ArticleLiterature Review

Glutamate cysteine ligase and the age-related decline in cellular glutathione: The therapeutic potential of γ-glutamylcysteine

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Abstract

A consistent underlying index of aging is a decline in the cellular levels of the tripeptide glutathione (GSH). GSH is an essential thiol antioxidant produced in the cytosol of all cells and plays a key role in protecting against oxidative stress by neutralising free radicals and reactive oxygen species (ROS). The decline in GSH has been associated with changes in the expression and activity of the rate-limiting enzyme glutamate cysteine ligase (GCL), which produces the intermediate dipeptide γ-glutamylcysteine (γ-GC). The molecular mechanisms that affect these age-related changes remain unclear due to the complexity of GCL regulation. Impairment of the transcriptional activity of Nrf2 has been demonstrated to contribute to GCL dysregulation in aged rats. However, considering the complex nature of GCL regulation, relatively little research has been conducted to investigate the age-associated post-transcriptional controls of the enzyme. Defining these unknown mechanisms may inform our understanding of the aetiology of many age-related diseases and assist in formulating appropriate therapeutic strategies. This review focuses on the suitability of treatment with exogenous γ-GC to raise GSH levels by circumventing the age-related dysregulation of the rate-limiting step of GSH, providing promise for future research for the treatment of chronic oxidative stress-related diseases.

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... While the capacity to defend against free radicals is quite complex, one major cellular pathway, the glutathione (GSH) system, has been investigated for its known changes occurring with age (Jones et al., 2002;Liu, 2002;Ghosh et al., 2014;Ferguson and Bridge, 2016). Cellular GSH regulation utilizes a series of GSH peroxidases (Gpx) to sequester different types of free radicals. ...
... GSH is a re-usable tripeptide antioxidant that is used as a substrate by Gpx to reduce radicals and is recycled back into a usable form by GSH reductase at the expense of NADPH. Advanced age diminishes cellular levels of both GSH as well as the availability of its amino-acid constituent, cysteine, within the plasma (Jones et al., 2002;Liu, 2002;Ghosh et al., 2014;Ferguson and Bridge, 2016). The ability to produce and maintain adequate cellular GSH levels is believed to be bottlenecked by either the availability of its cysteine substrate or the availability of the enzyme responsible for the first of two ligation reactions: glutamate-cysteine ligase (GCL) (Griffith, 1999;Lu, 2009). ...
... The ability to produce and maintain adequate cellular GSH levels is believed to be bottlenecked by either the availability of its cysteine substrate or the availability of the enzyme responsible for the first of two ligation reactions: glutamate-cysteine ligase (GCL) (Griffith, 1999;Lu, 2009). Both GSH and GCL abundance decrease with age in other organ systems (Liu, 2002;Ghosh et al., 2014;Ferguson and Bridge, 2016). Further, both GSH and GCL activity diminishes following SCI. ...
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Pre-clinical and clinical spinal cord injury (SCI) studies differ in study design, particularly in the demographic characteristics of the chosen population. In clinical study design, criteria such as such as motor scores, neurological level, and severity of injury are often key determinants for participant inclusion. Further, demographic variables in clinical trials often include individuals from a wide age range and typically include both sexes, albeit historically most cases of SCI occur in males. In contrast, pre-clinical SCI models predominately utilize young adult rodents and typically use only females. While it is often not feasible to power SCI clinical trials to test multi-variable designs such as contrasting different ages, recent pre-clinical findings in SCI animal models have emphasized the importance of considering age as a biological variable prior to human experiments. Emerging pre-clinical data have identified case examples of treatments that diverge in efficacy across different demographic variables and have elucidated several age-dependent effects in SCI. The extent to which these differing or diverging treatment responses manifest clinically can not only complicate statistical findings and trial interpretations but also may be predictive of worse outcomes in select clinical populations. This review highlights recent literature including age as a biological variable in pre-clinical studies and articulates the results with respect to implications for clinical trials. Based on emerging unpredictable treatment outcomes in older rodents, we argue for the importance of including age as a biological variable in pre-clinical animal models prior to clinical testing. We believe that careful analyses of how age interacts with SCI treatments and pathophysiology will help guide clinical trial design and may improve both the safety and outcomes of such important efforts.
... Glutation występuje w kilku postaciach redoksowych, a najważniejsze z nich to glutation zredukowany (GSH) i glutation utleniony (GSSG). W większości komórek eukariotyczych dominuje postać zredukowana GSH (>98%), która w warunkach stresu oksydacyjnego utleniana jest do GSSG w reakcji katalizowanej przez peroksydazę glutationową (glutathione peroxidase, GPx) [3]. Potencjał oksydacyjno-redukcyjny układu GSH/GSSG jest odpowiedzialny za antyoksydacyjne działanie glutationu, czyli neutralizację reaktywnych form tlenu i azotu (reactive oxygen and nitrogen species, RONS), nadtlenku wodoru (H 2 O 2 ), nadtlenków organicznych i związków elektrofilnych (detoksykacja ksenobiotyków) oraz za chelatowanie jonów metali [4]. ...
... Dzięki aktywności γ-GT, GSH uczestniczy w magazynowaniu i transporcie cysteiny, która jest aminokwasem limitującym syntezę GSH. Pozostałe aminokwasy (glutaminian i glicyna) nie odgrywają już tak ważnej roli, gdyż pozyskiwane są w wielu szlakach metabolicznych -a przez to stale dostępne w komórce [3]. Natomiast cysteina wytwarzana jest w wyniku transsulfuracji z dostarczanej z pożywieniem metioniny. ...
... Wewnątrzkomórkowe stężenie GSH warunkuje dynamiczna równowaga między jego syntezą, metabolizmem i transportem [3]. Istnieje kilka mechanizmów przez które komórki utrzymują wewnętrzną homeostazę GSH, tj. ...
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Abstrakt Glutation (γ-glutamylocysteinyloglicyna, GSH) jest najbardziej rozpowszechnionym tiolowym antyoksydantem wytwarzanym w cytozolu wszystkich komórek ssaków, który pełni ważną rolę ochronną przed stresem oksydacyjnym. GSH jest syntetyzowany de novo przez sekwencyjne działanie dwóch enzymów: ligazy γ-glutamylocysteiny (GCL) i syntetazy glutationowej (GS). GCL katalizuje pierwszy etap biosyntezy GSH, którego produktem jest γ-glutamylocysteina (γ-GC). GCL jest heterodimerycznym enzymem zbudowanym z podjednostki katalitycznej (GCLc) i modulatorowej (GCLm), kodowanych przez dwa różne geny. Podjednostki GCL podlegają złożonej regulacji zarówno na poziomie przed-, jak i potranslacyjnym. Zmiany w ekspresji i aktywności GCL mogą zaburzać poziom GSH i homeostazy redoks. Przyczyną wielu przewlekłych schorzeń związanych ze stresem oksydacyjnym jest upośledzenie aktywności katalitycznej GCL oraz spadek stężenia GSH. Badania przedkliniczne sugerują, że podawanie egzogennej γ-GC podwyższa wewnątrzkomórkowe GSH przez dostarczenie brakującego substratu i może wykazywać potencjał jako terapia uzupełniająca w chorobach związanych z deplecją GSH.
... Age-related decline of GSH levels is reported in mammalian cells, which increases the risk of oxidative tissue damages in the elderly [51]. The main reason for the declined GSH levels is reported to be the reduction of de novo synthesis of GSH in aged cells [51]. ...
... Thus, the adult mice exhibited higher resistance to apoptosis than the resistance exhibited by the weaning mice. Age-related decline of GSH levels is reported in mammalian cells, which increases the risk of oxidative tissue damages in the elderly [51]. The main reason for the declined GSH levels is reported to be the reduction of de novo synthesis of GSH in aged cells [51]. ...
... Age-related decline of GSH levels is reported in mammalian cells, which increases the risk of oxidative tissue damages in the elderly [51]. The main reason for the declined GSH levels is reported to be the reduction of de novo synthesis of GSH in aged cells [51]. GCL, the rate-limiting enzyme for GSH biosynthesis, condenses cysteine and glutamic acid to produce γ-glutamylcysteine, which reacts with glycine in the presence of GSH synthase to form GSH [32]. ...
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Age is a risk factor for drug-induced liver injury (DILI). However, there is a limited understanding of pediatric DILI. Here, 2-week-old weaning and 8-week-old adult male ICR mice were intraperitoneally injected with CCl4 (0.1 mmol/kg equal to 15.4 mg/kg) to comparatively evaluate the time-dependent liver damage and cellular events. CCl4 significantly enhanced the serum alanine aminotransferase/aspartate aminotransferase levels and hepatic centrilobular necrosis in the weaning mice, whereas it induced mild liver injury in the adult mice. CCl4-treated weaning mice exhibited higher hepatic levels of pro-apoptotic proteins (Bax, cleaved caspase-3, -7, and -9), activated MAPKs (p-JNK and p-Erk), and endoplasmic reticulum stress indicators (ATF6 and CHOP) and lower hepatic anti-apoptotic Bcl-2 levels than the adult mice. The weaning mice exhibited enhanced basal hepatic glutathione (GSH) levels due to high glutamate cysteine ligase (GCL) and low anti-cysteine dioxygenase (CDO) enzyme levels. However, CCl4 markedly reduced the hepatic GSH levels only in the weaning mice. Furthermore, higher hepatic levels of oxidative stress-induced malondialdehyde, 4-hydroxynonenal, nitrotyrosine-protein adducts, and oxidized proteins were observed in CCl4-treated weaning mice than in CCl4-treated adult mice. The enhanced levels of hepatic cytochrome P450 (CYP) 2E1 and CYP3A, and decreased hepatic GSH S-transferase (GST)-π and GSH reductase (GR) levels in the weaning mice may contribute to their enhanced susceptibility to liver damage.
... The very important role played by endogenous glutathione (GSH), known as the 'master antioxidant', is well known and need not be further elaborated. However, not so well known is the observation that the first enzyme in the two-step biosynthesis pathway for GSH is compromised as we get older, especially beyond the age of 60 [1,2]. Taking supplemental GSH orally is not an option, since oral GSH is rapidly degraded by the enzyme, gamma-glutamyl transpeptidase [2]. ...
... However, not so well known is the observation that the first enzyme in the two-step biosynthesis pathway for GSH is compromised as we get older, especially beyond the age of 60 [1,2]. Taking supplemental GSH orally is not an option, since oral GSH is rapidly degraded by the enzyme, gamma-glutamyl transpeptidase [2]. An orally bioavailable GSH has not yet been described in the literature; therefore, we now present an orally biooavailable GSH in the form of its prodrug. ...
