chloroplasts, and in turn leads to protein S-glutathionylation in modulating the structure and function of redox-sensitive enzymes such as 2-cysteine (Cys) peroxiredoxin A (2CPA), a recycler in the water-water cycle. GSH exchanges thiol-disulfides with the resolving CysR175, while donating an electron (e-, H+) to the peroxidatic CysP53, of 2CPA, which revives its reductase activity and fosters peroxide detoxification in photosynthesis. The electron flow protects photosynthetic processes (decreased total non-photochemical quenching, NPQ(T)) and maintains its efficiency (increased photosystem II quantum yield, ΦII). On the other hand, GSH also prompts retrograde signaling from the chloroplasts to the nucleus in adjusting OPDA-responsive gene expressions such as Glutathione S-Transferase 6 (GST6) and GST8, and actuating defense responses against various ecological constraints such as salinity, excess oxidants and light, as well as mechanical wounding. We thus propose that OPDA regulates a unique metabolic switch that interfaces light and defense signaling, where it links cellular and environmental cues to a multitude of plant physiological, e.g., growth, development, recovery, and acclimation, processes.

SlWRKY31 enhances chilling tolerance by interacting with SlSIZ1 in tomato fruit

Refrigeration can extend the storage life of postharvest fruits and vegetables. However, tomato fruit are susceptible to chilling injury (CI) under low temperatures, leading to economic losses. In the present work, SlWRKY31 facilitated to reduce the reactive oxygen species (ROS) content by improving the activity of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and production of glutathione (GSH) and ascorbic acid (AsA), thus inhibiting the CI index in tomato fruit. SlWRKY31 promoted the SAP AND MIZ1 DOMAIN-CONTAINING LIGASE1 (SlSIZ1) expression. Moreover, SlWRKY31 transactivated the SlSIZ1 by binding to the W-box motif derived from its promoter. SlSIZ1 contributed to repress the ROS generation by promoting the activity of SOD, POD and CAT and accumulation of GSH and AsA, and thereby boosted the chilling tolerance in tomatoes. SlSIZ1 enhanced the SlWRKY31 expression. The protein-protein interaction between SlSIZ1 and SlWRKY31 was occurred. Thus, SlWRKY31 retarded the CI by interacting with SlSIZ1 in tomato fruit.

Drought Stress and High Temperature Affect the Antioxidant Metabolism of Cotton (Gossypium hirsutum L.) Anthers and Reduce Pollen Fertility

Both drought and high temperature can influence the antioxidant metabolism of crop reproductive organs in different ways, affecting the fertility of reproductive organs and yield formation. However, the combined effects of drought stress and high temperature on the crop reproductive physiology have not yet been widely considered. In order to broaden our understanding of this mechanism of influence, a pond experiment was conducted using a cotton variety Yuzaomian 9110 divided into four treatment groups: control (CK), drought stress (DS), high temperature (HT), and drought stress coupled with high temperature (DS+HT). Results showed a significant negative correlation between pollen viability and superoxide anion (O2−) content, as well as hydrogen peroxide (H2O2). Compared with CK, DS did not alter O2− content in anthers, but HT treatment resulted in higher anther O2−. Compared with single-stress groups, DS+HT further promoted the formation of O2− in anthers, leading to more malondialdehyde in anthers. Moreover, a higher H2O2 content in anthers was found in DS and HT than in CK. DS+HT did not show altered H2O2 content relative to HT treatment, although its H2O2 was higher than in DS. Further analyses of the antioxidant enzyme system showed that DS had no significant effect on superoxide dismutase gene (GhCu/ZnSOD) expression, but HT and DS+HT significantly downregulated its expression. The expression of GhCu/ZnSOD was lower under DS+HT than HT, which might be why O2− content was not altered under DS treatment compared with CK and was higher in DS+HT than HT. DS and HT significantly downregulated the expression of the peroxidase gene (GhPOD) and catalase gene (GhCAT), which should be the main reason for the larger accumulation of H2O2 under drought stress and high-temperature conditions. Compared with single-stress groups, DS+HT had lower expression of GhCAT, resulting in a larger H2O2 content. Regarding the ascorbic acid–glutathione (AsA–GSH) cycle, DS and HT significantly downregulated the expression of monodehydroascorbate reductase gene (GhMDHAR) to hinder the production of AsA and upregulated the expression of ascorbate oxidase gene (GhAAO) to promote the oxidation of AsA, which was theoretically detrimental to AsA accumulation. However, HT downregulated the expression of the ascorbate peroxidase gene (GhAPX), hindering the reduction of H2O2 by AsA, which was the reason for AsA and H2O2 accumulation. Moreover, DS also significantly upregulated the expression of the dehydroascorbate reductase gene (GhDHAR2) to enhance the reduction of dehydroascorbate to form AsA, leading to a higher content of AsA under DS than HT. The combined stress significantly downregulated the expression of GhAAO to inhibit the oxidation of AsA but significantly upregulated the expression of GhMDHAR and GhDHAR2, promoting the AsA production, and downregulated the expression of GhAPX, hindering the reduction of H2O2 by AsA. All these resulted in increased AsA content under combined stresses. In addition, HT significantly downregulated the glutathione reductase gene (GhGR) expression, hindering the reduction of oxidized glutathione (GSSG), which led to the reduction of GSH. However, DS and DS+HT significantly downregulated the glutathione peroxidase gene (GhGPX) expression, resulting in the accumulation of GSH. Overall, compared with single-stress treatments, the effects of DS+HT on cotton pollen fertility and peroxide accumulation were more significant. The effects of DS+HT on the antioxidant enzyme system were mainly caused by high temperature, while the mechanism of abnormal accumulation of AsA and GSH caused by DS+HT was different from those of single-stress groups.

