Cysteine Ligase Catalytic Subunit Research Articles

Puerarin induces the upregulation of glutathione levels and nuclear translocation of Nrf2 through PI3K/Akt/GSK-3β signaling events in PC12 cells exposed to lead

Oxidative stress is thought to be involved in lead-induced toxicity, especially affecting the brain. We reported previously that puerarin possesses antioxidative properties in the nervous system. Therefore, the aim of the present study was to test the hypothesis that puerarin inhibits lead acetate-induced oxidative stress in PC12 cells by interrupting phosphatidylinositol-3 kinase (PI3K)/Akt signaling through increasing glutathione (GSH) synthesis. Our results showed that puerarin attenuates oxidative stress in a concentration-dependent manner in PC12 cells exposed to lead acetate demonstrated by scavenging reactive oxygen species (ROS) and reducing lipid peroxidation (LPO). Treatment with puerarin significantly up-regulates glutamate cysteine ligase catalytic subunit (GCLc) expression both at its mRNA and protein levels, but not glutamate cysteine ligase modifier (GCLm) subunit, accompanying the elevation of cellular glutathione level. The increased nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) was not because of increased transcription of Nrf2 as Nrf2 transcript levels did not change after puerarin treatment. The effects of puerarin could be partially blocked by pharmacologic inhibition of PI3K and the glycogen synthase kinase 3β (GSK-3β) pathways with LY294002 and LiCl, respectively. On the other hand, puerarin treatment promoted Akt and GSK-3β phosphorylation in PC12 cells exposed to lead acetate. Moreover, puerarin failed to modulate the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), p-c-Jun N-terminal kinases (JNK), and p-p38 mitogen-activated protein kinase (MAPK) demonstrating some specificity for its action on the PI3K/GSK-3β pathway. These findings suggest that puerarin as a phytoestrogen might be an attractive agent for prevention and treatment of chronic diseases related to lead neurotoxicity.

Hydrogen Sulfide Upregulates Glutamate–Cysteine Ligase Catalytic Subunit, Glutamate–Cysteine Ligase Modifier Subunit, and Glutathione and Inhibits Interleukin-1β Secretion in Monocytes Exposed to High Glucose Levels

Glutathione (GSH) deficiency and interleukin-1β (IL-1β) upregulation are linked to the progression of vascular inflammation and atherosclerosis. The consumption of sulfide-rich vegetables is known to lower the risk of atherosclerosis. This study examined the hypothesis that hydrogen sulfide (H2S) upregulates the glutamate-cysteine ligase catalytic subunit (GCLC) and GSH and inhibits IL-1β in a monocyte cell model. U937 monocytes were supplemented with H2S (0-12.5 μM) for 2 hr and then exposed to a control or high glucose (HG, 25 mM) for 22 hr. Levels of GCLC and glutamate-cysteine ligase modifier subunit (GCLM) expression were determined by western blotting and GSH using high-performance liquid chromatography (HPLC), and IL-1β using enzyme-linked immunoassay (ELISA). H2S significantly (P<0.05) upregulated expression of GCLC and GCLM, and formation of GSH, and inhibited IL-1β secretion in controls and HG-treated monocytes. This is the first demonstration of H2S upregulation of GCLC and GSH and inhibition of IL-1β levels, which may be what mediates the beneficial effects of H2S-rich compounds in mitigating the pathogenesis of metabolic syndrome and atherosclerosis.

Sex Differences in the Acute Nasal Antioxidant/Antielectrophilic Response of the Rat to Inhaled Naphthalene

Naphthalene is a nasal carcinogen, inducing respiratory adenomas in male and olfactory neuroblastomas in female rats, respectively. The reasons for the site and sex-specific tumorigenic response are unknown. Naphthalene is bioactivated to electrophilic metabolites; cytotoxicity followed by regenerative cell proliferation is likely involved in the tumorigenic response. To examine sex differences in the acute nasal response to naphthalene, male and female F344 rats were nose-only exposed to 0, 1, 3, 10, or 30 ppm naphthalene vapor for 4 or 6 h. Following exposure, respiratory/transitional mucosa (RTM) and olfactory mucosa (OM) were isolated and analyzed for markers of oxidant/electrophilic stress and/or toxicity, including reduced/oxidized glutathione levels (GSH/GSSG), mRNA levels of electrophile-responsive genes, and epithelial cytoxicity (as measured by membrane permeability to ethidium homodimer-1). Naphthalene caused significant depletion of GSH in RTM and OM with no increase in GSSG. Cytotoxicity was apparent at concentrations of 15 and 30 ppm. No consistent sex differences were observed in these responses. Sex differences were observed in the induction of antielectrophilic genes in OM: glutamyl cysteine ligase (catalytic subunit) (Gclc), NADPH quinone oxidase 1 (Nqo1), and heme oxygenase 1 (Hmox1) were all induced to a greater extent in the male OM compared with the female. No consistent sex differences were observed in the RTM. Although the mechanism of the sex difference in the RTM adenoma response remains enigmatic, sex differences in the induction of antioxidant/electrophile-responsive genes may contribute to the heightened sensitivity of the female OM to the carcinogenic effects of naphthalene.