... While mice and rat data are generally transferable to humans, it is incumbent that studies be conducted with CySSG in humans as soon as possible by clinician investigators relative to its oral bioavailability. GSH taken orally by humans is degraded by the enzyme, gamma-glutamyl transpeptidase [2], and is known not to be bioavailable [7]. In contrast, the mechanism that releases GSH from CySSG intracellularly is via a thiol-disulfide exchange reaction [5], and was shown here to be bioavailable in mice. ...
Article
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L-Cysteine-glutathione mixed disulfide (CySSG), a prodrug of glutathione (GSH), was found to be orally bioavailable in mice, and protected mice against a toxic dose of acetaminophen. If oral bioavailability can also be demonstrated in humans, a wide range of applicability for CySSG can be envisioned.
... Alternatively, GGC can be taken up by cells intact where it is converted to glutathione [16][17][18]. Furthermore, GGC itself is an antioxidant by acting as a cofactor for GPx where it is effectively comparable to GSH for hydrogen peroxide detoxification [19] Animal safety trials have demonstrated that GGC is safe at a repeated limit dosage of 1000 mg/kg/day over a 90-day period [20]. ...
... Furthermore, GGC itself is an antioxidant by acting as a cofactor for GPx where it is effectively comparable to GSH for hydrogen peroxide detoxification [19] Animal safety trials have demonstrated that GGC is safe at a repeated limit dosage of 1000 mg/kg/day over a 90-day period [20]. Exogenous supply of GGC, which is sold as a dietary supplement in the USA, has been reported to suppress oxidative injury and improve mitochondrial function both in in vitro and in vivo animal models of oxidative stress-induced tissue damage [18,[21][22][23][24]. In the context of lung disease, GGC has been tested in an LPS-induced mouse model of sepsis where it was demonstrated that GGC administration suppressed the production of LPS-induced inflammatory and oxidative mediators, which reduced lung tissue damage and sepsis lethality [24]. ...
... Despite multiple clinical trials with current glutathionebased therapies, improved clinical outcomes in CF have not been achieved [6,7]. As a precursor to glutathione, GGC has been consistently shown to increase intracellular levels of glutathione in both in vivo and in vitro models [16][17][18][21][22][23][24], and to reduce inflammation and oxidative stress [19,43,44]. Recently, it was shown that GGC exhibits anti-inflammatory effects in an in vivo and in vitro mouse sepsis model [24]. ...
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Systemic glutathione deficiency, inflammation, and oxidative stress are hallmarks of cystic fibrosis (CF), an inherited disease that causes persistent lung infections and severe damage to the respiratory system and many of the body organs. Improvements to current antioxidant therapeutic strategies are needed. The dietary supplement, γ-glutamylcysteine (GGC), which is the immediate precursor to glutathione, rapidly boosts cellular glutathione levels following a single dose in healthy individuals. Efficacy of GGC against oxidative stress induced by Pseudomonas aeruginosa, which is a common and chronic pathogen infecting lungs of CF patients, remains unassessed. Primary mucocilliary differentiated airway (bronchial and/or nasal) epithelial cells were created from four individuals with CF. Airway oxidative stress and inflammation was induced by P. aeruginosa lipopolysaccharide (LPS). Parameters including global proteomics alterations, cell redox state (glutathione, oxidative stress), pro-inflammatory mediators (IL-8, IDO-1), and cellular health (membrane integrity, stress granule formation, cell metabolic viability) were assayed under six experimental conditions: (1) Mock, (2) LPS-challenged (3) therapeutic, (4) prophylactic (5) therapeutic and prophylactic and (6) GGC alone. Proteomic analysis identified perturbation of several pathways related to cellular respiration and stress responses upon LPS challenge. Most of these were resolved when cells were treated with GGC. While GGC did not resolve LPS-induced IL-8 and IDO-1 activity, it effectively attenuated LPS-induced oxidative stress and stress granule formation, while significantly increasing total intracellular glutathione levels, metabolic viability and improving epithelial cell barrier integrity. Both therapeutic and prophylactic treatments were successful. Together, these findings indicate that GGC has therapeutic potential for treatment and prevention of oxidative stress-related damage to airways in cystic fibrosis.
... 9 However, its efficacy and safety are still questionable because of its low bioavailability and absorption ability. 5,10,11 Therefore, the clinical efficacy and safety of oral glutathione are unclear and limited. This study aimed to explore the clinical effect of glutathione on skin color and related skin conditions. ...
... 23,24 One important concern of oral glutathione supplementation is its limited bioavailability. Glutathione is rapidly degraded by intestinal and hepatic γ-glutamyl transpeptidase (γ-GT) 10,11,24 and eliminated from plasma. However, results from clinical studies showed contradictory findings with regard to glutathione bioavailability. ...
Article
Background Glutathione is one of agents which is commonly used to lighten skin color in Asia as a dietary supplement. Previous studies suggest its potential effect of glutathione on skin color. However, the clinical efficacy of glutathione in oral form is still questionable due to its limited absorption and bioavailability. Aim To determine the clinical effects of glutathione on skin color and related skin conditions. Patients/Methods A systematic review was conducted using PubMed, CINAHL, Scopus, EMBASE and Cochrane library were searched from inceptions to October 2017. All clinical studies evaluating the effect of glutathione on any skin effects in healthy volunteer were included. Results A total of four studies were included. Three studies were RCTs with placebo control, while one was a single‐arm trial. One study used topical form, while others used oral form of glutathione with 250 to 500 mg/day. We found that both oral glutathione with the dosage of 500 mg/day and topical 2.0% oxidized glutathione could brighten skin color in sun‐exposed area measured by skin melanin index. No significant differences in the reduction in skin melanin index were observed in sun‐protected area for any products. In addition, glutathione also has a trend to improve skin wrinkle, skin elasticity, and UV spots. Some adverse events but nonserious were reported. Conclusions Current evidence of the skin whitening effect of glutathione is still inconclusive due to the quality of included studies and inconsistent findings. However, there is a trend that glutathione might brighten skin color at skin‐exposed area.
... Le GS est composé de deux sous-unités identiques (~ 118 kDa). Il a été constaté que la surexpression du GS n'a pas un effet sur l'augmentation du niveau de GSH tandis que la surexpression du -GCS a un effet positif sur le niveau du GSH, dans la levure Saccharomyces cerevisiae et dans les cellules endothéliales de l'artère pulmonaire bovine (Grant et al., 1997;Shi et al., 1994), et d'autres études ont montré qu'une répression de l'expression des gènes codants les deux sousunités du -GCS aboutit à la diminution du niveau de GSH cellulaire (pour revue (Richter and Kietzmann, 2016)) (Ferguson and Bridge, 2016;Liu et al., 2012). ...
... La première étape de la synthèse est catalysée par l'enzyme gamma-glutamyl-cystéine synthétase (GCS), et la deuxième étape est médiée par le GSH synthétase (GS) (Meister and Anderson, 1983). Il a été démontré que la régulation de l'expression du gène codant le GS n'a pas un effet sur l'augmentation de niveau du GSH tandis que la régulation de celle du -GCS a un effet significatif sur le niveau du GSH, (Ferguson and Bridge, 2016;Grant et al., 1997;Liu et al., 2012;Shi et al., 1994) (pour revue (Richter and Kietzmann, 2016)). Une étude sur des souris transgéniques exprimant Tat du VIH-1, a montré le rôle de Tat dans la répression de l'expression de gènes codant le -GCS (Choi et al., 2000a). ...
Thesis
L'infection par le virus de l'immunodéficience humaine (VIH) est associée à la survenue de lymphomes B chez des patients infectés et l’incidence de certains lymphomes reste élevée même chez les individus infectés dont la fonction immunitaire est reconstituée sous traitement antirétroviral combiné. La contribution du VIH-1 à l'oncogenèse des cellules B reste donc énigmatique. Le VIH-1 induit un stress oxydant et des dommages à l'ADN (DA) dans les cellules infectées via de multiples mécanismes. Cependant il n'infecte pas les lymphocytes B. En revanche, la protéine virale Tat qui circule dans le sang des individus infectés est capable de pénétrer spontanément dans des cellules non infectables par VIH. Nous avons détecté des niveaux élevés d’espèces réactives de l’oxygène (ROS), principalement mitochondriales, et des DA dans les cellules B d'individus infectés par le VIH. Nous avons ainsi émis l'hypothèse que Tat pourrait induire des DA oxydants dans les cellules B et favoriser ainsi l'instabilité génétique et la transformation maligne de ces cellules.Dans des cellules B isolées à partir du sang périphérique de donneurs sains et incubées en présence de protéine Tat recombinante, un stress oxydant a été induit, la capacité antioxydante a diminué avec la diminution de taux du glutathion, le facteur de transcription NF-κB a été activé, et sont apparus des DA accompagnés d'aberrations chromosomiques. En outre, tous les effets induits par Tat dépendaient de son activité transcriptionnelle. Dans le but de mieux comprendre le(s) mécanisme(s) d’action de Tat chez les patients séropositifs, des extraits bruts de plantes endémiques du Liban ont été utilisés pour leur potentiel antioxydant. L’effet pro-oxydant de Tat a été contrecarré, le stress oxydant inhibé ainsi que les DA induits par la protéine virale. En conclusion, nous proposons que les dommages oxydants causés à l’ADN et les aberrations chromosomiques induites par Tat correspondent à de nouveaux facteurs oncogéniques favorisant le développement de lymphomes B chez les individus infectés par le VIH-1.
... In the progression of oxidative stress during aging, an important role is played by the suppression of the antioxidant defense system. This is evidenced by a signifi cant decrease in the content and synthesis of glutathione in the tissues of the cardiovascular system [4][5][6]. The levels of glutathione correlate positively with the endothelial-depended vasodilatation, which is greatly depressed with aging and hypertensive patients [7][8][9]. ...
... Although the values of prothrombin index before and after therapy were within the range of reference values, this suggests the possibility of garlic usage as a mild antithrombotic agent in patients with hypertension and hypercholesterolemia. Our data correlate with those obtained earlier in hypertensive patients of younger age (20-39 years old) who showed H 2 S content in blood plasma of healthy donors(1), hypertensive patients at the initial examination (2), hypertensive patients after 10 days of standard treatment (3), hypertensive patients after 10 days of standard treatment and 14 days of garlic supplement consumption (4), hypertensive patients after 10 days of standard treatment and 30 days of garlic supplement consumption(5). The comparison between healthy donors and hypertensive patients was made by unpaired t test. The comparison in group of hypertensive patients was made by paired t test. ...
... When GLC is impaired, this leads to decreased GSH biosynthesis, reduced cellular antioxidant capacity and the induction of oxidative stress. GSH concentration and γ-GC ligase activity in the central nervous system decline with age in association with increased oxidative stress (24) . ...
... Improvement was seen in five of the six mood parameters measured: anger, confusion, depression, fatigue, and tension; only vigour was unchanged. All the parameters in the untreated patients remained unchanged (24) . ...