Exogenous glutathione maintains the postharvest quality of mango fruit by modulating the ascorbate-glutathione cycle.

Mango fruit is prone to decay after harvest and premature senescence, which significantly lowers its quality and commercial value. The mango fruit (Mangifera indica L.cv. Guixiang) was treated with 0 (control), 2, 5, and 8 mM of reduced glutathione (GSH) after harvest. The fruit was stored at 25 ± 1 °C for 12 days to observe the changes in the antioxidant capacity and postharvest quality. Compared with the control, the 5 mM GSH treatment significantly decreased the weight loss by 44.0% and 24.4%, total soluble solids content by 25.1% and 4.5%, and soluble sugar content by 19.0% and 27.0%. Conversely, the 5 mM GSH treatment increased the firmness by 25.9% and 30.7% on days 4 and 8, respectively, and the titratable acidity content by 115.1% on day 8. Additionally, the 5 mM GSH treatment decreased the malondialdehyde and hydrogen peroxide contents and improved the antioxidant capacity of mango fruit by increasing the superoxide dismutase and peroxidase activities and upregulating the expression of the encoding genes. Meanwhile, the higher levels of monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase enzyme activities and gene expressions accelerated the AsA-GSH cycle, thereby increasing the accumulation of AsA and GSH and maintaining the redox balance. Overall, the experimental results suggest that 5 mM GSH maintains high antioxidant capacity and postharvest quality of mangoes and can use as an effective preservation technique for postharvest mangoes.

Mechanisms associated with the synergistic induction of resistance to tobacco black shank in tobacco by arbuscular mycorrhizal fungi and β-aminobutyric acid.

Tobacco black shank (TBS), caused by Phytophthora nicotianae, is one of the most harmful diseases of tobacco. There are many studies have examined the mechanism underlying the induction of disease resistance by arbuscular mycorrhizal fungi (AMF) and β-aminobutyric acid (BABA) alone, but the synergistic effects of AMF and BABA on disease resistance have not yet been studied. This study examined the synergistic effects of BABA application and AMF inoculation on the immune response to TBS in tobacco. The results showed that spraying BABA on leaves could increase the colonization rate of AMF, the disease index of tobacco infected by P.nicotianae treated with AMF and BABA was lower than that of P.nicotianae alone. The control effect of AMF and BABA on tobacco infected by P.nicotianae was higher than that of AMF or BABA and P.nicotianae alone. Joint application of AMF and BABA significantly increased the content of N, P, and K in the leaves and roots, in the joint AMF and BABA treatment than in the sole P. nicotianae treatment. The dry weight of plants treated with AMF and BABA was 22.3% higher than that treated with P.nicotianae alone. In comparison to P. nicotianae alone, the combination treatment with AMF and BABA had increased Pn, Gs, Tr, and root activity, while P. nicotianae alone had reduced Ci, H2O2 content, and MDA levels. SOD, POD, CAT, APX, and Ph activity and expression levels were increased under the combined treatment of AMF and BABA than in P.nicotianae alone. In comparison to the treatment of P.nicotianae alone, the combined use of AMF and BABA increased the accumulation of GSH, proline, total phenols, and flavonoids. Therefore, the joint application of AMF and BABA can enhance the TBS resistance of tobacco plants to a greater degree than the application of either AMF or BABA alone. In summary, the application of defense-related amino acids, combined with inoculation with AMF, significantly promoted immune responses in tobacco. Our findings provide new insights that will aid the development and use of green disease control agents.

