Glutamate Cysteine Ligase Activity Research Articles

Pharmacological elevation of glutathione inhibits status epilepticus-induced neuroinflammation and oxidative injury

Glutathione (GSH) is a major endogenous antioxidant, and its depletion has been observed in several brain diseases including epilepsy. Previous studies in our laboratory have shown that dimercaprol (DMP) can elevate GSH via post-translational activation of glutamate cysteine ligase (GCL), the rate limiting GSH biosynthetic enzyme and inhibit neuroinflammation in vitro. Here we determined 1) the role of cysteamine as a new mechanism by which DMP increases GSH biosynthesis and 2) its ability to inhibit neuroinflammation and neuronal injury in the rat kainate model of epilepsy. DMP depleted cysteamine in a time- and concentration-dependent manner in a cell free system. To guide the in vivo administration of DMP, its pharmacokinetic profile was determined in the plasma, liver, and brain. The results confirmed DMP's ability to cross the blood-brain-barrier. Treatment of rats with DMP (30 mg/kg) depleted cysteamine in the liver and hippocampus that was associated with increased GCL activity in these tissues. GSH levels were significantly increased (20 %) in the hippocampus 1 h after 30 mg/kg DMP administration. Following DMP (30 mg/kg) administration once daily, a marked attenuation of GSH depletion was seen in the SE model. SE-induced inflammatory markers including cytokine release, microglial activation, and neuronal death were significantly attenuated in the hippocampus with DMP treatment. Taken together, these results highlight the importance of restoring redox status with rescue of GSH depletion by DMP in post epileptogenic insults.

262 Weaning-Induced Changes in Tissue Cysteine Utilization in Pigs

Abstract Weaning stress in pigs is associated with low and variable feed intake, poor nutrient utilization, and high feed costs. Cysteine is a sulfur amino acid with key roles in pig production, but how cysteine metabolism and requirements are affected by weaning stress should be better defined. The objective of this study was to determine weaning-induced changes in tissue cysteine metabolism. Pigs (initial BW = 7.23 ± 0.68 kg) were either weaned at 21 d age and euthanized at 23 d age (W; n = 9) or remained with the sow and euthanized at 23 d age (NW; n = 9). Weaned pigs were fasted to simulate acute nutritional stress but had free access to water. At euthanasia, blood, liver, jejunum, and ileum tissues were collected. Plasma and tissue amino acids and amino thiols were analyzed by HPLC. The abundance of proteins central to liver cysteine utilization were determined by immunoblot and enzyme activity assays. Glutathione (GSH) concentrations were less in jejunum (1,379 versus 1,720 ± 70 nmol/g; P < 0.01) and ileum (1,497 versus 1,740 ± 74 nmol/g; P < 0.05) in W than NW pigs. However, liver GSH concentration was not different between W and NW pigs (P > 0.10). While cysteine in jejunum and ileum were not affected by weaning (P > 0.10), γ-glutamylcysteine (γ-GC), the immediate precursor of GSH, declined in both jejunum (9.7 versus 14.3 ± 1.4 nmol/g; P < 0.01) and ileum (6.4 versus 11.2 ± 0.8 nmol/g; P < 0.001) in W compared with NW pigs. In liver, cysteine concentration was greater in W compared with NW pigs (417 versus 298 ± 33 nmol/g; P < 0.05). Although liver cysteine was greater in W pigs, liver activity of glutamate cysteine ligase (GCL; 2.49 versus 4.15 ± 0.25 nmol γ-GC/(mg protein•min); P < 0.001) and glutathione synthetase (GSS; 1.54 versus 1.92 nmol GSH/(mg protein•min); P < 0.001), enzymes needed for GSH production, were less in W than NW pigs. Immunoblot analysis of the catalytic subunit of GCL (GCLC; P < 0.05) and modifier subunit of GCL (GCLM; P = 0.057) were also lower in W compared with NW pigs, but GSS was not different between groups (P > 0.10). While plasma taurine was lower in W compared with NW pigs (64 versus 128 ± 7 µmol/L, P < 0.001), liver taurine was greater in W compared with NW pigs (4,726 versus 2,250 ± 471 nmol/g; P < 0.01). Despite an increase systemic and liver availability of cysteine, the rate-limiting amino acid for GSH synthesis, small intestine GSH concentrations decline. This suggests that systemic cysteine is not effectively utilized for gut GSH production in pigs after weaning.