... A decreased concentration of gluta thione has been reported in erythrocytes and lymphocytes of healthy older people, and potentially could also occur in hepatocytes. 41 This decrease in glutathione concen tration suggests that older patients could be more prone to elevated oxidative stress than younger patients, which in conjunction with drug treatment might initiate DILI development. As cellular stress reaches a critical hepatocyte injury threshold, cell death signalling pathways are induced (eg, activation of the mitogen-activated protein kinase JNK), eventually causing apoptosis and necrosis through mitochondrial permea bility transition and outer membrane permea bilisation. ...
Article
Drug-induced liver injury (DILI) is a rare, unpredictable, and potentially serious adverse reaction. It is induced by many drugs, herbs, and dietary supplements and represents a diagnostic challenge to clinicians. Older people (aged 65 years and older) are often polymedicated, and their declining physiological function affects drug pharmacokinetics. There is no consistent evidence that age is a general risk factor for DILI; however, age might be a risk factor with specific medications, with antimicrobials and cardiovascular drugs being the most likely medications to cause DILI in older people. Ageing influences DILI phenotypes, making cholestatic damage and chronic DILI more likely. In older people with DILI, comorbidities act as confounding causes and account for higher mortality unrelated to the liver. There are no specific therapies for DILI and supportive measures are still the mainstay of management. This Review highlights current advances and gaps in DILI epidemiology, mechanisms, and diagnosis that are pertinent to older individuals.
... The presence of glycine also proved to ~ 22 ~ be important in the interaction of GSH with its related enzymes, e.g. the absence of glycine in γ-glutamylcysteine dramatically reduces the binding affinity of GST [143,144]. This example supports that, although γ-glutamylcysteine could partially substitute for GSH [145,146], the glycine moiety of GSH is pivotal for enzyme catalysis [143,144]. ...
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Background: Weaning is known to result in barrier dysfunction and villus atrophy in the immediate post-weaning phase, and the magnitude of these responses is hypothesized to correlate with changes in the glutathione (GSH) redox system. Therefore, these parameters were simultaneously measured throughout the weaning phase, in piglets differing in birth weight category and weaning age, as these pre-weaning factors are important determinants for the weaning transition. Low birth weight (LBW) and normal birth weight (NBW) littermates were assigned to one of three weaning treatments; i.e. weaning at 3 weeks of age (3w), weaning at 4 weeks of age (4w) and removal from the sow at 3 d of age and fed a milk replacer until weaning at 3 weeks of age (3d3w). For each of these treatments, six LBW and six NBW piglets were euthanized at 0, 2, 5, 12 or 28 d post-weaning piglets, adding up 180 piglets. Results: Weaning increased the glutathione peroxidase activity on d 5 post-weaning in plasma, and duodenal and jejunal mucosa. Small intestinal glutathione-S-transferase activity gradually increased until d 12 post-weaning, and this was combined with a progressive rise of mucosal GSH up till d 12 post-weaning. Oxidation of the GSH redox status (GSH/GSSG Eh) was only observed in the small intestinal mucosa of 3d3w weaned piglets at d 5 post-weaning. These piglets also demonstrated increased fluorescein isothiocyanate dextran (FD4) and horseradish peroxidase fluxes in the duodenum and distal jejunum during the experiment, and specifically demonstrated increased FD4 fluxes at d 2 to d 5 post-weaning. On the other hand, profound villus atrophy was observed during the weaning transition for all weaning treatments. Finally, LBW and NBW piglets did not demonstrate notable differences in GSH redox status, small intestinal barrier function and histo-morphology throughout the experiment. Conclusion: Although moderate changes in the GSH redox system were observed upon weaning, the GSH redox status remained at a steady state level in 3w and 4w weaned piglets and was therefore not associated with weaning induced villus atrophy. Conversely, 3d3w weaned piglets demonstrated GSH redox imbalance in the small intestinal mucosa, and this co-occurred with a temporal malfunction of their intestinal barrier function.
... The latter modulates the catalytic properties of GCLC by lowering its sensitivity to the inhibition of GSH and by increasing its affinity to glutamate. Without the presence of GCLM, GCLC would exert its function poorly in vivo 22 . ...
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Purpose: To investigated the effects of ginsenoside Rb1 on diabetic retinopathy in streptozotocin-induced diabetic rats. Methods: Diabetes was induced by a single intraperitoneal injection of streptozotocin (80 mg/kg) in male Wistar rats. Ginsenoside Rb1 (20, 40 mg/kg) was injected (i.p.) once a day for 4 weeks. Then, using fundus photography, the diameter and vascular permeability of retinal vessels were investigated. Retinal histopathology was undertaken. Contents of malondialdehyde (MDA) and glutathione (GSH) in retinas were assayed. Levels of nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione cysteine ligase catalytic subunit (GCLC), and glutathione cysteine ligase modulatory subunit (GCLM) were measured. Results: Treatment with ginsenoside Rb1 attenuated the diabetes-induced increase in the diameter of retinal blood vessels. Ginsenoside Rb1 reduced extravasation of Evans Blue dye from retinal blood vessels. Ginsenoside Rb1 partially inhibited the increase in MDA content and decrease in GSH level in rat retinas. Nrf2 levels in the nuclei of retinal cells and expression of GCLC and GCLM were increased significantly in rats treated with ginsenoside Rb1. Conclusion: These findings suggest that ginsenoside Rb1 can attenuate diabetic retinopathy by regulating the antioxidative function in rat retinas.
... Furthermore, as (15). More recently, shifts in the redox state of glutathione have been used as a more sensitive indicator of oxidative stress (16). Pro-oxidizing shifts have been shown in brain regions (17), and have the ability to impair redox-dependent cellular processes and contribute to the accumulation of oxidative damage (18). ...
Article
Background: N-acetyl cysteine (NAC) is a thiolic antioxidant that is thought to increase cellular glutathione (GSH) by augmenting the concentration of available cysteine, an essential precursor to GSH production. Manipulating redox status can affect brain function, and NAC intake has been associated with improving brain function in models of neurodegenerative diseases. Objectives: The objective of the study was to determine if short-term dietary supplementation with NAC could ameliorate functional impairment associated with aging. Methods: C57BL/6J male mice aged 6, 12, or 24 mo were fed a control diet or the control diet supplemented with 0.3% NAC for a total of 12 wk. After 4 wk of dietary supplementation, mice began a series of behavioral tests to measure spontaneous activity (locomotor activity test), psychomotor performance (bridge-walking and coordinated running), and cognitive capacity (Morris water maze and discriminated active avoidance). The performance of the mice on these tests was analyzed through the use of analyses of variance with Age and Diet as factors. Results: Supplementation of NAC improved peak motor performance in a coordinated running task by 14% (P < 0.05), and increased the time spent around the platform by 24% in a Morris water maze at age 6 mo. However, the supplementation had no to minimal effect on the motor and cognitive functions of 12- and 24-mo-old mice. Conclusions: The findings of this preclinical study support the claim that NAC has nootropic properties in 6-mo-old mice, but suggest that it may not be useful for improving motor and cognitive impairments in older mice.
... Among these amino acids, cysteine is the rate-limiting substrate for enzyme activity for GSH synthesis, because the other precursor amino acids, glutamate and glycine, have much higher intracellular concentrations than cysteine [90,94]. In agreement with these findings, it has been shown that GSH concentrations can only be elevated by increasing cytosolic cysteine availability [95]. The biosynthesis of GSH occurs via a two-step ATP-requiring enzymatic process that is catalyzed by glutamate-cysteine ligase (GCL; also known as γ-glutamylcysteine synthetase) and glutathione synthetase (GSS) [96,97]. ...
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Multiple system atrophy (MSA) is a rare neurodegenerative disease characterized by parkinsonism, cerebellar impairment, and autonomic failure. Although the causes of MSA onset and progression remain uncertain, its pathogenesis may involve oxidative stress via the generation of excess reactive oxygen species and/or destruction of the antioxidant system. One of the most powerful antioxidants is glutathione, which plays essential roles as an antioxidant enzyme cofactor, cysteine-storage molecule, major redox buffer, and neuromodulator, in addition to being a key antioxidant in the central nervous system. Glutathione levels are known to be reduced in neurodegenerative diseases. In addition, genes regulating redox states have been shown to be post-transcriptionally modified by microRNA (miRNA), one of the most important types of non-coding RNA. miRNAs have been reported to be dysregulated in several diseases, including MSA. In this review, we focused on the relation between glutathione deficiency, miRNA dysregulation and oxidative stress and their close relation with MSA pathology.
... Soon this valuable precursor will be commercialized and utilized as an active ingredient in food supplements, cosmetics including anti-aging creams and will be routinely used in different therapeutics. Ferguson and Bridge presented in detail the potential of γ-Glutamylcysteine (γ-GC) in elevating the levels of glutathione implicated in age-related disorders and avoiding the defective or impaired glutamate cysteine ligase (GCL) activity as well as rate-limiting step of this enzyme [96]. A study conducted by Pileblad and Magnijsson in the year 1992 provided an evidence that the administration of γ-glutamylcysteine in the rat brain induced glutathione levels significantly in the brain regions [97]. ...
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Current treatments aimed at controlling and alleviating the fatal symptoms of cystic fibrosis (CF) have directed their attention to draw valid inferences, by employing different strategies and agents for the absolute prevention of disease. Variety of CF clinical studies exploits the master antioxidant known as glutathione (GSH) and its precursors to improve the health status of CF patients and to develop the ultimate role of therapeutics. The practical usage of GSH in various clinical trials is assessed by its ability to improve numerous clinical endpoints and surrogate markers of CF, which are yet to be determined. More recently a precursor of GSH known as Gemma-glutamylcysteine (GGC) is gaining larger acceptance, ascertained by its efficacious role in ameliorating the health status of various patients. This review has two main focuses: to identify different clinical trials elucidating the role of GSH in the etiology of CF. Secondly to unravel the beneficial effects of N-Acetylcysteine (NAC) and GGC to discern novel clinical interventions and therapies to annihilate the disastrous effects of CF successfully. © 2018, International Journal of Scientific and Technology Research. All rights reserved.
... (NAC) to treat sepsis both directly as a GSH substitute and indirectly as a GSH precursor, clinical outcomes are controversial [13]. γ-GC, as an immediate precursor of GSH that differs from GSH in the absence of thermodynamic limitations regarding transmembrane transport, can be readily taken up by many cell types to promote GSH synthesis [14][15][16]. ...