Sustainable and efficient method utilizing N-acetyl-L-cysteine for complete and enhanced ochratoxin A clearance by antagonistic yeast

With the increasing global climate change, ochratoxin A (OTA) pollution in food and environment has become a serious and potential risk element threatening food safety and human health. Biodegradation of mycotoxin is an eco-friendly and efficient control strategy. Still, research works are warranted to develop low-cost, efficient, and sustainable approaches to enhance the mycotoxin degradation efficiency of microorganisms. In this study, the activities of N-acetyl-L-cysteine (NAC) against OTA toxicity were evidenced, and its positive effects on the OTA degradation efficiency of antagonistic yeast, Cryptococcus podzolicus Y3 were verified. Co-culturing C. podzolicus Y3 with 10 mM NAC improved 100% and 92.6% OTA degradation rate into ochratoxin α (OTα) at 1 d and 2 d. The excellent promotion role of NAC on OTA degradation was observed even at low temperatures and alkaline conditions. C. podzolicus Y3 treated with OTA or OTA+NAC promoted reduced glutathione (GSH) accumulation. GSS and GSR genes were highly expressed after OTA and OTA+NAC treatment, contributing to GSH accumulation. In the early stages of NAC treatment, yeast viability and cell membrane were reduced, but the antioxidant property of NAC prevented lipid peroxidation. Our finding provides a sustainable and efficient new strategy to improve mycotoxin degradation by antagonistic yeasts, which could be applied to mycotoxin clearance.

Effects of ozone treatment on gene profiling involved in ASA-GSH cycle in postharvest cantaloupe

Ozone has been proven to be an environmental-friendly method in postharvest storage of cantaloupe with the elimination of reactive oxygen species (ROS), but the mechanism is not clear. In this study, the activity of key enzymes, transcriptomics, and the content of Ascorbic acid(ASA), Reduced glutathione (GSH), H2O2, and Malondialdehyde (MDA) were measured as typical indicators to investigate the effect of ozone treatment on the ascorbic acid-glutathione (ASA-GSH) cycle metabolism of the antioxidant defense system of cantaloupe after harvest. The results showed that the formation of H2O2 and MDA and the accumulation of ASA and GSH in cantaloupe were reduced and increased in the ozone treatment group (OT), respectively. Ozone promoted the activity of the key enzymes of the ASA-GSH cycle in cantaloupe during storage, particularly Ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR), which was confirmed with the variation of H2O2, MDA, ASA, and GSH. The 14 genes associated with the ASA-GSH cycle were identified using transcriptomics, and the FPKM in GR-2, MDHAR-3, and MDHAR-4 were greater in OT than in the control group (CK) on the 10th day, all AO genes were stimulated in OT compared to CK on the 30th day, indicating that AO and MDHAR genes of ASA-GSH cycle may be the target genes induced by ozone treatment in the postharvest cantaloupe. Correlation analysis indicated that the ability of ozone-treated cantaloupe to regulate ROS was a synergistic effect of enzyme activity and gene expression related to the ASA-GSH cycle. This study was to lay a foundation for future studies on the possible effects of ozone on the ASA-GSH cycle of many fruit and vegetable.

Sodium nitroprusside ameliorates lead toxicity in rice (Oryza sativa L.) by modulating the antioxidant scavenging system, nitrogen metabolism, lead sequestration mechanism, and proline metabolism.