Covalent Targeting of Glutamate Cysteine Ligase to Inhibit Glutathione Synthesis.

Dysregulated oxidative stress plays a major role in cancer pathogenesis and some types of cancer cells are particularly vulnerable to inhibition of their cellular antioxidant capacity. Glutamate-cysteine ligase (GCL) is the first and rate-limiting step in the synthesis of the major cellular antioxidant glutathione (GSH). Developing a GCL inhibitor may be an attractive therapeutic strategy for certain cancer types that are particularly sensitive to oxidative stress. In this study, we reveal a cysteine-reactive ligand, EN25, that covalently targets an allosteric cysteine C114 on GCLM, the modifier subunit of GCL, and leads to inhibition of GCL activity. This interaction also leads to reduced cellular GSH levels and impaired cell viability in ARID1A-deficient cancer cells, which are particularly vulnerable to glutathione depletion, but not in ARID1A-positive cancer cells. Our studies uncover a novel potential ligandable site within GCLM that can be targeted to inhibit GSH synthesis in vulnerable cancer cell types.

Copper Exposure Induced Chicken Hepatotoxicity: Involvement of Ferroptosis Mediated by Lipid Peroxidation, Ferritinophagy, and Inhibition of FSP1-CoQ10 and Nrf2/SLC7A11/GPX4 Axis.

Copper (Cu) is one of the most significant trace elements in the body, but it is also a widespread environmental toxicant health. Ferroptosis is a newly identified programmed cell death, which involves various heavy metal-induced organ toxicity. Nevertheless, the role of ferroptosis in Cu-induced hepatotoxicity remains poorly understood. In this study, we found that 330 mg/kg Cu could disrupt the liver structure and cause characteristic morphological changes in mitochondria associated with ferroptosis. Additionally, Cu treatment increased MDA (malondialdehyde) and LPO (lipid peroxide) production while reducing GSH (reduced glutathione) content and GCL (glutamate cysteine ligase) activity. However, it is noticeable that there were no appreciable differences in liver iron content and key indicators of iron metabolism. Meanwhile, our further investigation found that 330 mg/kg Cu-exposure changed multiple ferroptosis-related indicators in chicken livers, including inhibition of the expression of SLC7A11, GPX4, FSP1, and COQ10B, whereas enhances the levels of ACLS4, LPCAT3, and LOXHD1. Furthermore, the changes in the expression of NCOA4, TXNIP, and Nrf2/Keap1 signaling pathway-related genes and proteins also further confirmed 330 mg/kg Cu exposure-induced ferroptosis. In conclusion, our results indicated that ferroptosis may play essential roles in Cu overload-induced liver damage, which offered new insights into the pathogenesis of Cu-induced hepatotoxicity.

Simulation modeling of glutamate cysteine ligase activity

L-Y-glutamyl-L-cysteinyl glycine, or glutathione, as one of the basic intracellular antioxidants, plays a vital role in cellular metabolism. In mammalian cells, glutathione is synthesized via two steps. The first step that is considered rate limiting is catalyzed by glutamate cysteine ligase. In this work, a stochastic algorithm based on continuous-time Markov chains was used to simulate the activity of glutamate-cysteine ligase. Several different mechanisms of enzymatic activity including reversible inhibition of glutathione, and an ATP binding motif have been considered. Based on physiological metabolite measurements made for human erythrocytes, the activity of glutamate cysteine ligase was determined. There are many possible ways for substrates to bind to an active site of the studied enzyme, but, only the mechanism by which primary binding to ATP can occur makes it possible to obtain the catalytic rate value similar to that of the experimentally measured glutamatecysteine ligase activity relative to physiological concentrations of substrates. In other cases, the values differ by more than one order of magnitude. The performed analysis allows the conclusion that when models for glutathione biosynthesis are constructed in vivo conditions, the ATP concentration and reversible inhibition of glutathione should be taken into account.