Article
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Sepsis is a life-threatening organ dysfunction caused by dysregulated host response to infection and characterized by redox imbalance and severe oxidative stress. Glutathione (GSH) serves several vital functions, including scavenging free radicals and maintaining intracellular redox balance. Extracellular GSH is unable to be taken into the majority of human cells, and the GSH prodrug N-acetyl-L-cysteine (NAC) does not exhibit promising clinical effects. γ-glutamylcysteine (γ-GC), an intermediate dipeptide of the GSH-synthesis pathway and harboring anti-inflammatory properties, represents a relatively unexplored option for sepsis treatment. The anti-inflammatory efficiency of γ-GC and the associated molecular mechanism need to be explored. In vivo investigation showed that γ-GC reduced sepsis lethality and attenuated systemic inflammatory responses in mice, as well as inhibited lipopolysaccharide (LPS)-stimulated production of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), high-mobility group box 1 (HMGB1), and nitric oxide (NO) and the expression of inducible NO synthase and cyclooxygenase 2 in RAW264.7 cells. Moreover, both in vivo and in vitro experiments demonstrated that γ-GC exhibited better therapeutic effects against inflammation compared with N-acetyl-L-cysteine (NAC) and GSH. Mechanistically, γ-GC suppressed LPS-induced reactive oxygen species accumulation and GSH depletion. Inflammatory stimuli, such as LPS treatment, upregulated the expression of glutathione synthetase via activating nuclear factor-erythroid 2-related factor (Nrf2) and nuclear factor kappa B (NF-κB) pathways, thereby promoting synthesis of GSH from γ-GC. These findings suggested that γ-GC might represent a potential therapeutic agent for sepsis treatment.
... These two transcriptional factors, therefore, affect cellular GSH levels as well ( Figure 1). NF-κB stimulates GCLC and GCLM transcription, the first by binding to the gclc promoter and the second indirectly, by activating AP-1, which, in turn, activates the gclm promoter (16,(58)(59)(60). ...
Article
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Glutathione (γ-glutamyl-cysteinyl-glycine) is an intracellular thiol molecule and a potent antioxidant that participates in the toxic metabolism phase II biotransformation of xenobiotics. It can bind to a variety of proteins in a process known as glutathionylation. Protein glutathionylation is now recognised as one of important posttranslational regulatory mechanisms in cell and tissue physiology. Direct and indirect regulatory roles in physiological processes include glutathionylation of major transcriptional factors, eicosanoids, cytokines, and nitric oxide (NO). This review looks into these regulatory mechanisms through examples of glutathione regulation in apoptosis, vascularisation, metabolic processes, mitochondrial integrity, immune system, and neural physiology. The focus is on the physiological roles of glutathione beyond biotransformational metabolism.
... The indirect antioxidant enzymes contribute to reduction of free radicals by indirect means. An example is glutamate-cysteine ligase and CSE, which are required for biosynthesis of GSH and cysteine, respectively (145,382). The antioxidant enzymes play significant roles in maintenance of redox balance and probably constitute the most important arm of antioxidant defense mechanisms. ...
Article
Significance: Once considered to be mere by-products of metabolism, reactive oxygen, nitrogen and sulfur species are now recognized to play important roles in diverse cellular processes such as response to pathogens and regulation of cellular differentiation. It is becoming increasingly evident that redox imbalance can impact several signaling pathways. For instance, disturbances of redox regulation in the brain mediate neurodegeneration and alter normal cytoprotective responses to stress. Very often small disturbances in redox signaling processes, which are reversible, precede damage in neurodegeneration. Recent Advances: The identification of redox-regulated processes, such as regulation of biochemical pathways involved in the maintenance of redox homeostasis in the brain has provided deeper insights into mechanisms of neuroprotection and neurodegeneration. Recent studies have also identified several post-translational modifications involving reactive cysteine residues, such as nitrosylation and sulfhydration, which fine-tune redox regulation. Thus, the study of mechanisms via which cell death occurs in several neurodegenerative disorders, reveal several similarities and dissimilarities. Here, we review redox regulated events that are disrupted in neurodegenerative disorders and whose modulation affords therapeutic opportunities. Critical issues: Although accumulating evidence suggests that redox imbalance plays a significant role in progression of several neurodegenerative diseases, precise understanding of redox regulated events is lacking. Probes and methodologies that can precisely detect and quantify in vivo levels of reactive oxygen, nitrogen and sulfur species are not available. Future directions: Due to the importance of redox control in physiologic processes, organisms have evolved multiple pathways to counteract redox imbalance and maintain homeostasis. Cells and tissues address stress by harnessing an array of both endogenous and exogenous redox active substances. Targeting these pathways can help mitigate symptoms associated with neurodegeneration and may provide avenues for novel therapeutics. Antioxid. Redox Signal. 00, 000-000.
... When we approached the oxidative stress response from the antioxidant perspective, we observed an important decrease in GSH content at baseline in almost all subsets. An age-related decrease of GSH content has been already described in the lymphocyte population [26,48], and the decline has been associated with a decrease in the γ-glutamil cysteine ligase (GCL), an important enzyme in the GSH biosynthesis, expression, and activity [49]. Notably, OFF showed the same GSH level as CENT, and much lower than the CTRL group. ...
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The production of reactive oxygen species (ROS) may promote immunosenescence if not counterbalanced by the antioxidant systems. Cell membranes, proteins, and nucleic acids become the target of ROS and progressively lose their structure and functions. This process could lead to an impairment of the immune response. However, little is known about the capability of the immune cells of elderly individuals to dynamically counteract the oxidative stress. Here, the response of the main lymphocyte subsets to the induced oxidative stress in semisupercentenarians (CENT), their offspring (OFF), elderly controls (CTRL), and young individuals (YO) was analyzed using flow cytometry. The results showed that the ratio of the ROS levels between the induced and noninduced (I/NI) oxidative stress conditions was higher in CTRL and OFF than in CENT and YO, in almost all T, B, and NK subsets. Moreover, the ratio of reduced glutathione levels between I/NI conditions was higher in OFF and CENT compared to the other groups in almost all the subsets. Finally, we observed significant correlations between the response to the induced oxidative stress and the degree of methylation in specific genes on the oxidative stress pathway. Globally, these data suggest that the capability to buffer dynamic changes in the oxidative environment could be a hallmark of longevity in humans.
... An age-related decline in GSH has been observed in a number of organisms as a result of the decreased rate of GSH synthesis (Ferguson and Bridge, 2016). Therefore, GSH synthesis in the elderly during basal and induced hormetic responses may not be as active as that in young persons. ...
Article
Although the toxicity of synthetic chemicals at high doses is well known, chronic exposure to low-dose chemical mixtures has only recently been linked to many age-related diseases. However, it is nearly impossible to avoid the exposure to these low-dose chemical mixtures as humans are exposed to a myriad of synthetic chemicals as a part of their daily lives. Therefore, coping with possible harms due to low dose chemical mixtures is challenging. Interestingly, within the range of environmental exposure, disease risk does not increase linearly with increasing dose of chemicals, but often tends to plateau or even decrease with increasing dose. Hormesis, the over-compensation of various adaptive responses through cellular stresses, is one possible mechanism for this non-linearity. Although the hormetic effects of synthetic chemicals or radiation have long been debated in the field of toxicology, the hormesis concept has recently been generalized in the field of molecular biology; similar to responses to synthetic chemicals, mild to moderate intermittent stressors from any source can induce hormetic responses. Examples of stressors are exercise, calorie restriction, intermittent fasting, cognitive stimulation, and phytochemicals. Mitohormesis is hormesis induced by such stressors through mitochondrial retrograde signalling including the increased production of mild reactive oxygen species. Xenohormesis is phytochemical-induced hormesis, reflective of a mutualistic relationship between plant and animals. As humans had repeated exposure to all of these stressors during their evolution, the hormetic effects of these health behaviours may be considered to be evolutionarily adapted. Although hormesis induced by synthetic chemicals occurs in humans, such hormesis may not be recommended to the public due to unresolved issues on safety including the impossibility of control exposure. However, the use of personal health behaviors which enhance mitohormetic- or xenohormetic-stress can be readily incorporated into everyone's daily lives as a practical way to counteract harmful effects of unavoidable low-dose chemical mixtures.
... Should cellular GSH depletion arise as a result of damaged regulatory control of GCL activity, NAC or other cysteine prodrugs would theoretically not be expected to be effective in elevating GSH levels above the lowered homeostasis [25]. On the other hand, exogenous γ-GC taken up intact, should feed directly into the unregulated GS enzyme and potentially increase GSH levels above homeostatic levels [26]. ...
Article
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Objective: To determine if orally dosed γ-glutamylcysteine (γ-GC) can increase cellular glutathione (GSH) levels above homeostasis. Many chronic and age-related disorders are associated with down-regulation, or impairment, of glutamate cysteine ligase (GCL). This suggests that γ-GC supply may become limiting for the maintenance of cellular GSH at the normal levels required to effectively protect against oxidative stress and any resulting physiological damage. Methods: GSH levels were measured in lymphocytes of healthy, non-fasting participants before and after single oral doses (2 and 4g) of γ-GC. Blood samples were immediately processed using high speed fluorescence-activated cell sorting to isolate 10(6) lymphocytes that were then assayed for GSH content. Results: A single 2g dose of γ-GC increased lymphocyte GSH content above basal levels (53±47%, p<0.01, n=14) within 90min of administration. A randomized dosage (2 and 4g γ-GC) crossover design was used to explore the pharmacokinetics of this GSH increase. In general, for both dose levels (n=9), GSH increased from initial basal levels over 3h (tmax) before reaching maximum GSH concentrations (Cmax) that were near two (2g γ-GC) to three (4g γ-GC) fold basal levels (0.4 nmol/10(6) lymphocytes). Beyond tmax, GSH levels progressively declined reaching near basal levels by 5h. The GSH half-life was between 2 and 3h with exposure (AUC) to increased GSH levels of 0.7 (2g γ-GC) and 1.8 (4g γ-GC) nmol.h/10(6) lymphocytes. Conclusions: Oral γ-GC is a non-toxic form of cysteine that can be directly taken up by cells and transiently increase lymphocyte GSH above homeostatic levels. Our findings that γ-GC can increase GSH levels in healthy subjects suggests that it may have potential as an adjunct for treating diseases associated with chronic GSH depletion. This trial was registered at anzctr.org.au as ACTRN12612000952842.
... Although to different extent, all of these treatments were able to prevent or revert SOD1 aggregation in neuronal cells, ameliorating mutant G93A-SOD1 protein solubility, preserving mitochondrial function and preventing apoptosis, thus suggesting that modulation of the redox state of cysteine residues in specific compartments could be a significant therapeutic strategy for ALS. Interestingly, that glutathione deficiency leads to mitochondrial damage in the brain had been already reported in a seminal article by Alton Meister more than 25 years ago (Mårtensson et al., 1990) and it is known that GSH decreases with age (Ferguson and Bridge, 2016). ...