Proteomics-Based Identification of Differentially Abundant Proteins from Human Keratinocytes Exposed to Arsenic Trioxide.

IntroductionArsenic is a widely distributed environmental toxicant that can cause multi-tissue pathologies. Proteomic assays allow for the identification of biological processes modulated by arsenic in diverse tissue types.MethodThe altered abundance of proteins from HaCaT human keratinocyte cell line exposed to arsenic was quantified using a label-free LC-MS/MS mass spectrometry workflow. Selected proteomics results were validated using western blot and RT-PCR. A functional annotation analytics strategy that included visual analytical integration of heterogeneous data sets was developed to elucidate functional categories. The annotations integrated were mainly tissue localization, biological process and gene family.ResultThe abundance of 173 proteins was altered in keratinocytes exposed to arsenic; in which 96 proteins had increased abundance while 77 proteins had decreased abundance. These proteins were also classified into 69 Gene Ontology biological process terms. The increased abundance of transferrin receptor protein (TFRC) was validated and also annotated to participate in response to hypoxia. A total of 33 proteins (11 increased abundance and 22 decreased abundance) were associated with 18 metabolic process terms. The Glutamate--cysteine ligase catalytic subunit (GCLC), the only protein annotated with the term sulfur amino acid metabolism process, had increased abundance while succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial precursor (SDHB), a tumor suppressor, had decreased abundance.ConclusionA list of 173 differentially abundant proteins in response to arsenic trioxide was grouped using three major functional annotations covering tissue localization, biological process and protein families. A possible explanation for hyperpigmentation pathologies observed in arsenic toxicity is that arsenic exposure leads to increased iron uptake in the normally hypoxic human skin. The proteins mapped to metabolic process terms and differentially abundant are candidates for evaluating metabolic pathways perturbed by arsenicals.

Isothiocyanates protect against oxidized LDL-induced endothelial dysfunction by upregulating Nrf2-dependent antioxidation and suppressing NFκB activation

Oxidative stress plays a pivotal role in the pathophysiology of cardiovascular diseases. Oxidized low-density lipoprotein (oxLDL) is a key contributor to atherogenesis through multiple mechanisms. In this study, we investigated the protection by three structurally related isothiocyanates, i.e., sulforaphane (SFN), benzyl isothiocyanate (BITC), and phenethyl isocyanate (PEITC), against oxLDL-induced leukocyte adhesion to vascular endothelium and the mechanism involved. The protection against oxLDL-induced endothelial dysfunction by isothiocyanates was studied in human umbilical vein endothelial cells (HUVECs). oxLDL increased reactive oxygen species (ROS) production, stimulated nuclear factor-kappaB (NFκB) activation, and enhanced intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin expression in HUVECs, which led to promotion of monocyte adhesion to HUVECs. Treatment with SFN, BITC, and PEITC (0-10 μM) dose-dependently induced heme oxygenase (HO)-1, glutamate cysteine ligase (GCL) catalytic and modifier subunit expression, intracellular glutathione content, and antioxidant response element (ARE)-luciferase reporter activity. SFN, BITC, and PEITC pretreatment reversed oxLDL-induced ROS production, NFκB nuclear translocation, κB-reporter activity, ICAM-1, VCAM-1, and E-selectin expression, and monocyte adhesion to endothelial cells. Both heme oxygenase 1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) knockdown attenuated the isothiocyanate inhibition of oxLDL-induced ROS production, κB-reporter activity, and adhesion molecule expression. SFN, BITC, and PEITC protect against oxLDL-induced endothelial damage by upregulating Nrf2-dependent HO-1 and GCL expression, which leads to inhibition of NFκB activation and ICAM-1, VCAM-1, and E-selectin expression.