Azithromycin ameliorated cigarette smoke-induced airway epithelial barrier dysfunction by activating Nrf2/GCL/GSH signaling pathway

BackgroundAirway epithelium is the first barrier against environmental insults, and epithelial barrier dysfunction caused by cigarette smoke (CS) is particularly relevant to chronic obstructive pulmonary disease (COPD) progression. Our study was to determine whether Azithromycin (AZI) ameliorates CS-induced airway epithelial barrier dysfunction and the underlying mechanisms.MethodsPrimary bronchial epithelial cells (PBECs), human bronchial epithelial cells (HBECs), Sprague Dawley rats and nuclear factor erythroid 2-related factor 2 (Nrf2)−/− mice were pretreated with AZI and subsequently exposed to CS. Transepithelial electronic resistance (TEER), junction proteins as well as pro-inflammatory cytokines and apoptosis markers were examined to assess epithelial barrier dysfunction. Metabolomics study was applied to explore the underlying mechanism of AZI.ResultsCS-induced TEER decline and intercellular junction destruction, accompanied with inflammatory response and cell apoptosis in PBECs were restored by AZI dose-dependently, which were also observed in CS-exposed rats. Mechanistically, GSH metabolism pathway was identified as the top differentially impacted pathway and AZI treatment upregulated the activities of glutamate cysteine ligase (GCL) and the contents of metabolites in GSH metabolic pathway. Furthermore, AZI apparently reversed CS-induced Nrf2 suppression, and similar effects on airway epithelial barrier dysfunction were also found for Nrf2 agonist tert-butylhydroquinone and vitamin C. Finally, deletion of Nrf2 in both HBECs and C57BL/6N mice aggravated CS-induced GSH metabolism imbalance to disrupt airway epithelial barrier and partially deprived the effects of AZI.ConclusionThese findings suggest that the clinical benefits of AZI for COPD management are related with the protection of CS-induced airway epithelial barrier dysfunction via activating Nrf2/GCL/GSH pathway, providing potential therapeutic strategies for COPD.Graphical abstract

Redox regulation of enzymes involved in sulfate assimilation and in the synthesis of sulfur-containing amino acids and glutathione in plants.

Sulfur is essential in plants because of its presence in numerous molecules including the two amino acids, cysteine, and methionine. Cysteine serves also for the synthesis of glutathione and provides sulfur to many other molecules including protein cofactors or vitamins. Plants absorb sulfate from their environment and assimilate it via a reductive pathway which involves, respectively, a series of transporters and enzymes belonging to multigenic families. A tight control is needed to adjust each enzymatic step to the cellular requirements because the whole pathway consumes energy and produces toxic/reactive compounds, notably sulfite and sulfide. Glutathione is known to regulate the activity of some intermediate enzymes. In particular, it provides electrons to adenosine 5′-phosphosulfate reductases but also regulates the activity of glutamate-cysteine ligase by reducing a regulatory disulfide. Recent proteomic data suggest a more extended post-translational redox control of the sulfate assimilation pathway enzymes and of some associated reactions, including the synthesis of both sulfur-containing amino acids, cysteine and methionine, and of glutathione. We have summarized in this review the known oxidative modifications affecting cysteine residues of the enzymes involved. In particular, a prominent regulatory role of protein persulfidation seems apparent, perhaps because sulfide produced by this pathway may react with oxidized thiol groups. However, the effect of persulfidation has almost not yet been explored.