Article
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Several proteins are found misfolded and aggregated in sporadic and genetic forms of amyotrophic lateral sclerosis (ALS). These include superoxide dismutase (SOD1), transactive response DNA-binding protein (TDP-43), fused in sarcoma/translocated in liposarcoma protein (FUS/TLS), p62, vasolin-containing protein (VCP), Ubiquilin-2 and dipeptide repeats produced by unconventional RAN-translation of the GGGGCC expansion in C9ORF72. Up to date, functional studies have not yet revealed a common mechanism for the formation of such diverse protein inclusions. Consolidated studies have demonstrated a fundamental role of cysteine residues in the aggregation process of SOD1 and TDP43, but disturbance of protein thiols homeostatic factors such as protein disulfide isomerases (PDI), glutathione, cysteine oxidation or palmitoylation might contribute to a general aberration of cysteine residues proteostasis in ALS. In this article we review the evidence that cysteine modifications may have a central role in many, if not all, forms of this disease.
... While the underlying mechanisms associated with declining motor and cognitive function remain unclear, it is known that the brain is particularly vulnerable to oxidative stress due to its high aerobic metabolism rate, abundance of redox modifiable substrates like iron and polyunsaturated fatty acids, relatively low antioxidant capacity, and limited cell turnover and neuroplasticity (Evans, 1993;Klempin and Kempermann, 2007). While some controversy has emerged (Perez et al., 2009), the oxidative stress theory remains an active explanation of the mechanisms underlying aging processes in multiple species and systems (Ferguson and Bridge, 2016;Huang et al., 2015;Sohal and Forster, 2014). In humans and in rodents, studies have provided evidence of increased oxidative stress and oxidative damage in the aging brain (Grimm et al., 2011;Perluigi et al., 2010), and that damage accumulation varied in different regions of the brain . ...
Article
Age-related declines in motor and cognitive function have been associated with increases in oxidative stress. Accordingly, interventions capable of reducing the oxidative burden would be capable of preventing or reducing functional declines occurring during aging. Popular interventions such as antioxidant intake and moderate exercise are often recommended to attain healthy aging and have the capacity to alter redox burden. This review is intended to summarize the outcomes of antioxidant supplementation (more specifically of vitamins C and E) and exercise training on motor and cognitive declines during aging, and on measures of oxidative stress. Additionally, we will address whether co-implementation of these two types of interventions can potentially further their individual benefits. Together, these studies highlight the importance of using translationally-relevant parameters for interventions and to study their combined outcomes on healthy brain aging.
... A limitation of this study is that the effect of AEE upon Nrf2 activation was not directly elucidated by suppressing Nrf2 transcriptional activity using either small interfering RNA or an inhibitor. However, as Nrf2 is a central controlling factor required for mediating positive regulation of NQO1 and GCLM gene expression [53,54], the observation that elevated mRNA levels of NQO1 and GCLM occurred upon AEE treatment support possible involvement of Nrf2 in the protective effect of AEE. ...
Article
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Background Acanthus ebracteatus (AE), an herb native to Asia, has been recognized in traditional folk medicine not only for its antioxidant properties and various pharmacological activities but also as an ingredient of longevity formulas. However, its anti-neurodegenerative potential is not yet clearly known. This work aimed to evaluate the protective effect of AE leaf extract against glutamate-induced oxidative damage in mouse hippocampal HT22 cells, a neurodegenerative model system due to a reduction in glutathione levels and an increase in reactive oxygen species (ROS). Methods Cell viability, apoptosis, and ROS assays were performed to assess the protective effect of AE leaf extract against glutamate-induced oxidative toxicity in HT22 cells. The antioxidant capacity of AE was evaluated using in vitro radical scavenging assays. The subcellular localization of apoptosis-inducing factor (AIF) and the mRNA and protein levels of genes associated with the nuclear factor erythroid 2–related factor 2 (Nrf2) antioxidant system were determined to elucidate the mechanisms underlying the neuroprotective effect of AE leaf extract. Results We demonstrated that AE leaf extract is capable of attenuating the intracellular ROS generation and HT22 cell death induced by glutamate in a concentration-dependent manner. Co-treatment of glutamate with the extract significantly reduced apoptotic cell death via inhibition of AIF nuclear translocation. The increases in Nrf2 levels in the nucleus and gene expression levels of antioxidant-related downstream genes under Nrf2 control were found to be significant in cells treated with the extract. Conclusions The results suggested that AE leaf extract possesses neuroprotective activity against glutamate-induced oxidative injury and may have therapeutic potential for the treatment of neurodegenerative diseases associated with oxidative stress.
... For example, GCLC was generally not found at the cell periphery. As the serum concentration of its product, cGC, is higher at 7 μM than its cytosolic level (42), cGC enters cells to participate in GSH synthesis (4,21,42,53,63). Moreover, cell-surface c-glutamyl transpeptidase (GGT), in the presence of a c-glutamyl donor such as glutamine, synthesizes cGC ( Fig. 1) (53,63). ...
Article
Some patients treated for ductal carcinoma in situ (DCIS) of the breast will experience cancer recurrences, whereas other patients will not. Unfortunately, current techniques cannot identify which pre-invasive lesions will lead to recurrent cancer. Because the mechanism of cancer recurrence is unknown, it is difficult to design a test that detects its activity. We propose that certain pentose phosphate pathway enzymes, glutathione synthesis enzymes, and RhoA cluster at the epithelial cell periphery during cancer recurrences. Enzyme clustering enhances metabolic flux. Using fluorescence microscopy we show that phosphophorylated glucose transporter type-1, transketolase-like protein-1, glutathione synthetase, GTP-loaded RhoA, and RhoA accumulate at the epithelial cell periphery in biopsies of women who will suffer recurrences, but not in samples from women who will not experience recurrences as judged using 2x2 contingency tables. A machine study of individual cribriform, papillary, micropapillary, and comedo forms of DCIS demonstrated 97% precision and 95% recall in the detection of samples from women will not experience a recurrence and a 90% precision and 95% recall in the detection of lesions that become recurrent. A holdout study of these patients showed 73% true negatives, 18% true positives, 4% false positives, and 4% false negatives at a 50% threshold. This work suggests mechanistic features of recurrences that may contribute to a new clinical test distinguishing high from low recurrence risk DCIS patients.
... The enzyme γ-glutamylcysteine ligase is the rate-limiting enzyme of de novo synthesis of glutathione, which catalyzes the synthesis of intermediate dipeptide γ-glutamylcysteine by ligating glutamic acid and cysteine. 108 Inhibition of γ-glutamylcysteine ligase by Buthionine sulfoximine causes a large drop in the amount of cellular glutathione and increases the level of ROS. Many types of cancer show elevation of glutathione levels and resistance to oxidative stress. ...
Article
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Although the terms "excessive reactive oxygen species (ROS)" and "oxidative stress" are widely used, the role of oxidative stress in biochemistry are often misunderstood. ROS are not a single species but a variety of compounds, each with unique biochemical properties and abilities to react with molecules. Excessive ROS causes activation of growth signals through thiol oxidation and may lead to DNA damage. In this review, we first discuss a conceptual framework for the interplay of ROS species and antioxidants and describe the effects of ROS on FLT3-mediated growth signaling. We then focus on ROS-mediated DNA damage. High concentrations of ROS result in various DNA lesions, including 8-oxo-7,8-dihydroguanine, oxazolone, DNA-protein crosslinks, and hydantoins, that have unique biological impacts. Here we describe nine well-characterized DNA lesions, the types of repair mechanisms, the mutations induced, and their effects on transcription and replication. Finally, this review will discuss biochemically inspired implications for cancer therapy. Several teams have put forward plans to harness the excessive ROS and burdened DNA repair systems of tumor cells to treat cancer.
... In addition, consuming pre-formed glutathione is an inefficient way to raise cellular levels of glutathione since published clinical studies demonstrate that it may take weeks or months to impact cellular levels when elevated levels are most needed acutely. [31][32][33][34][35][36][37][38][39][40][41][42][43] concept should be considered and tested using this approach in conjunction with measuring blood biomarkers that reflect individual and pathway proteins that correspond to inflammation and oxidative stress. ...
... A possible mechanism may be medicated through the induction of Nrf2 and its downtown genes by BDC to suppress oxidative stress, just like curcumin. g-GCL is a key rate-limiting enzyme in de novo synthesis cascade of GSH (Ferguson and Bridge, 2016). g-GCL consists of a catalytic subunit g-GCLc, and a modulatory subunit g-GCLm. ...
Article
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This study was conducted to investigate the beneficial effects of bisdemethoxycurcumin (BDC) on growth performance, glutathione (GSH) redox potential, antioxidant enzyme defense, and gene expression in lipopolysaccharide (LPS)-challenged broilers. A total of 320, male, 1-day-old broilers were randomly assigned to 4 treatment groups including 8 replicates with 10 birds per cage in a 2 × 2 factorial arrangement: BDC supplementation (a basal diet with 0 or 150 mg/kg BDC) and LPS challenge (intraperitoneal injection of 1 mg/kg body weight saline or LPS at 16, 18, and 20 d of age). Results showed that dietary BDC supplementation prevented the LPS-induced decrease in ADG of broilers (P
... 5) For many disorders, diseases, and aging, it is suggested that GSH depletion is associated with the onset of dysfunctional GCL regulation with insufficient γ-EC synthesis to supply GSH and maintain appropriate homeostasis. [6][7][8] However, exogenous GSH administration could not help alleviate those disorders and disease states, as the ability to ensure homeostatic regulation of GSH levels is decreased in those cases. 9) On the other hand, recent studies have revealed that the administration of γ-EC increases intracellular GSH levels in a human trial pilot study. ...
Article
Gamma-glutamylcysteine (γ-EC) is an intermediate generated in the de novo synthesis of glutathione (GSH). Recent studies have revealed that the administration of γ-EC shows neuroprotective effects against oxidative stress in age-related disorders and chronic diseases like Alzhiemer’s disease in model animals, which is not expected function in GSH. A phytochelatin synthase-like enzyme derived from Nostoc sp. (NsPCS) mediates γ-EC synthesis from GSH. To achieve low-cost and stable commercial level supply, the availability of immobilized NsPCS for γ-EC production was investigated in this study. Among the tested immobilization techniques, covalent binding to the cellulose carrier was most effective, and could convert GSH completely to γ-EC without decreasing the yield. The stable conversion of γ-EC from 100 mM GSH was achieved by both batch repeated and continuous reactions using the immobilized NsPCS on cellulose sheet and column shape monolith, respectively. The immobilization of NsPCS on those carriers is promising alternative technique for high-yielding and cost-effective production of γ-EC on its commercial applications. Fullsize Image
... The enzyme GCL that catalyzes the first and rate-limiting step plays a decisive role in GSH synthesis. 76 Thus, selectively blocking the rate-limiting step of GSH synthesis using specific drugs is a potential method for reducing GSH content in cancer cells. While several effective inhibitors of GSH synthesis are now utilized, the most anticipated inhibitor has been L-buthionine sulfoximine (BSO), which is commercially available. ...