Protective effect of AVS073, a polyherbal formula, against UVA-induced melanogenesis through a redox mechanism involving glutathione-related antioxidant defense

BackgroundAyurved Siriraj Brand Wattana formula (AVS073), a Thai herbal formula, has traditionally been used for health promotion and prevention of age-related problems. Ultraviolet A (UVA) is recognized to play a vital role in stimulation of melanin synthesis responsible for abnormal skin pigmentation possibly mediated by photooxidative stress. We thus aimed to study the inhibitory effect of AVS073 extracts on UVA-induced melanogenesis via a redox mechanism involving glutathione (GSH) synthesis and glutathione S-transferase (GST) using human melanoma (G361) cell culture.MethodsThe standardization of AVS073 extracts was carried out by TLC and UHPLC to obtain fingerprinting profiles of the formula, which identified several phenolic compounds including gallic acid (GA) in the formula. Antimelanogenic actions of AVS073 (up to 60 μg/ml) and GA (up to 10 μg/ml) were investigated by measuring tyrosinase activity and mRNA as well as melanin level in G361 cells irradiated with UVA. Moreover, antioxidant actions of the herbal formula and GA were determined by evaluating oxidant formation and modulation of GSH-related antioxidant defenses including GSH content, GST activity and mRNA level of γ-glutamate cysteine ligase catalytic (γ-GCLC) and modifier (γ-GCLM) subunit and GST.ResultsAVS073 extracts and GA, used as a reference compound, suppressed UVA-augmented tyrosinase activity and mRNA and melanin formation. In addition, pretreatment with AVS073 and GA was able to inhibit cellular oxidative stress, GSH depletion, GST inactivation and downregulation of γ-GCLC, γ-GCLM and GST mRNA in G361 cells exposed to UVA radiation.ConclusionsAVS073 formula exerted antimelanogenic effects possibly through improving the redox state by upregulation of GSH and GST. Moreover, pharmacological activity of the polyherbal formula would be attributed to combined action of different phenolic compounds present in the formula.

Utilization of 6-(methylsulfinyl)hexyl isothiocyanate for sensitization of tumor cells to antitumor agents in combination therapies

In the present study, we performed in silico and in vitro analyses to evaluate the chemosensitizing effects of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) on tumor cells. Our in silico analyses of the ligand–receptor interactions between 6-MITC and the glutamate cysteine ligase (GCL) catalytic subunit (GCLC) revealed that 6-MITC possibly inhibited GCL enzyme activity, and that Cys-249 and Gln-251 were important residues for stable binding of ligands to GCLC. It was further found that 6-MITC interfered with the hydrogen bonds of the cysteinyl and glutamyl moieties of GSH with Cys-249 and Gln-251, respectively, and possibly overrode the feedback inhibition of GCL enzyme activity by GSH. To the best of our knowledge, this is the first in silico analysis to suggest an overriding effect of 6-MITC on GSH-induced feedback inhibition of GCL. In our in vitro analyses, combined treatment with 6-MITC and l-buthionine-S,R-sulfoximine (BSO) depleted GSH within 4h in tumorigenic human c-Ha-ras and mouse c-myc-cotransfected highly metastatic serum-free mouse embryo-1 (r/m HM-SFME-1) cells, but did not deplete GSH in normal SFME cells. Furthermore, exposure to 6-MITC plus BSO for 4h, followed by glycyrrhetinic acid (GA) treatment for 3h, eradicated the tumor cells with minimal damage to the normal cells. The present findings suggest that 6-MITC in combination therapies could be used to sensitize tumor cells to antitumor agents, thereby leading to their eradication.

Development and systematic oxidative stress of a rat model of chronic bronchitis and emphysema induced by biomass smoke