As a toxic anthropogenic pollutant, lead (Pb) can be harmful to both plants and animals. Here, the effects of the application of nitric oxide (NO) donor, sodium nitroprusside (SNP, 0, 50, and 100μM), on the morphological, biochemical, and molecular responses of rice plants under Pb (0, 150, and 300μM) toxicity in hydroponic conditions were investigated. Pb stress decreased biomass, photosynthetic pigments, Fv/Fm value, and nitrogen (N) and increased the accumulation of hydrogen peroxide (H2O2), methylglyoxal (MG), malondialdehyde (MDA), and electrolyte leakage (EL) in rice seedlings. However, by improving the metabolism of chlorophyll and proline, SNP increased the content of chlorophyll and proline, restored the performance of the photosynthetic apparatus, and stimulated the growth of Pb-stressed rice seedlings. SNP by reducing the expression of HMA2 and increasing the expression of HMA3 and HMA4 caused the immobilization of Pb in the roots and reduced its transfer to the leaves. Adding SNP increased the activity of antioxidant enzymes and glyoxalase cycle and decreased H2O2, MG, MDA, and EL in the leaves of Pb-stressed rice seedlings. By upregulating the expression of genes GSH1, PCS, and ABCC1, SNP increased the accumulation of GSH and PCs in the roots and leaves and increased the plant's tolerance to Pb stress. By modulating the activity of enzymes involved in N metabolism, SNP increased the concentration of N and nitrate and decreased the concentration of ammonium in the leaves of Pb-stressed seedlings. Our study provides evidence that NO may become a promising tool for increasing the tolerance of rice plants to Pb toxicity.

Transcriptional and metabolic remodeling in clear cell renal cell carcinoma caused by ATF4 activation and the integrated stress response (ISR).

Research has shown extensive metabolic remodeling in clear cell renal cell carcinoma (ccRCC), with increased glutathione (GSH) levels. We hypothesized that activating transcription factor-4 (ATF4) and the integrated stress response (ISR) induce a metabolic shift, including increased GSH accumulation, and that Vitamin A deficiency (VAD), found in ccRCCs, can also activate ATF4 signaling in the kidney. To determine the role of ATF4, we used publicly available RNA sequencing (RNA-seq) data sets from The Cancer Genomics Atlas. Subsequently, we performed RNA-seq and liquid chromatography-mass spectrometry-based metabolomics analysis of the murine TRAnsgenic Cancer of the Kidney (TRACK) model for early-stage ccRCC. To validate our findings, we generated RCC4 cell lines with ATF4 gene edits (ATF4-knockout [KO]) and subjected these cells to metabolic isotope tracing. Analysis of variance, the two-sided Student's t test, and gene set enrichment analysis were used (p < 0.05) to determine statistical significance. Here we show that most human ccRCC tumors exhibit activation of the transcription factor ATF4. Activation of ATF4 is concomitant with enrichment of the ATF4 gene set and elevated expression of ATF4 target genes ASNS, ALDH1L2, MTHFD2, DDIT3 (CHOP), DDIT4, TRIB3, EIF4EBP1, SLC7A11, and PPP1R15A (GADD34). Transcript profiling and metabolomics analyses show that activated hypoxia-inducible factor-1α (HIF1α) signaling in our TRACK ccRCC murine model also induces an ATF4-mediated ISR. Notably, both normoxic HIF1α signaling in TRACK kidneys and VAD in wild-type kidneys diminish amino acid levels, increase ASNS, TRIB3, and MTHFD2 messenger RNA levels, and increase levels of lipids and GSH. By metabolic isotope tracing in human RCC4 kidney cancer parental and ATF4 gene-edited (ATF4-KO) cell lines, we show that ATF4 increases GSH accumulation in part via activation of the mitochondrial one-carbon metabolism pathway. Our results demonstrate for the first time that activation of ATF4 enhances GSH accumulation, increases purine and pyrimidine biosynthesis, and contributes to transcriptional and metabolic remodeling in ccRCC. Moreover, constitutive HIF1α expressed only in murine kidney proximal tubules activates ATF4, leading to the metabolic changes associated with the ISR. Our data indicate that HIF1α can promote ccRCC via ATF4 activation. Moreover, lack of Vitamin A in the kidney recapitulates aspects of the ISR.