Article
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Glutathione (GSH), the main redox buffer, has long been recognized as a pivotal modulator of tumor initiation, progression and metastasis. It is also implicated in the resistance of platinum-based chemotherapy and radiation therapy. Therefore, depleting intracellular GSH was considered a potent solution to combating cancer. However, reducing GSH within cancer cells alone always failed to yield desirable therapeutic effects. In this regard, the convergence of GSH-scavenging agents with therapeutic drugs has thus been pursued in clinical practice. Unfortunately, the therapeutic outcomes are still unsatisfactory due to untargeted drug delivery. Advanced nanomedicine of synergistic GSH depletion and cancer treatment has attracted tremendous interest because they promise to deliver superior therapeutic benefits while alleviating life-threatening side effects. In the past five years, the authors and others have demonstrated that numerous nanomedicines, by simultaneously delivering GSH-depleting agents and therapeutic components, boost not only traditional chemotherapy and radiotherapy but also multifarious emerging treatment modalities, including photodynamic therapy, sonodynamic therapy, chemodynamic therapy, ferroptosis, and immunotherapy, to name a few, and achieved decent treatment outcomes in a large number of rodent tumor models. In this review, we summarize the most recent progress in engineering nanomedicine for GSH depletion-enhanced cancer therapies. Biosynthesis of GSH and various types of GSH-consuming strategies will be briefly introduced. The challenges and perspectives of leveraging nanomedicine for GSH consumption-augmented cancer therapies will be discussed at the end.
... γ-glutamylcysteine is the dipeptide and precursor of glutathione (Anderson & Meister, 1983). Unlike GSH, as a direct substrate for the production of GSH, supplemental γ-GC can be utilized by cells (Ferguson & Bridge, 2016). It is reported that oral administration of γ-GC could increase intracellular glutathione levels (Zarka & Bridge, 2017). ...
Article
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Alcohol abuse is a major cause of alcoholic liver disease (ALD) and can result in fibrosis and cirrhosis. γ‐glutamylcysteine (γ‐GC) is a precursor of glutathione (GSH) with antioxidant and anti‐inflammatory properties. Our research aimed to explore the protective impact of γ‐GC on ALD and its potential mechanisms of efficiency in vitro and in vivo. L02 cells were pretreated with γ‐GC (20, 40, and 80 μM) for 2 h and exposed to ethanol for 24 h. Cell viability, apoptosis, oxidative stress, and inflammatory levels were measured. The expression of protein cleaved caspase‐3 and cleaved PARP and flow cytometry results indicated that γ‐GC decreases apoptosis on L02 cells after ethanol treatment. Moreover, γ‐GC also attenuated oxidative stress and mitochondrial damage in hepatocytes caused by ethanol via increasing cellular GSH, SOD activity, and mitochondrial membrane potential. In vivo experiments, γ‐GC effectively reduced the AST, ALT, and TG levels in mice. The inflammation of ALD was alleviated by γ‐GC both in vivo and in vitro. Additionally, histopathological examination demonstrated that γ‐GC treatment lessened lipid droplet formation and inflammatory damage. In conclusion, these results showed that γ‐GC has anti‐inflammatory and anti‐apoptotic effects on ALD because it could help hepatocytes maintain sufficient GSH levels to combat the excess reactive oxygen species (ROS) generated during ethanol metabolism. Practical applications Alcohol intake is the fifth highest risk factor for premature death and disability among all risk variables. However, few medicines are both safe and effective for the treatment of ALD. As a direct precursor of GSH, γ‐GC has a broad variety of potential antioxidant and anti‐inflammatory applications for the treatment of numerous medical conditions. In conclusion, these results showed that γ‐GC could protect cells from ALD via suppressing oxidative stress, alleviating inflammation, and preventing apoptosis.
Article
Aging is a natural process accompanied by inflammation and oxidative stress and is closely associated with age-related diseases. As a direct precursor of glutathione, γ-glutamylcysteine (γ-GC) possesses antioxidant and anti-inflammatory properties; however, whether γ-GC plays an important role in anti-aging remains unknown. Here, we investigated the protective effects and mechanisms of γ-GC in D-galactose (D-gal)-induced senescence in PC12 cells and aging mice. Our results showed that γ-GC treatment significantly reduced the percentage of senescence-associated-β-galactosidase (SA-β-Gal)-positive cells and inhibited D-gal-induced cell cycle arrest in PC12 cells. The results of Nissl and hematoxylin and eosin (H&E) staining in mouse brain showed that γ-GC treatment markedly reversed the damage in the hippocampus of D-gal-induced aging mice. Moreover, γ-GC increased the phosphorylation of AMP-activated protein kinase (AMPK) to promote the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) while inhibiting the nuclear translocation of deleted in breast cancer 1 (DBC1), which leads to the activation of sirtuin 1 (SIRT1) and deacetylation of p53 in the nucleus. Therefore, γ-GC may be a potential therapeutic candidate compound for the prevention and treatment of age-related diseases.
Article
The present study aimed to evaluate the anti-aging effect of the leaves of Scutellaria baicalensis Georgi (LSBG) and investigate its mechanisms. For this purpose, SD rats were received D-galactose (D-gal) subcutaneously (0.3 g/kg) and LSBG intragastrically (0.4 g/kg or 0.8 g/kg) for 7 weeks. Behavior tests were conducted to evaluate the cognitive function of all rats. Results showed that memory impairment was reversed by LSBG. Then, metabolomics of the cortex and hippocampus were used to investigate the potential mechanisms. 21 metabolites in the cortex and 22 metabolites in the hippocampus of aging rats were altered, respectively. Additionally, results showed that the content of key metabolites and activities of enzymes in glutamate metabolism and its downstream metabolism (glutathione metabolism) could be regulated by the LSBG. Additionally, proteins in the Nrf2 signaling pathway were analyzed by western blot. And the protein expression levels of Nrf2, GCLC, HO-1, NQO-1 were significantly regulated by the LSBG in the cortex and hippocampus. Above all, the anti-aging effects of the LSBG were involved in regulating the glutamate metabolism and Nrf2 signaling pathway.
Article
Silymarin (SM) is a well-known antioxidant, anti-inflammatory and anti-cancer compound extracted from the milk thistle. Here, we investigated the protective effect of SM against acrylamide (AA)-induced neurotoxicity, mainly caused by oxidative stress, via activation of the nuclear transcription factor E2-related factor 2 (Nrf2) signalling pathway in PC12 cells. The MTT reduction assay was used to measure cell viability in various drug-treated groups and demonstrated that SM could increase cell viability in AA-treated PC12 cells. We then measured the reactive oxygen species (ROS) levels by the peroxide-sensitive fluorescent probe DCFH-DA and intracellular glutathione (GSH) and malondialdehyde (MDA) levels by absorption spectrophotometry. Our data revealed that SM could reduce ROS and MDA levels and increase GSH levels in AA-induced PC12 cells. To identify a potential mechanism for SM-induced protection, we measured the mRNA and protein expression levels of Nrf2 and its downstream target antioxidants glutathione peroxidase (Gpx), glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modifier subunit (GCLM) by quantitative real-time PCR and Western blot, respectively. The results suggested that SM could activate Nrf2 signalling and increase the expression of Nrf2, Gpx, GCLC and GCLM in AA-treated PC12 cells. In conclusion, SM can effectively alleviate AA-induced neurotoxicity in PC12 cells.
Article
Background/aims: In diabetic patients, reduced urinary pH (UpH) is a predictive factor for cardiorenal-vascular disorders. Synthesis of glutathione, an anti-oxidative stress substance, is induced to counteract renal oxidative stress. UpH declines as glutamate is consumed, as does the synthesis of ammonia from glutamate. Glutathione is synthesized from glutamate and cysteine; however, in diabetes, the relationship between lowered UpH and the roles of renal amino acids is unknown. We, therefore, examined the relationship between amino-acid kinetics, UpH, and renal function. Methods: This cross-sectional study targeted 100 non-diabetic obese individuals (OG: obese group) and 100 diabetics (DG: diabetic group). We investigated their blood amino acids, urinary amino-acid excretion, the reabsorption rates of various amino acids, and their relationship with the UpH and estimated glomerular filtration rate (eGFR). Results: The DG subjects showed higher blood cysteine concentration, urinary glutamate, and cysteine excretions than the OG subjects. Although the glutamate reabsorption rate declined in the DG subjects, that of cysteine increased due to the lowered eGFR. The DG subjects' urinary cysteine excretion correlated positively with UpH, making this urinary cysteine excretion the sole independent risk factor for lower UpH. Conclusion: In patients with diabetes, the reabsorbed amount of cysteine, not glutamate, regulates the amount of glutathione synthesis in the kidneys. The more an amount of cysteine reabsorption increases concurrently with a decline in eGFR, the more its urinary excretion decreases. Under these conditions, concurrently, the glutamate consumption then increases, resulting in decreased ammonia synthesis and UpH.
Article
Glutaredoxins (Grxs) are ubiquitous thioltransferases and members of the thioredoxin (Trx) fold superfamily. They have multiple functions in cells including oxidative stress responses and cell signaling. A novel glutaredoxin from buckwheat (rbGrx) with higher catalytic activity was identified, cloned, and purified. The structures of glutathionylated rbGrx and an rbGrx mutant, in which cysteine 39 was mutated to alanine, were solved by x-ray diffraction at a resolution of 2.05 Å and 2.29 Å, respectively. In rbGrx, GSH (glutathione) is bound at the conserved GSH-binding site, and its structure shows that it has the potential to function as a scaffold protein for the assembly and delivery of GSH. The crystal structure shows that GSH does not bind to the C39A rbGrx mutant, and the C39A mutant had no catalytic activity, indicating that C39 is a key residue that is involved in both the binding of rbGrx to GSH and the regulation of its catalytic activity. The model showing the binding of GSH with rbGrx provides a basis for understanding its molecular function and its potential future applications in medicinal food science.