Gclc overexpression inhibits apoptosis of bone marrow mesenchymal stem cells through the PI3K/AKT/Foxo1 pathway to alleviate inflammation in acute lung injury

BackgroundAcute lung injury (ALI) represents a serious heterogenous pulmonary disorder with high mortality. Bone marrow mesenchymal stem cells (BMSCs) have a good therapeutic effect on ALI, but their survival rate in vivo is not high. GCLc has all the activities of Glutamate cysteine ligase (GCL) and can reduce reactive oxygen species, antioxidant stress response and improve cell survival. Therefore, in our study, overexpressing GCLc BMSCs were constructed by lentiviral transduction and intratracheally transplanted into ALI mice to evaluate their therapeutic effects, and we explored the mechanism of anti-apoptosis of GCLc in BMSCs. MethodsOverexpressing GCLc hBMSCs were constructed using lentiviral vectors. The cell viability of MSCs was detected by CCK-8 assay. GSH, MDA, SOD and ROS were detected by the manufacturer's kit. Western blot and RT-qPCR were used to detect the expression of GCLc, bax, bcl2, cleaved-caspase 3, caspase 3, cleaved-caspase 9, caspase 9 and Foxo1 in BMSCs stimulated by H2O2. Apoptosis of BMSCs was analyzed by flow cytometry, JC-1 and TUNEL method. Confocal microscopy was to observe the nuclear extracellular migration of Foxo1. We then examined the expression levels of the pathway proteins by Western blot. In ALI animal model, we evaluated the therapeutic effect of the overexpressing GCLc BMSCs by H&E staining, in vitro imaging, wet/dry weight ratio of lung tissue, and extraction of bronchoalveolar lavage fluid from mice to analyze protein concentrations, neutrophil, leukocyte and macrophage counts and ELISA for inflammatory factors. ResultsWe demonstrated that overexpression of GCLc reduced MDA and ROS and increased GSH and SOD, while GCLc reduced the expression of pro-apoptotic proteins (bax, cleaved-caspase 3, caspase 3, cleaved-caspase 9, caspase 9) and elevated the expression of anti-apoptotic proteins (bcl-2) in BMSCs. We verified that it acts through the PI3K/AKT/Foxo1 pathway. In ALI vivo, overexpression of GCLc BMSCs had a longer retention time in the lung compared to vector BMSC and improved pulmonary edema, decreased alveolar protein concentration and reduced TNF-α, IL-1β, IL-6 levels and increased IL-10 levels in the lung. ConclusionsThese results show that GCLc overexpressing BMSCs with anti-apoptotic effects significantly improve acute lung injury.

N-cystaminylbiguanide MC001 prevents neuron cell death and alleviates motor deficits in the MPTP-model of Parkinson’s disease

Parkinson’s disease (PD) is a common neurodegenerative disease characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra (SN), which is highly associated with oxidative stress. Antioxidants are therefore considered as potential therapies in PD treatment. In this study, we examined the neuroprotective effect of a cysteamine-based biguanide N-cystaminylbiguanide (MC001) in the MPTP mouse model of PD. The results showed that MC001 prevented neuron cell death and alleviated motor deficits in the MPTP mouse model of PD. Both in vitro and in vivo data indicated that MC001 may exert its neuroprotective effect by alleviating ROS production, suppressing neuroinflammation, and upregulating BDNF expression. Further mechanistic studies revealed that MC001 promoted GSH synthesis by inducing the expression of Glutamate-cysteine ligase catalytic subunit (Gclc) and enhancing the activity of Glutamate-cysteine ligase (Gcl). Our results suggest that MC001 warrants further investigation as a potential candidate for the treatment of PD.

Elevated Temperature and Exposure to Copper Leads to Changes in the Antioxidant Defense System of the Reef-Building Coral Mussismilia harttii.