ABSTRACTBackground: Epidemiological research and meta-analyses of published data have shown that biomass smoke (BS) is a risk factor for chronic obstructive pulmonary disease (COPD). However, the link between BS and COPD lacks experimental confirmation. Objectives: To verify whether BS can induce pathologic changes and systemic oxidative stress, which may be relevant to the development of emphysema and chronic bronchitis in rats. Methods: Rats were exposed to BS, cigarette smoke (CS), or clean air (sham) for 14 weeks. During the exposure, the O2, SO2, and CO levels were monitored. Pathological changes in the lungs, systemic oxidative stress, and inflammation biomarkers, together with GSTM1 and GSTP1 mRNA expression in the lung were measured. The glutamate–cysteine ligase catalytic subunit (GCLC) protein expression in the lung was measured using immunohistochemistry and western blotting. Results: The O2, CO, and SO2 levels were 20.31 ± 0.03%, 981.72 ± 64.76, and 2.59 ± 0.26 mg/m3 for the BS group, respectively, while their levels in the CS group were 20.28 ± 0.15%, 745.56 ± 30.83, and 12.64 ± 0.591 mg/m3 respectively. As with the rats exposed to CS, the BS rats showed an increased number of inflammatory cells in the bronchoalveolar lavage fluid, an increased pulmonary mean linear intercept and a decreased pulmonary mean alveolar number. Characteristics of chronic bronchitis and peribronchial fibrosis were also found in the BS-exposed rat lungs. Reduced body weight, systemic oxidative stress, and increased GCLC protein expression in the lungs were observed in the rats exposed to BS and CS. Conclusions: BS can cause emphysema and chronic bronchitis similar to that caused by CS, which is accompanied by systemic oxidative stress and inflammation.

N-Acetylcysteine Protects Murine Alveolar Type II Cells from Cigarette Smoke Injury in a Nuclear Erythroid 2–Related Factor–2–Independent Manner

Emphysema is caused by the cigarette smoke (CS)-induced destruction of alveolar wall septa, and CS is the main risk factor for chronic obstructive pulmonary disease (COPD). To study the mechanisms of response to this insult, we focused on oxidant-induced lung injury and the potential role of nuclear erythroid 2-related factor-2 (Nrf2), which is a key regulator of the antioxidant defense system. We studied the protective role of N-acetylcysteine (NAC) against the injury of alveolar type II (ATII) cells induced by CS in vivo and in vitro. ATII cells were isolated and purified using magnetic MicroBeads (Miltenyi Biotec, Auburn, CA) from Nrf2(-/-) mice and wild-type mice. We analyzed pulmonary injury, inflammation, glutathione (GSH) concentrations, the expression of glutathione cysteine ligase catalytic subunit mRNA, glutathione cysteine ligase modifier subunit mRNA, and glutathione reductase mRNA, and Nrf2, heme oxygenase-1, and nicotinamide adenine dinucleotide phosphate-reduced:quinone oxireductase levels by Western blotting, TUNEL assay, and immunocytofluorescence for 4-hydroxynonenal as a marker of oxidative stress. We found that CS induced greater injury in ATII cells obtained from Nrf2(-/-) mice than from wild-type mice. Furthermore, NAC attenuated the injuries by CS in ATII cells obtained from wild-type mice both in vivo and in vitro. Moreover, NAC decreased the injury of ATII cells obtained from Nrf2(-/-) mice. Our results suggest that Nrf2-GSH signaling is important for the protective activity of NAC. In addition, in ATII cells deficient in Nrf2, this compound can provide partial protection through its reactive oxygen species-scavenging activities. Targeting the antioxidant system regulated by Nrf2 may provide an effective strategy against lung injury in COPD.

MicroRNA Expression in Response to Controlled Exposure to Diesel Exhaust: Attenuation by the Antioxidant N -Acetylcysteine in a Randomized Crossover Study

Background: Adverse health effects associated with diesel exhaust (DE) are thought to be mediated in part by oxidative stress, but the detailed mechanisms are largely unknown. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and may respond to exposures such as DE.Objectives: We profiled peripheral blood cellular miRNAs in participants with mild asthma who were exposed to controlled DE with and without antioxidant supplementation.Methods: Thirteen participants with asthma underwent controlled inhalation of filtered air and DE in a double-blinded, randomized crossover study of three conditions: a) DE plus placebo (DEP), b) filtered air plus placebo (FAP), or c) DE with N-acetylcysteine supplementation (DEN). Total cellular RNA was extracted from blood drawn before exposure and 6 hr after exposure for miRNA profiling by the NanoString nCounter assay. MiRNAs significantly associated with DEP exposure and a predicted target [nuclear factor (erythroid-derived 2)-like 2 (NRF2)] as well as antioxidant enzyme genes were assessed by reverse transcription–quantitative polymerase chain reaction (RT-qPCR) for validation, and we also assessed the ability of N-acetylcysteine supplementation to block the effect of DE on these specific miRNAs. 8-hydroxy-2´-deoxyguanosine (8-OHdG) was measured in plasma as a systemic oxidative stress marker.Results: Expression of miR-21, miR-30e, miR-215, and miR-144 was significantly associated with DEP. The change in miR-144 was validated by RT-qPCR. NRF2 and its downstream antioxidant genes [glutamate cysteine ligase catalytic subunit (GCLC) and NAD(P)H:quinone oxidoreductase 1 (NQO1)] were negatively associated with miR-144 levels. Increases in miR-144 and miR-21 were associated with plasma 8-hydroxydeoxyguanosine 8-OHdG level and were blunted by antioxidant (i.e, DEN).Conclusions: Systemic miRNAs with plausible biological function are altered by acute moderate-dose DE exposure. Oxidative stress appears to mediate DE-associated changes in miR-144.