Flurochloridone induces responses of free radical reactions and energy metabolism disorders to BRL-3A cell

Flurochloridone (FLC), a wildly used herbicide, could induce hepatotoxicity after long-term exposure to male rat, in addition to its reactive oxygen species (ROS)-dependent reproductive toxicity. The hepatotoxicity effect and mechanism was investigeted using 1, 10 and 100 μmol L−1 FLC treated BRL-3A liver cell in this study. The function of mitochondrial respiration, glycolysis rate and real time ATP production rate are determined by seahorse XF analyzer, and the bio-transformers of FLC, intermediates of TCA cycle and glycolysis, and related amino acids are determined and identified by [U-13C] Glucose metabolic flux technology based on UPLC-HRMS. The mRNA expression of cytochrome P450s and the key regulatory enzymes of glucose metabolism and γ- glutamyl cycle pathway. The protein expressions of protein kinase B (AKT) and glycogen synthase kinase-3 beta (GSK-3β) were determined. The results show dechlorination and glutathione (GSH) conjugate products of FLC are predominant bio-transformmers after 24 h treatment in BRL-3A cell. FLC could enhance glycolysis function and inhibit mitochondrial aerobic respiratory, which is accompanied by the decreased total ATP level and ATP produced rate. Increased glucose-6-phosphate, fructose-6-phosphate, pyruvate and lactate levels, and elevated level of GSH and its precursor 5-glutamate-cysteine (γ-Glu-Cys) are observed in FLC treated cells, which indicates that energy metabolism dysfunction and GSH accumulation could be potentially mediated by activating γ- Glutamyl cycle pathway. Conclusively, FLC induced hepatotoxicity could be potentially related to some free radical reactions, including inhibiting mitochondrial function, glucose metabolism via glycolysis, regulating γ- glutamyl cycle pathway to promote reactive oxygen species (ROS) level, and then induced cell apoptosis by inhibiting AKT/GSK-3β signal.

Glutathione production by non-Saccharomyces yeasts and its impact on winemaking: A review

Glutathione (GSH) is a non-protein thiol naturally present in grape berries and produced by yeasts during fermentation. It has a strong antioxidant activity; thus, the addition of pure GSH during winemaking is recommended to limit the oxidative phenomena of wine, preserving sensory characteristics and stability, ultimately promoting a healthier product by reducing the need for SO2 addition. A promising alternative approach considers the use of yeast starter cultures high producers of this compound in situ, during the fermentation process, in substitution of external GSH addition. Recent research showed that multistarter fermentations with non-Saccharomyces yeasts produce even higher concentrations of GSH compared to single Saccharomyces cerevisiae. Accumulation of GSH in yeast cells is also considered valuable during the growth and dehydration of biomass for starter production, aiding strains to overcome the stressful conditions of industrial process. Moreover, a current trend in oenology is the use during fermentation of inactivated dry yeasts preparations as a source of nutrients, and many of them contain GSH-enriched cells. The aim of this review was to assess the significance of GSH production for the exploitation of wine-related non-Saccharomyces yeasts, both in starter biomass production and during fermentations, which were until now studied in detail for S. cerevisiae. This compendium highlights an interesting new feature of non-conventional yeasts and upgrade the strategy of multistarter fermentation as a valuable tool to positively modulate wine composition.

Centri‐Voltammetric GSH Detection with PDI‐C4SH as a Carrier Material

AbstractIn this study, we developed a centri‐voltammetric electrochemical sensor for glutathione (GSH) detection. For this purpose, N,N′‐2‐octyloxy‐1‐methyl‐2‐oxoethyl)‐(‐2,4‐thiolbutyloxy)‐1‐methyl‐2‐oxoethyl)‐3,4 : 9,10‐perylenetetracarboxyldiimide (PDI‐C4SH) molecule was used as a carrier molecule for the first time. This molecule, interacted with the thiol ends of GSH and formed a disulfide bridge. By this way, PDI‐C4SH improved the accumulation of GSH onto the electrode surface and increased the sensitivity of the detection protocol. All the electrochemical measurements were carried out with differential pulse voltammetry by using a carbon paste electrode. The linear response was obtained in the range of 0.05 mM and 5 mM for GSH concentration (R2=0.9966) while limit of detection value was calculated as 0.017 mM. For the sample application experiments, developed system was applied to the determination of GSH in red wine samples by using the standard addition method, and an acceptable recovery value of 99.03 % was obtained.