The frequency and severity of coral bleaching events have increased in recent years. Global warming and contamination are primarily responsible for triggering these responses in corals. Thus, the objective of this study was to evaluate the isolated and combined effects of elevated temperature and exposure to copper (Cu) on responses of the antioxidant defense system of coral Mussismilia harttii. In a marine mesocosm, fragments of the coral were exposed to three temperatures (25.0, 26.6, and 27.3°C) and three concentrations of Cu (2.9, 5.4, and 8.6 μg/L) for up to 12 days. Levels of reduced glutathione (GSH) and the activity of enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glutamate cysteine ligase (GCL), were evaluated on the corals and symbionts. The short exposure to isolated and combined stressors caused a reduction in GSH levels and inhibition of the activity of antioxidant enzymes. After prolonged exposure, the combination of stressors continued to reduce GSH levels and SOD, CAT, and GCL activity in symbionts and GST activity in host corals. GCL activity was the parameter most affected by stressors, remaining inhibited after 12-days exposure. Interesting that long-term exposure to stressors stimulated antioxidant defense proteins in M. harttii, demonstrating a counteracting response that may beneficiate the oxidative state. These results, combined with other studies already published suggest that the antioxidant system should be further studied in order to understand the mechanisms of tolerance of South Atlantic reefs.

Non-canonical Glutamate-Cysteine Ligase Activity Protects against Ferroptosis

Cysteine is required for maintaining cellular redox homeostasis in both normal and transformed cells. Deprivation of cysteine induces the iron-dependent form of cell death known as ferroptosis; however, the metabolic consequences of cysteine starvation beyond impairment of glutathione synthesis are poorly characterized. Here, we find that cystine starvation of non-small-cell lung cancer cell lines induces an unexpected accumulation of γ-glutamyl-peptides, which are produced due to a non-canonical activity of glutamate-cysteine ligase catalytic subunit (GCLC). This activity is enriched in cell lines with high levels of NRF2, a key transcriptional regulator of GCLC, but is also inducible in healthy murine tissues following cysteine limitation. γ-glutamyl-peptide synthesis limits the accumulation of glutamate, thereby protecting against ferroptosis. These results indicate that GCLC has a glutathione-independent, non-canonical role in the protection against ferroptosis by maintaining glutamate homeostasis under cystine starvation.

Chlorothalonil causes redox state change leading to oxidative stress generation in Danio rerio

A diverse range of chemicals are used in agriculture to increase food production on a large scale, and among them is the use of pesticides such as chlorothalonil, a broad-spectrum fungicide used in the control of foliar fungal diseases. This study aimed to elucidate the effects of chlorothalonil on biochemical biomarkers of oxidative stress in tissues of the fish Danio rerio. To achieve this, animals were exposed for 4 and 7 days, to nominal concentrations of chlorothalonil at 0 μg/L (DMSO, 0.001%), 0.1 μg/L and 10 μg/L, and after the exposure period, the tissues (gills and liver) were removed for biochemical analysis. Antioxidant capacity against peroxyl radicals (ACAP) and enzyme activities, such as superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and glutamate cysteine ligase (GCL), were evaluated in both tissues. In addition, the concentration of reactive oxygen species (ROS), reduced glutathione (GSH) and lipid peroxidation (LPO) levels were also analysed. A significant increase in ROS concentration, ACAP levels, GST and GCL activities and a significant reduction of LPO levels in gills exposed to the highest concentration were observed after 4 days. However, there was a significant reduction of ACAP and CAT activity, as well as a significant increase of GST activity and LPO levels in gills exposed to the lower concentration after 7 days. The liver was less affected, presenting a significant reduction in CAT activity and LPO levels after 4 days. However, a significant increase in SOD activity and LPO levels occurred after 7 days. These results indicate that chlorothalonil, after 4 days, caused activation of the antioxidant defence system in gills of animals exposed to the highest concentration. However, after 7 days, the lowest concentration of this compound caused oxidative stress in this same organ. Also, the results show that gills were more affected than the liver, probably because gills can be involved in chlorothalonil metabolisation. Therefore, it is possible that the liver could be exposed to lower chlorothalonil concentrations or less toxic metabolites due to the metabolism taking place in the gills.