Effect of Glutathione Depletion on Nrf2/ARE Activation by Deltamethrin in PC12 Cells

Transcription factor NF-E2-related factor 2 (Nrf2) is important for cell protection against chemical-induced oxidative stress. Previously, we have reported that in PC12 cells, Nrf2 can be triggered by deltamethrin (DM), a commonly used pyrethroid insecticide. Molecular mechanisms behind Nrf2 activation by DM are still unclear. Here we studied the effects of cell glutathione (GSH) depletion on Nrf2 activation by DM. We found that DM enhanced Nrf2 expression at the mRNA and protein levels and increased nuclear Nrf2 levels. Activation of Nrf2 was associated with activation of its downstream targets, such as heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit (GCLC). In contrast, DL-buthionine-[S,R]- sulfoximine (BSO), a known GSH-depleting agent, did not increase Nrf2 protein expression or cause its nuclear accumulation. However, pre-treatment with BSO triggered mRNA expression of HO-1 and GCLC. Furthermore, BSO pre-treatment suppressed DM-induced Nrf2 upregulation and activation and lowered mRNA expression of HO-1 and GCLC upon DM treatment. These data demonstrate that GSH depletion is not necessary for the activation of Nrf2/ARE by DM in PC12 cells, and that GCLC and HO-1 expression can increase through other signalling pathways.

Seaweed extracts and unsaturated fatty acid constituents from the green alga Ulva lactuca as activators of the cytoprotective Nrf2–ARE pathway

Increased amounts of reactive oxygen species (ROS) have been implicated in many pathological conditions, including cancer. The major machinery that the cell employs to neutralize excess ROS is through the activation of the antioxidant-response element (ARE) that controls the activation of many phase II detoxification enzymes. The transcription factor that recognizes the ARE, Nrf2, can be activated by a variety of small molecules, most of which contain an α,β-unsaturated carbonyl system. In the pursuit of chemopreventive agents from marine organisms, we built, fractionated, and screened a library of 30 field-collected eukaryotic algae from Florida. An edible green alga, Ulva lactuca, yielded multiple active fractions by ARE–luciferase reporter assay. We isolated three monounsaturated fatty acid (MUFA) derivatives as active components, including a new keto-type C18 fatty acid (1), the corresponding shorter chain C16 acid (2), and an amide derivative (3) of the C18 acid. Their chemical structures were elucidated by NMR and mass spectrometry. All three contain the conjugated enone motif between C7 and C9, which is thought to be responsible for the ARE activity. Subsequent biological studies focused on 1, the most active and abundant ARE activator isolated. C18 acid 1 induced the expression of ARE-regulated cytoprotective genes, including NAD(P)H:quinone oxidoreductase 1, heme oxygenase 1, thioredoxin reductase 1, both subunits of the glutamate–cysteine ligase (catalytic subunit and modifier subunit), and the cystine/glutamate exchange transporter, in IMR-32 human neuroblastoma cells. Its cellular activity requires the presence of Nrf2 and PI3K function, based on RNA interference and pharmacological inhibitor studies, respectively. Treatment with 1 led only to Nrf2 activation, and not the increase in production of NRF2 mRNA. To test its ARE activity and cytoprotective potential in vivo, we treated mice with a single dose of a U. lactuca fraction that was enriched with 1, which showed ARE-activating effects similar to those observed in vitro. This could be owing to this fraction's ability to stabilize Nrf2 through inhibition of Keap1-mediated Nrf2 ubiquitination and the subsequent accumulation and nuclear translocation of Nrf2. The induction of many ARE-driven antioxidant genes in vivo and most prominently in the heart agreed with the commonly recognized cardioprotective properties of MUFAs. A significant increase in Nqo1 transcript levels was also found in other mouse tissues such as the brain, lung, and stomach. Collectively, this study provides new insight into why consumption of dietary seaweed may have health benefits, and the identified compounds add to the list of chemopreventive dietary unsaturated fatty acids.