TaWRKY74 participates copper tolerance through regulation of TaGST1 expression and GSH content in wheat

Glutathione S-transferase (GST) is the key enzyme in glutathione (GSH) synthesis, and plays a crucial role in copper (Cu) detoxification. Nonetheless, its regulatory mechanisms remain largely unclear. In this study, we identified a Cu-induced glutathione S-transferase 1 (TaGST1) gene in wheat. Yeast one-hybrid (Y1H) screened out TaWRKY74, which was one member from the WRKY transcription factor family. The bindings between TaGST1 promoter and TaWRKY74 were further verified by using another Y1H and luciferase assays. Expression of TaWRKY74 was induced more than 30-folds by Cu stress. Functions of TaWRKY74 were tested by using transiently silence methods. In transiently TaWRKY74-silenced wheat plants, TaWRKY74 and TaGST1 expression, GST activity, and GSH content was significantly inhibited by 25.68%, 19.88%, 27.66%, and 12.68% in shoots, and 53.81%, 52.11%, 23.47%, and 17.11% in roots, respectively. However, contents of hydrogen peroxide, malondialdehyde, or Cu were significantly increased by 2.58%, 12.45%, or 37.74% in shoots, and 25.24%, 53.84%, and 103.99% in roots, respectively. Notably, exogenous application of GSH reversed the adverse effects of transiently TaWRKY74-silenced wheat plants during Cu stress. Taken together, our results suggesting that TaWRKY74 regulated TaGST1 expression and affected GSH accumulation under Cu stress, and could be useful to ameliorate Cu toxicity for crop food safety.

The selective cytotoxicity of silver thiosulfate, a silver complex, on MCF-7 breast cancer cells through ROS-induced cell death

BackgroundSilver is a transition metal that is known to be less toxic than platinum. However, only few studies have reported the anticancer effects of some silver complexes and their possibility as an alternative to platinum complex. This study investigated the anticancer effects of the silver thiosulfate complex (STS), [Ag(S2O3)2]3−, consisting of silver and sodium thiosulfate.MethodsIn vitro cytotoxic activity of STS was investigated comparatively in human cancer cell lines (K562 and MCF-7) and normal human cells (mesenchymal stem cells and mammary epithelial cells). For its anticancer effects, cell cycle, mode of cell death, morphological changes, and accumulation of intracellular ROS and GSH were evaluated in MCF-7 to provide mechanistic insights.ResultsSTS showed a concentration-dependent cytotoxicity in MCF-7 cell, which was abolished by pretreatment with N-acetylcysteine, suggesting ROS accumulation by STS. Moreover, STS caused cell cycle arrest at the G1 phase, decrease in the GSH levels, and morphological changes in MCF-7. Direct measurement of ROS demonstrated the elevation of intracellular ROS accumulation in cancer cells treated with STS; however, neither cytotoxicity nor ROS accumulation was observed in normal human cells.ConclusionThe results obtained here are the first evidence to show that STS exhibited an anticancer activity through ROS-induced mechanisms, and that its cytotoxicity is highly selective to cancer cells. The results of the present study warrant further investigation on the detailed mechanism of STS actions, as well as its in vivo effectiveness and safety for clinical application.

Вплив екзогенного глутатіону на кардіодинаміку і відкривання мітохондріальної пори при ішемії–реперфузії серця щурів

The effect of post-conditioning with reduced glutathione (GSH, hepaval Italy/Ukraine) on myocardial contractility, oxygen cost, and mitochondrial factor release as a marker of mitochondrial permeability transition pore (MPTP) opening was studied in ischemia–reperfusion model at Langendorffisolated rat heart. It was found that reperfusion with KrebsHenseleit solution containing GSH provided more complete restoration of the left ventricle developed pressure (70.2 and 56% at 5th and 40th min of reperfusion against 23.6 and 30.9% in control, P &lt; 0.05 for both), reduced oxygen cost of myocardial work (184 and 157% at 5th and 40th min of reperfusion against 413 and 216% in control, P &lt; 0.05 for both), and decreased the value of mitochondrial factor by 3 times, indicating inhibition of MPTP. It was shown that the level of GSH in cardiac tissues was significantly increased by 1.5 times 30 min after administration of hepaval (52 mg per kg) intraperitoneally, indicating accumulation of GSH from the bloodstream. Thus, we have shown that post-conditioning with GSH had cardioprotective effect, inhibited the formation of MPTP and can be used as a tool for correction of post-ischemic disturbances of heart function.