Article
Our previous study showed that 2,2’-,4,4’-tetrabromodiphenyl ether (BDE-47) is cytotoxic and induces apoptosis in Neuro-2a cells. In the present study, we aimed to investigate whether nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an antioxidant transcriptional regulator of oxidative stress and apoptosis, is involved in this process. The results of toxicological experiments showed that BDE-47 decreased the cellular mitochondrial membrane potential (MMP) and increased cytochrome c release to the cytoplasm, followed by an increase in intracellular caspase-9 and caspase-3 activity, suggesting that a mitochondrial pathway was involved in the apoptotic process. Intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) contents as well as the oxidized/reduced glutathione (GSSG/GSH) ratio were elevated simultaneously in a concentration-dependent manner, indicating that BDE-47 induced oxidative stress. The ROS scavenger N-acetyl-L-cysteine (NAC) not only alleviated the oxidative stress but also blocked apoptosis and the decrease in MMP induced by BDE-47, indicating that the overproduction of ROS participates in a mitochondria-mediated apoptotic pathway. Moreover, BDE-47 stimulated the transcriptional induction of the Nrf-2 gene and induced mRNA expression of the main antioxidant response genes in the Nrf-2 pathway, including heme oxygenase 1 (HO-1), NAD(P)H/quinone oxidoreductase-1 (NQO1), glutamate-cysteine ligase modifier (GCLM) and glutathione peroxidase (GPX). Additionally, NAC and the p38 mitogen activated protein kinase (MAPK) signaling pathway inhibitor SB 203580 greatly reduced Nrf2 and HO-1 induction. We hypothesized that the ROS mediated mitochondrial pathway is involved in the BDE-47-induced apoptosis in Neuro-2a cells and that the Nrf2 pathway helps protect Neuro-2a cells from BDE-47-induced apoptosis.
Article
Background Acute 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. Methods Overexpressing 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. Results We 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. Conclusions These results show that GCLc overexpressing BMSCs with anti-apoptotic effects significantly improve acute lung injury.
Article
Introduction The accumulation of oxidative stress, neuroinflammation and abnormal aggregation of amyloid β-peptide (Aβ) have been shown to induce synaptic dysfunction and memory deficits in Alzheimer’s disease (AD). Cellular depletion of the major endogenous antioxidant Glutathione (GSH) has been linked to cognitive decline and the development of AD pathology. Supplementation with γ-glutamylcysteine (γ-GC), the immediate precursor and the limiting substrate for GSH biosynthesis, can transiently augment cellular GSH levels by bypassing the regulation of GSH homeostasis. Methods In the present study, we investigated the effect of dietary supplementation of γ-GC on oxidative stress and Aβ pathology in the brains of APP/PS1 mice. The APP/PS1 mice were fed γ-GC from 3 months of age with biomarkers of apoptosis and cell death, oxidative stress, neuroinflammation and Aβ load being assessed at 6 months of age. Results Our data showed that supplementation with γ-GC lowered the levels of brain lipid peroxidation, protein carbonyls and apoptosis, increased both total GSH and the glutathione/glutathione disulphide (GSH/GSSG) ratio and replenished ATP and the activities of the antioxidant enzymes (superoxide dismutase (SOD), catalase, glutamine synthetase and glutathione peroxidase (GPX)), the latter being a key regulator of ferroptosis. Brain Aβ load was lower and acetylcholinesterase (AChE) activity was markedly improved compared to APP/PS1 mice fed a standard chow diet. Alteration in brain cytokine levels and matrix metalloproteinase enzymes MMP-2 and MMP-9 suggested that γ-GC may lower inflammation and enhance Aβ plaque clearance in vivo. Spatial memory was also improved by γ-GC as determined using the Morris water maze. Conclusion Our data collectively suggested that supplementation with γ-GC may represent a novel strategy for the treatment and/or prevention of cognitive impairment and neurodegeneration.
Article
Alzheimer's disease (AD) is the most prevalent neurogenerative disease, characterized by progressive memory loss and cognitive deficits. Intracellular neurofibrillary tangles (NFTs) and amyloid-β (Aβ)-formed neuritic plaques are major pathological features of AD. Aβ evokes activation of microglia to release inflammatory mediators and ROS to induce neurotoxicity, leading to neurodegeneration. γ-Glutamylcysteine (γ-GC), an intermediate dipeptide of the GSH-synthesis pathway with anti-inflammatory and anti-oxidative properties, represents a relatively unexplored option for AD treatment. In the present study, we investigated the anti-inflammatory effect of γ-GC on Aβ oligomer (AβO)-induced neuroinflammation and the associated molecular mechanism in microglia. The results showed that γ-GC reduced AβO-induced release of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and nitric oxide (NO), and the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). γ-GC decreased ROS and MDA production and increased the GSH level, GSH/GSSG ratio, and SOD activity in AβO-treated microglia. Mechanistically, γ-GC inhibited activation of nuclear factor kappa B (NF-κB), and upregulated the nuclear receptor-related 1 (Nurr1) protein expression to suppress the transcriptional effect of NF-κB on the inflammatory genes. Besides, γ-GC suppressed the AβO-induced neuroinflammation in mice. These findings suggested that γ-GC might represent a potential therapeutic agent for anti-neuroinflammation.
Article
Glutathione (GSH) is a tripeptide that is readily synthesized intracellularly in humans and other mammals. More than a century of research suggests that GSH has numerous biological functions, including protection from the potential adverse events associated with reactive oxygen species (ROS) and related redox reactions that may induce oxidative stress, and that may be linked to innate detoxification processes. Normal tissue and plasma levels of GSH decline through the aging process and decrease during various disease states. While the health value of dietary GSH remains controversial, there is evidence that some metabolic intermediates, such as γ-glutamylcysteine (GGC) may function to preserve adequate GSH levels when the synthetic pathways decline in activity, and the innate antioxidant system is challenged. It is also important to recognize that among the thousands of protein-coding human genes and their respective polymorphisms, at least two genes (Gclc and Gclm) are directly involved with GSH synthesis via glutamate-cysteine ligase. This commentary examines the classic biochemistry, toxicology, safety, and clinical value of GSH and its intermediates that may be modulated by dietary supplementation.
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Background: Ultraviolet (UV) radiation-induced oxidative stress is the main cause of photodamage to the skin. Glutathione (GSH) serves important physiological functions, including scavenging oxygen free radicals and maintaining intracellular redox balance. γ-glutamylcysteine (γ-GC), as an immediate precursor of GSH and harboring antioxidant and anti-inflammatory properties, represents an unexplored option for skin photodamage treatment. Purpose: The purpose of this study was to investigate whether γ-GC can reduce UVB-induced NIH-3T3 cell damage. Methods: The experimental groups were as follows: control, UVB radiation, UVB radiation after pretreatment with γ-GC. Cell counting kit-8 (CCK-8) assays were used to measure cell proliferation, flow cytometry, and immunoblotting to detect the apoptosis rate and apoptosis-associated proteins. The levels of Reactive Oxygen Species (ROS), Superoxide Dismutase (SOD) and GSH/GSSG (oxidized GSH) were measured to assess oxidative stress. Immunoblotting and immunofluorescence were used to detect DNA damage. The members of the MAPK signaling pathways were detected by immunoblotting. Results: UVB irradiation significantly reduced cell viability, destroyed the oxidative defense system. Pretreatment with γ-GC reduced UVB-induced cytotoxicity, restored the oxidation defense system, and inhibited activation of the MAPK pathway. It also reduced the apoptosis rate, downregulated the levels of cleaved caspase 3 and cleaved PARP. Furthermore, pretreatment with γ-GC reduced the accumulation of γH2AX after UVB radiation exposure, indicating that γ-GC could protect cells from DNA damage. Conclusion: γ-GC protected NIH-3T3 from damage caused by UVB irradiation. The photoprotective effect of γ-GC is mediated via strengthening the endogenous antioxidant defense system, which prevents DNA damage and inhibits the activation of the MAPK pathway.
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Gamma-glutamyl-cysteine (γ-EC) is a precursor of glutathione (GSH) biosynthesis. We investigated whether it functions as a substrate for three intracellular and one extracellular GSH metabolic enzymes, which mediate the antioxidant defense function of GSH. Among them, glutathione peroxidase, glutathione S-transferase, and γ-glutamyl transferase (GGT) exhibited substrate specificity for γ-EC, whereas glutathione reductase did not. The specificities of γ-EC and its disulfide form to GGT were comparable to GSH and its oxidized form, GSSG, respectively. These result indicate that they can supply GSH constituent amino acids, glutamate, cysteine, and cystine through degradation by GGT. γ-EC may contribute valuable antioxidant defense properties as a food and cosmetic additive.
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Cadmium (Cd) is a highly toxic environmental pollutant, leading to the occurrence and development of multiple neurological diseases. γ-glutamylcysteine (γ-GC) is a dipeptide formed by the condensation of L-glutamic acid and L-cysteine, which has antioxidant, anti-inflammatory, and chelating properties. The purpose of this study is to investigate the effect of γ-GC on Cd-induced apoptosis in PC12 cells. PC12 cells were pretreated with or without γ-GC (2 mM or 4 mM) for 2 h and exposed to Cd (10 μM) for 12 h, and survival, apoptosis, and oxidative stress of PC12 cells were detected after different treatments. The results showed that γ-GC significantly inhibited cell viability reduction, apoptosis, and depolarization of mitochondrial transmembrane potential in Cd-treated PC12 cells, as indicated by CCK-8 assay, flow cytometry, TUNEL staining, and JC-1 detection. Western blot showed that γ-GC down-regulated the ratio of Bax/Bcl-2 and the protein levels of cytosolic cytopigment c, cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP. Mechanistically, γ-GC suppressed Cd-induced ROS production, MDA accumulation, and GSH depletion, and increased the activity of antioxidant enzymes. Cd-induced activation of MAPK and PI3K/CAT signaling pathways were inhibited by γ-GC treatment, while sustained phosphorylation of JNK, p38, or Akt reversed anti-apoptotic effects of γ-GC. These results suggested that γ-GC inhibited Cd-induced apoptosis in PC12 cells through decreasing oxidative stress and inhibiting the activation of MAPK and PI3K/Akt signaling pathways. γ-GC could be used as a potential protective agent against Cd neurotoxicity.
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Recently, combination therapy has proven to be an effective strategy for treating polygenic/multifactorial/complex disorder such as Parkinson's disease (PD). Here, we hypothesized that dual up-regulation of glutamate cysteine ligase (GCL) catalytic subunit (GCLc) and GCL modifier subunit (GCLm) via nuclear factor E2-related factor (Nrf2) contribute to the antioxidant effect of paeoniflorin (PF) synergistically with glycyrrhetinic acid (GA) (henceforth called PF/GA) in the context of MPP+/MPTP neurotoxicity. Expectedly, CompuSyn synergism/antagonism analysis showed that PF/GA exerts synergistic neuroprotection. Moreover, the antioxidant effect of PF was significantly enhanced by the combined administration of GA, although GA alone did not confer the effect. Mechanistically, PF triggered extracellular signal-regulated kinase (ERK1/2) phosphorylation, resulting in Nrf2 nuclear translocation from cytoplasmic pool via de novo synthesis in MPP+-challenged SH-SY5Y cells. Concomitantly, GA activates Akt which in turn induces nuclear accumulation of Nrf2. Especially, PF/GA up-regulated glutamate-cysteine ligase catalytic subunit (Gclc) and glutamate-cysteine ligase modifier subunit (Gclm) are formed via two separate pathways. Furthermore, these results were confirmed through pathway blockade assays using PD98059 (ERK1/2 inhibitor), LY294002 (phosphatidylinositol-3-kinase inhibitor), and shRNA-induced Nrf2 knockdown. Additionally, using a mouse MPTP-induced model of PD, we demonstrated that PF/GA synergistically ameliorates both motor deficits and oxidative stress in the ventral midbrain. In parallel, PF/GA also up-regulated both GCLc and GCLm expression at levels of transcription and translation. Conversely, antiparkinsonism and antioxidant effects of PF/GA were not observed in Nrf2-knockout MPTP-mice. Collectively, these results show that ERK1/2 and Akt activation contribute to the synergistic antioxidant effect of PF/GA. Hence, PF/GA regimen warrants further preclinical and possible clinical study for PD.