Activation of denovo GSH synthesis pathway in mouse spleen after long term low-dose γ-ray irradiation

Glutathione (GSH) is an important cellular antioxidant and has a critical role in maintaining the balance of cellular redox. In this study, we investigated the GSH biosynthesis genes involved in the elevation of endogenous GSH levels using an irradiation system with an irradiation dose rate of 1.78 mGy/h, which was about 40,000 times less than the dose rates used in other studies. The results showed that GSH levels were significantly increased in the low-dose (0.02 and 0.2 Gy) irradiated group compared to those in the non-irradiated group, but enzymatic antioxidants such as superoxide dismutase and catalase were not induced at any doses tested. The elevation in GSH was accompanied by elevated expression of glutamate–cysteine ligase modifier subunit, but no changes were observed in the expression of glutamate–cysteine ligase catalytic subunit and thioredoxin in de novo GSH synthesis. In the case of genes involved in the GSH regeneration cycle, the expression of glutathione reductase was not changed after irradiation, whereas glutathione peroxidase was only increased in the 0.2 Gy irradiated group. Collectively, our results suggest that the de novo pathway, rather than the regeneration cycle, may be mainly switched on in response to stimulation with long-term low-dose radiation in the spleen.

Role of Nrf2 antioxidant defense in mitigating cadmium-induced oxidative stress in the olfactory system of zebrafish

Exposure to trace metals can disrupt olfactory function in fish leading to a loss of behaviors critical to survival. Cadmium (Cd) is an olfactory toxicant that elicits cellular oxidative stress as a mechanism of toxicity while also inducing protective cellular antioxidant genes via activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. However, the molecular mechanisms of Cd-induced olfactory injury have not been characterized. In the present study, we investigated the role of the Nrf2-mediated antioxidant defense pathway in protecting against Cd-induced olfactory injury in zebrafish. A dose-dependent induction of Nrf2-regulated antioxidant genes associated with cellular responses to oxidative stress was observed in the olfactory system of adult zebrafish following 24h Cd exposure. Zebrafish larvae exposed to Cd for 3h showed increased glutathione S-transferase pi (gst pi), glutamate–cysteine ligase catalytic subunit (gclc), heme oxygenase 1 (hmox1) and peroxiredoxin 1 (prdx1) mRNA levels indicative of Nrf2 activation, and which were blocked by morpholino-mediated Nrf2 knockdown. The inhibition of antioxidant gene induction in Cd-exposed Nrf2 morphants was associated with disruption of olfactory driven behaviors, increased cell death and loss of olfactory sensory neurons (OSNs). Nrf2 morphants also exhibited a downregulation of OSN-specific genes after Cd exposure. Pre-incubation of embryos with sulforaphane (SFN) partially protected against Cd-induced olfactory tissue damage. Collectively, our results indicate that oxidative stress is an important mechanism of Cd-mediated injury in the zebrafish olfactory system. Moreover, the Nrf2 pathway plays a protective role against cellular oxidative damage and is important in maintaining zebrafish olfactory function.

Rice protein improves oxidative stress by regulating glutathione metabolism and attenuating oxidative damage to lipids and proteins in rats

AimsTo evaluate the effects of rice protein (RP) on glutathione metabolism and oxidative damage. Main methodsSeven-week-old male Wistar rats were fed diets containing casein and RP without cholesterol for 3weeks. Plasma and liver lipid levels, hepatic accumulation of total glutathione (T-GSH), oxidized glutathione (GSSG), reduced glutathione (GSH), malondialdehyde (MDA) and protein carbonyl (PCO) were measured. In the liver, the total antioxidative capacity (T-AOC), mRNA levels of glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modulatory subunit (GCLM), and the activities of hepatic catalase (CAT), total superoxide dismutase (T-SOD), γ-glutamylcysteine synthetase (γ-GCS), glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GSHPx) were also measured. Key findingsT-AOC, GCLC and GCLM mRNA levels, antioxidative enzyme activities (T-SOD and CAT) and glutathione metabolism related enzyme activities (γ-GCS, GST, GR and GSHPx) were effectively stimulated by RP feeding compared to casein, and RP significantly reduced the hepatic accumulation of MDA and PCO in rats. These results indicate that lipid-lowering activity was induced by RP feeding. SignificanceThe present study demonstrates that RP improves oxidative stress primarily through enzymatic and non-enzymatic antioxidative defense mechanisms, reflected by enhancing the antioxidative status and attenuating the oxidative damage to lipids and proteins. These results suggest that RP can prevent hyperlipidemia in part through modifying glutathione metabolism, and sulfur amino acids may be the main modulator of this antioxidative mechanism.