Anti-inflammatory mechanism and active ingredients of the Chinese tallow tree.

The leaf of the Chinese tallow tree (CTT, Sapium sebiferum (L.) Roxb) has been used in Traditional Chinese Medicine (TCM) to treat eczema, shingles, edema, swelling, ascites, scabs, and snakebites. The present work aimed to explore the antioxidant-related anti-inflammatory mechanisms of CTT leaf and to further investigate their possible active ingredients. The anti-inflammatory activities of different fractions were determined using a 12-O-tetradecanoylphorbol-13-acetate (TPA) induced model of acute edema in mouse ears. The SOD, CAT and GCL activities and the GSH content of the ear tissue were measured using kits, and the ratio of the treated and control ears was calculated. The anti-inflammatory activities of each single compound and those of a mixture of the compounds were also determined using the TPA-induced model. The anti-inflammatory effects of the three fractions were positively correlated with their increasing GSH capacities. Although the GSH levels decreased during TPA-induced acute edema, the CTT leaf extract could recover these levels by increasing the glutamate cysteine ligase activity. The mixture of ellagic acid, isoquercitrin and astragalin showed an anti-inflammatory effect similar to that of the CTT leaf extract. However, none of these three individual compounds showed comparable activity alone. These results demonstrated that increasing GSH is an antioxidant-related anti-inflammatory mechanism of CTT leaves. In addition, ellagic acid, isoquercitrin and astragalin were found to be jointly responsible for this bioactivity.

Aqueous particulate matter (PM2.5) from Brazil alters antioxidant profile responses and causes oxidative stress

In vitro assays were used to determine the effects of aqueous fine particulate matter (PM2.5) extracts on different homogenates of rat organs. The PM2.5 collected from two regions (Seropédica and Duque de Caxias) in Rio de Janeiro, Brazil, were used to evaluate different oxidative damages and redox state. Two PM2.5 extract concentrations (concentrate and 10x dilution) were evaluated using rat tissue homogenates (heart, lung, liver, and kidney). Measurements of total antioxidant capacity (ACAP), lipid peroxidation levels (LPO), reduced glutathione concentrations (GSH), activity of glutamate cysteine ligase (GCL), activity of glutathione S-transferase (GST) and catalase (CAT) were performed. Most PM2.5 aqueous extract concentrations contain numerous metals (e.g. Li, Ti, Fe, Cu, Cd, Sr, among others), which reduced ACAP and generated LPO in most tissue homogenates. Besides this, GST activity decreased in liver and lung, increasing in kidney and heart homogenates. GCL activity increased in lung, liver and kidney and undergo decrease in the heart homogenate. All concentrations of PM2.5 evaluated decreased GSH levels. PM2.5 concentrations for both sites (regions) raised catalase activity in most tissue homogenates except in the liver. In vitro toxicity of the PM2.5 in different tissues, presented an ability to interact with cellular antioxidant defense system, causing depletion of the sulfhydryl stocks, resulting in the damage of biological molecules and inducing oxidative imbalance.

The impact of polar fraction of the fine particulate matter on redox responses in different rat tissues.

Particulate matter (PM) contains different chemical substances that have been associated with health effects and an increased risk of mortality due to their toxicity. In this study, fine particulate matter (PM2.5) samples were collected in a region with rural characteristics (Seropédica (Se)) and another with some industries (Duque de Caxias (DC)) (Brazil, RJ). Rats were exposed to PM2.5 extracts daily for 25 days at different dilutions: 10×, 5×, and a concentrated solution (CS). Biochemical analyses were investigated for total antioxidant capacity (ACAP), lipid peroxidation (LPO) levels, reduced glutathione (GSH) concentration, activity of glutamate cysteine ligase (GCL), and activity of glutathione S-transferase (GST). The liver showed a significant increase in GCL (DC-5×, DC-CS and Se-CS) and GST activities (DC-CS and Se-CS) in both regions when compared to the control group. In the renal cortex, GCL activity decreased in most of the tested groups while GST activity increased only in the 5× groups of both regions (DC and Se). In the renal medulla, GCL activity decreased for Se-10× and DC-CS but increased for Se-5×, and GST activity increased in the Se-10×, DC-5×, and DC-CS groups. Lung GCL increased in all groups for both regions. Moreover, this organ also showed an increase in GST activity when higher metal concentrations were present (5× and CS). TBARS levels were increased for all tissues in most tested concentrations. These data indicate that soluble compounds (e.g., metals) from PM2.5 sampled in areas with different pollution indexes can change the redox status and cause damage to different tissues.

Multigenerational effects of ecotoxicological interaction between arsenic and silver nanoparticles

Despite arsenic being one of the most widely distributed toxicants over the world, its reported occurrence in Latin America is relatively low when compared to North America, Europe, and Asia. Regardless, it still requires proper assessment; especially considering its potential interaction with other new contaminants, such as nanomaterials. In the present study, a brief introduction to arsenic contamination in Latin America is made, its main methods of intake by humans are described, and concerns regarding its interaction with other contaminants, especially nanomaterials, are presented. A multigenerational approach through five generations of Caenorhabditis elegans was performed to assess, at ecotoxicological relevant concentrations, the toxic effects of arsenic/nanomaterial interaction through the measurement of biochemical and physiological parameters. The reactive oxygen species (ROS) and lipid peroxidation levels were assessed, levels of reduced glutathione (GSH) determined, and the enzyme activities of glutamate cysteine ligase (GCL), glutathione-S-transferase (GST), and catalase (CAT) were measured. Fertility, reproduction, and growth parameters were also assessed and compared. A nanomaterial modulatory effect was found to accentuate the toxicity of arsenic on some of the most important enzymes in the antioxidant defense system; deleterious effects were also found in the development and reproduction of the animals throughout the generations. This work shows that multigenerational approaches can unveil toxic effects that would remain cryptic when evaluated in only one generation.

The Effect of Dietary Quercetin on the Glutathione Redox System and Small Intestinal Functionality of Weaned Piglets.

Quercetin has been shown to alleviate mucosal damage and modulate the glutathione (GSH) redox system in the colon of rodents. In the current study, we assessed whether quercetin was able to mitigate small intestinal dysfunction in weaned pigs. Here, 224 weaned piglets were fed a diet containing quercetin at either 0, 100, 300, or 900 mg/kg diet until d14 post-weaning, followed by a common basal diet until d42. Eight animals per treatment were sampled at d5 and d14 post-weaning. In these animals, the small intestinal histomorphology, barrier function, and protein abundance of occludin, caspase-3, and proliferating cell nuclear antigen were assessed. None of these parameters were affected, and neither did quercetin improve performance up to d42 post-weaning. The GSH redox system was evaluated in blood, small intestinal mucosa, and liver. Quercetin did not affect the glutathione peroxidase, glutathione reductase, and glutamate–cysteine ligase activity in these tissues. In contrast, the hepatic glutathione transferase (GST) activity was significantly increased by quercetin supplementation at d5 post-weaning of 100, 300, and 900 mg/kg. Importantly, d5 was characterized by a more oxidized GSH redox status. To conclude, dietary quercetin had little effect on the small intestine, but did upregulate hepatic GST in the occurrence of redox disturbance.