Protection by chrysin, apigenin, and luteolin against oxidative stress is mediated by the Nrf2-dependent up-regulation of heme oxygenase 1 and glutamate cysteine ligase in rat primary hepatocytes

Chrysin, apigenin, and luteolin are flavones that differ in their number of hydroxyl groups in the B ring. In this study, we investigated the protection by chrysin, apigenin, and luteolin against tert-butyl hydroperoxide (tBHP)-induced oxidative stress and the possible mechanisms involved in rat primary hepatocytes. Chrysin, apigenin, and luteolin dose-dependently up-regulated the protein expression of heme oxygenase 1 (HO-1) and glutamate cysteine ligase (GCL) catalytic (GCLC) and modifier subunit (GCLM) and increased the intracellular glutathione (GSH) content and the ratio of GSH to oxidized GSH. Among the flavones studied, chrysin showed the greatest induction of HO-1, GCLC, and GCLM protein expression and GSH content. Cellular reactive oxygen species production induced by tBHP was attenuated by pretreatment with chrysin, apigenin, and luteolin (P<.05), and this protection was reversed by the GCL inhibitor l-buthionine-S-sulfoximine and the HO-1 inhibitor zinc protoporphyrin. Chrysin, apigenin, and luteolin activated extracellular signal-regulated protein kinase 2 (ERK2), nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, nuclear Nrf2-antioxidant responsive element (ARE) binding activity, and ARE-dependent luciferase activity. Both ERK2 and Nrf2 siRNAs attenuated chrysin-induced HO-1, GCLC, and GCLM protein expression. Taken together, these results suggest that chrysin, apigenin, and luteolin inhibit tBHP-induced oxidative stress by up-regulating HO-1, GCLC, and GCLM gene transcription via the ERK2/Nrf2/ARE signaling pathways in rat primary hepatocytes.

Polymorphism in glutamate cysteine ligase catalytic subunit (GCLC) is associated with sulfamethoxazole-induced hypersensitivity in HIV/AIDS patients

BackgroundSulfamethoxazole (SMX) is a commonly used antibiotic for prevention of infectious diseases associated with HIV/AIDS and immune-compromised states. SMX-induced hypersensitivity is an idiosyncratic cutaneous drug reaction with genetic components. Here, we tested association of candidate genes involved in SMX bioactivation and antioxidant defense with SMX-induced hypersensitivity.ResultsSeventy seven single nucleotide polymorphisms (SNPs) from 14 candidate genes were genotyped and assessed for association with SMX-induced hypersensitivity, in a cohort of 171 HIV/AIDS patients. SNP rs761142 T > G, in glutamate cysteine ligase catalytic subunit (GCLC), was significantly associated with SMX-induced hypersensitivity, with an adjusted p value of 0.045. This result was replicated in a second cohort of 249 patients (p = 0.025). In the combined cohort, heterozygous and homozygous carriers of the minor G allele were at increased risk of developing hypersensitivity (GT vs TT, odds ratio = 2.2, 95% CL 1.4-3.7, p = 0.0014; GG vs TT, odds ratio = 3.3, 95% CL 1.6 – 6.8, p = 0.0010). Each minor allele copy increased risk of developing hypersensitivity 1.9 fold (95% CL 1.4 – 2.6, p = 0.00012). Moreover, in 91 human livers and 84 B-lymphocytes samples, SNP rs761142 homozygous G allele carriers expressed significantly less GCLC mRNA than homozygous TT carriers (p < 0.05).Conclusionsrs761142 in GCLC was found to be associated with reduced GCLC mRNA expression and with SMX-induced hypersensitivity in HIV/AIDS patients. Catalyzing a critical step in glutathione biosynthesis, GCLC may play a broad role in idiosyncratic drug reactions.

Effect of p62 on tau hyperphosphorylation in a rat model of Alzheimer's disease.