Down-regulation of GCLC is involved in microcystin-LR-induced malignant transformation of human liver cells

Microcystin-LR (MCLR) is a potent hepatotoxin which could lead to the development of hepatocellular carcinoma. However, the mechanisms of its carcinogenic action remain obscure. The catalytic subunit of glutamylcysteine ligase (GCLC) primarily regulates de novo synthesis of glutathione and is central to the antioxidant capacity of the cell, but emerging data suggest that the GCLC expression is associated with cancer development. The purpose of this study was to investigate the role and molecular mechanisms of GCLC in MCLR-induced malignant transformation of a human liver cell line WRL68. During MCLR-induced cell transformation, the expression of GCLC and activity of glutamate-cysteine ligase (GCL) decreased continuously, accompanied with consistent low levels of glutathione (GSH) but high levels of oxidative DNA damages. Furthermore, MCLR markedly inhibited protein phosphatase 2 A (PP2 A), and increased the level of GCLC phosphorylation. In contrast, overexpression of GCLC significantly enhanced the levels of GSH, inhibited oxidative DNA damages, and suppressed MCLR-induced cell invasion and migration, as well as tumor growth in nude mice. GCLC overexpression partially attenuated MCLR-induced PP2 A inhibition. Together, the current results suggest that down-regulation of GCLC is involved in MCLR-induced malignant transformation of human liver cells by inducing oxidative stress.

NOX1/NADPH oxidase regulates the expression of multidrug resistance-associated protein 1 and maintains intracellular glutathione levels.

The involvement of superoxide-generating NADPH oxidase (NOX) in the cytotoxic effects of cigarette smoke extracts has been documented. However, the underlying molecular mechanisms and NOX isoform involved have not been fully clarified. Among the different NADPH oxidase isoforms identified so far, NOX1 and NOX4 were found to be expressed in rat H9c2 cardiomyocytes. When H9c2 cells were exposed to acrolein or methyl vinyl ketone (MVK), major toxic components of cigarette smoke extracts, a dose-dependentdecline in cell viability was observed. Unexpectedly, disruption of Nox1 as well as Nox4 significantly exacerbated cytotoxicity induced by acrolein or MVK. Compared with Nox4-disrupted cells, Nox1-disrupted cells were more vulnerable to acrolein and MVK at lower concentrations. Disruption of Nox1 markedly attenuated the levels of total and reduced glutathione (GSH) in H9c2 clones. Reduction in the cystine level in the culture medium to deplete intracellular GSH significantly exacerbated acrolein or MVK-induced cytotoxicity. Nox1 disruption neither attenuated the level of glutamate-cystine antiporter protein nor the activity of glutamate-cysteine ligase, both rate-limiting factors for GSH synthesis. On the other hand, increased expression of multidrug resistance-associated protein 1 (MRP1), which mediates glutathione efflux, was demonstrated in Nox1-disrupted cells. The augmented toxicity of acrolein and MVK in these cells was partially but significantly blunted in the presence of an MRP1 inhibitor, reversan. Taken together, these results show that NOX1/NADPH oxidase regulates the expression of MRP1 to maintain intracellular GSH levels in cardiomyocytes and protect against cytotoxic components of cigarette smoke extracts. A novel crosstalk between NOX1 and MRP1 was demonstrated in this study.

Growth, antioxidant system, and immunological status of shrimp in bioflocs and clear water culture systems

Abstract: The objective of this work was to evaluate the effect of the traditional culture system in clear water and of the one in microbial flocs on the antioxidant and immunological status of Litopenaeus vannamei shrimp. Samples (gill, hemolymph, and hepatopancreas) were collected on days 15, 30, and 60 of the experimental period. The following immunological parameters were analyzed at each evaluation time: granular and hyaline hemocytes, total protein, and apoptosis. Assays on glutamate-cysteine ligase (GCL) activity and on the concentrations of reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were also performed. The evaluated immunological parameters did not differ significantly between treatments. Shrimp reared in clear water showed higher levels of lipid peroxidation in the gills and of GCL activity in the hemolymph on days 15 and 30. Shrimp in microbial floc had a higher survival rate, and the water did not have to be renewed during the experimental period. The microbial floc system improves production levels and provides a healthier culture environment.