Tau hyperphosphorylation is a main cause of neuronal loss in Alzheimer's disease, which can be caused by many factors, including oxidative stress. The multifunctional protein p62, which exists in neurofibrillary tangles and causes aggregation of hyperphosphorylated tau, not only serves as a receptor in selective autophagy, but also regulates oxidative stress. However, whether p62 participates in oxidative stress-induced tau hyperphosphorylation remains unclear. In this study, we produced an Alzheimer's disease rat model by injecting β-amyloid protein into the hippocampus and β-galactose intraperitoneally. Hematoxylin-eosin staining was used for morphological analysis of brain tissue, and western blotting, immunohistochemistry and reverse transcription-PCR were employed to study p62 and autophagy related proteins, antioxidant defense system kelch-like ECH-associated protein 1-NF-E2-related factor 2 related proteins and hyperphosphorylated tau, respectively. The number of neurons in the brain decreased in Alzheimer's disease rats, and the autophagy related proteins Atg12-Atg5, microtubule-associated protein 1 light chain 3-phosphatidylethanolamine and Beclin1 increased significantly, while p62 expression reduced. Expression of kelch-like ECH-associated protein 1 increased, NF-E2-related factor 2 protein and the downstream gene products of glutamate cysteine ligase catalytic subunit and glutamate cysteine ligase modulatory subunit decreased, and hyperphosphorylated tau increased. These findings demonstrate that autophagy levels increased and p62 levels decreased in the brains of Alzheimer's disease rats. Moreover, the anti-oxidative capability of the NF-E2-related factor 2-antioxidant response element pathway was decreased, which may be the cause of tau hyperphosphorylation in Alzheimer's disease brain tissue and the subsequent structural and functional damage to neurons.

Crosstalk between Nrf2 and the proteasome: Therapeutic potential of Nrf2 inducers in vascular disease and aging

Excessive reactive oxygen species (ROS) generation is as a major cause of oxidative stress and is implicated in cellular dysfunction in aging, cardiovascular disease and other pathologies. As antioxidant trials have largely failed to provide protection in humans, research focus has shifted to activating endogenous antioxidant defenses. In vascular models, activators of the transcription factor NF-E2 related factor 2 (Nrf2) pathway have been shown to restore redox homeostasis by increasing antioxidant/electrophilic response element-mediated (ARE/EpRE) expression of phase II and antioxidant enzymes, including NAD(P)H:quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1) and γ-glutamate cysteine ligase catalytic subunit (GCLC). Nrf2 activators disrupt basal ubiquitin-dependent degradation of Nrf2 by the 26S proteasome, leading to nuclear Nrf2 accumulation and gene induction. This review examines the evidence for crosstalk between Nrf2 and the proteasome, highlighting the mechanisms by which select Nrf2 activators regulate stress-induced proteasomal activity and removal of oxidized proteins. Exploiting the dual action of natural Nrf2 inducers may provide a novel therapeutic strategy for restoring cellular redox homeostasis in aging and cardiovascular related diseases such diabetes, atherosclerosis and stroke.

High expression of GCLC is associated with malignant melanoma of low oxidative phenotype and predicts a better prognosis

Reactive oxygen species (ROS) are strongly implicated in melanoma development, and treatment with antioxidants has shown efficacy in suppressing malignant transition and progression. Here, we investigated the significance of the glutamate-L: -cysteine ligase catalytic subunit (GCLC) expression, a key regulator of glutathione synthesis, for malignant melanoma. A large set of melanoma cell lines (n = 36) was analyzed, and higher GCLC levels were associated with lower presence of intracellular ROS and interestingly also lower rates of cell proliferation. Moreover, treatment with the antioxidant N-acetylcysteine efficiently reduced the growth speed of several investigated malignant cells. In addition GCLC expression was significantly linked to a prominent set of cellular antioxidants, accounting for the observed lower basal levels of oxidative stress and higher antioxidative capacity. Key attributes defining the malignant phenotype of melanoma cells including survival, invasiveness, and switch from E-cadherin to N-cadherin expression were more prominent in cells with lower GCLC expression. Our findings were further corroborated by observations in Rag2(-/-)γc(-/-)mice, in which melanoma cells with lower GCLC expression depicted a dramatically stronger tumor growth. Furthermore, prognostic significance of GCLC expression was investigated in patients (n = 28) with advanced malignant melanoma. High tumor immunoreactivity for GCLC was a significant determinant for better 5-year overall survival. Conclusively, we show for the first time that GCLC may serve a dual role, as a surrogate marker for cellular redox state as well as malignant potential of melanoma cells. These promising results regarding its prognostic significance as well as its potential as a pharmacological target require further in-depth investigations.