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With the development of society, physical activity has come to be an effective means by which people pursue good health to improve the quality of life. However, with the increase of intensity and the passage of time, exercise injury has become a hazard that can no longer be ignored. It is imperative to find effective ways to inhibit or reduce the negative effects of exercise. Mitochondria are important organelles involved in exercise and play an important role in exercise injury and prevention. Studies have found that exercise preconditioning and increased mitochondrial nutrition can effectively decrease mitochondrial damage after exercise. Against this background, some of the newest developments in this important field are reviewed here. The results discussed indicate that exercise preconditioning and supplement mitochondrial nutrition need to be increased to prevent exercise-related injuries.
Chapter
Glutathione is a very ancient molecule widely distributed in aerobic cells and organisms, either prokaryotes or eukaryotes. Since glutathione in not found in anaerobic cells it could have evolved in the course of the adaptation to the presence of oxygen in the atmosphere. Glutathione is the major non-protein low molecular weight antioxidant and the most important cellular thiol reducing agent. Glutathione biosynthesis occurs in the cytosol from its constituent amino acids; GSH is present also in the most important cellular districts like mitochondria and nucleus to indicate its central role in several metabolic pathways and protective mechanisms. There are several glutathione dependent enzymes involved in various steps of cell metabolism. GSH is a key antioxidant that modulates various cellular processes and therefore is determinant for redox signaling, xenobiotics’s detoxication, regulation of cell proliferation, apoptosis and immune functions. Glutathione concentration and redox state is due to a complex interaction between biosynthesis, utilization, degradation, and transport. All these factors are of great importance for understanding the significance of cellular redox balance and its correlation with pathological conditions.
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Health is usually defined as the absence of pathology. Here, we endeavor to define health as a compendium of organizational and dynamic features that maintain physiology. The biological causes or hallmarks of health include features of spatial compartmentalization (integrity of barriers and containment of local perturbations), maintenance of homeostasis over time (recycling and turnover, integration of circuitries, and rhythmic oscillations), and an array of adequate responses to stress (homeostatic resilience, hormetic regulation, and repair and regeneration). Disruption of any of these interlocked features is broadly pathogenic, causing an acute or progressive derailment of the system coupled to the loss of numerous stigmata of health.
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Polyphenols in red wine are bioactive compounds with positive effects on health and disease prevention. White grapes musts and wines have a lower concentration of phenolic compounds compared to the red ones and are therefore considered less beneficial to health. In Andalucia, a region located in the South West of Spain, the Pedro Ximenez white grapes are desiccated under the sun for a week before they are pressed and the juice (must) obtained. This ancient procedure increases the variety and content of polyphenols present in the Pedro Ximenez must (PXM). We have incorporated PXM to the daily diet of aged Mus spretus mice (24 months) and investigated their properties by comparing several parameters determined in these old mice with those measured in young mice (two months). Biochemical, histological and transcriptional analyses indicated that PXM exhibits potent antioxidant properties, promotes the normalization of the biotransforming ability of several cytochromes P450 in the liver, and regularizes the hepatic apoptosis, promoting proliferation instead. Our data indicate that PXM possesses a profound ability to promote liver regeneration of both the structure and the function, contributing to a healthy aging process.
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gamma-Glutamylcysteine synthetase (rat kidney; Mr approximately 104,000) is composed of 2 nonidentical subunits. In the present work, a procedure was developed for the reversible dissociation of the enzyme into its subunits (Mr = 73,000 and 27,700) under nondenaturing conditions. Students in which gel electrophoresis was used, in conjunction with an enzyme activity stain and elution and re-electrophoresis of protein bands, showed that the heavy subunit contains all of the structural requirements for enzymatic activity and also for feedback inhibition of the enzyme activity by glutathione. The light subunit, which may be formed from a precursor protein, has a significantly lower content of Trp, Phe, Tyr, Val, and Ala residues than the heavy subunit, while its content of Lys, His, Met, and Asx residues is higher.
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Nrf1 is a member of the CNC-basic leucine zipper (CNC-bZIP) family of transcription factors. CNC bZIP factors, together with small Maf proteins, bind as heterodimers to the NF-E2/AP-1 element. Similarity between the NF-E2/AP-1 element and the antioxidant response element identified in a number of promoters of genes involved in detoxification and antioxidant response raises the possibility that Nrf1 plays a role in mediating the antioxidant response element response. In this study, we exploited the availability of cells from Nrf1 knockout mice to study the role of Nrf1 transcription factor in the regulation of antioxidant gene expression and in cellular antioxidant response. Fibroblast cells derived from Nrf1 null embryos showed lower levels of glutathione and enhanced sensitivity to the toxic effects of oxidant compounds. Our results indicate that Nrf1 plays a role in the regulation of genes involved in glutathione synthesis and suggest a basis for a correspondingly low GSH concentration and reduced stress response.
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The aim of the present study was to evaluate the effect that a dietary intake of resveratrol (RSV) had on the expression of glutamate cysteine ligase (GCL) in the kidneys of aged rats. Young, middle-aged and aged rats were each randomly divided into two groups. The control groups were fed a controlled diet and the experimental groups received a controlled diet supplemented with RSV. GCL activity levels in the kidneys were determined. Protein content and relative gene expression levels of the two subunits of GCL were evaluated by western blot analysis and quantitative polymerase chain reaction, respectively. GCL activity levels significantly increased in the kidneys of aged rats fed the RSV-supplemented diet. In addition, RSV markedly increased the protein content and relative mRNA expression levels of the GCL subunits in the kidneys of aged rats. These observations have important implications for the development of therapeutic agents for the kidneys that may enable the elderly population to combat oxidative stress.
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Background Glutathione (GSH) is present in all mammalian tissues as the most abundant non-protein thiol that defends against oxidative stress. GSH is also a key determinant of redox signaling, vital in detoxification of xenobiotics, and regulates cell proliferation, apoptosis, immune function, and fibrogenesis. Biosynthesis of GSH occurs in the cytosol in a tightly regulated manner. Key determinants of GSH synthesis are the availability of the sulfur amino acid precursor, cysteine, and the activity of the rate-limiting enzyme, glutamate cysteine ligase (GCL), which is composed of a catalytic (GCLC) and a modifier (GCLM) subunit. The second enzyme of GSH synthesis is GSH synthetase (GS).Scope of reviewThis review summarizes key functions of GSH and focuses on factors that regulate the biosynthesis of GSH, including pathological conditions where GSH synthesis is dysregulated.Major conclusionsGCL subunits and GS are regulated at multiple levels and often in a coordinated manner. Key transcription factors that regulate the expression of these genes include NF-E2 related factor 2 (Nrf2) via the antioxidant response element (ARE), AP-1, and nuclear factor kappa B (NFκB). There is increasing evidence that dysregulation of GSH synthesis contributes to the pathogenesis of many pathological conditions. These include diabetes mellitus, pulmonary and liver fibrosis, alcoholic liver disease, cholestatic liver injury, endotoxemia and drug-resistant tumor cells.General significanceGSH is a key antioxidant that also modulates diverse cellular processes. A better understanding of how its synthesis is regulated and dysregulated in disease states may lead to improvement in the treatment of these disorders. This article is part of a Special Issue entitled Cellular functions of glutathione.
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Background X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder of X-linked inheritance caused by a mutation in the ABCD1 gene which determines an accumulation of long-chain fatty acids in plasma and tissues. Recent evidence shows that oxidative stress may be a hallmark in the pathogenesis of X-ALD and glutathione plays an important role in the defense against free radicals. In this study we have analyzed glutathione homeostasis in lymphocytes of 14 patients with X-ALD and evaluated the balance between oxidized and reduced forms of glutathione, in order to define the role of this crucial redox marker in this condition. Methods Lymphocytes, plasma and erythrocytes were obtained from the whole blood of 14 subjects with X-ALD and in 30 healthy subjects. Total, reduced and protein-bound glutathione levels were measured in lymphocytes by HPLC analysis. Erythrocyte free glutathione and antioxidant enzyme activities, plasma thiols and carbonyl content were determined by spectrophotometric assays. Results A significant decrease of total and reduced glutathione was found in lymphocytes of patients, associated to high levels of all oxidized glutathione forms. A decline of free glutathione was particularly significant in erythrocytes. The increased oxidative stress in X-ALD was additionally confirmed by the decrease of plasma thiols and the high level of carbonyls. Conclusion Our results strongly support a role for oxidative stress in the pathophysiology of X-ALD and strengthen the importance of the balance among glutathione forms as a hallmark and a potential biomarker of the disease.
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The transcription factor Nrf2 (NF-E2-related factor 2) plays a vital role in maintaining cellular homeostasis, especially upon the exposure of cells to chemical or oxidative stress, through its ability to regulate the basal and inducible expression of a multitude of antioxidant proteins, detoxification enzymes and xenobiotic transporters. In addition, Nrf2 contributes to diverse cellular functions including differentiation, proliferation, inflammation and lipid synthesis and there is an increasing association of aberrant expression and/or function of Nrf2 with pathologies including cancer, neurodegeneration and cardiovascular disease. The activity of Nrf2 is primarily regulated via its interaction with Keap1 (Kelch-like ECH-associated protein 1), which directs the transcription factor for proteasomal degradation. Although it is generally accepted that modification (e.g. chemical adduction, oxidation, nitrosylation or glutathionylation) of one or more critical cysteine residues in Keap1 represents a likely chemico-biological trigger for the activation of Nrf2, unequivocal evidence for such a phenomenon remains elusive. An increasing body of literature has revealed alternative mechanisms of Nrf2 regulation, including phosphorylation of Nrf2 by various protein kinases (PKC, PI3K/Akt, GSK-3β, JNK), interaction with other protein partners (p21, caveolin-1) and epigenetic factors (micro-RNAs -144, -28 and -200a, and promoter methylation). These and other processes are potentially important determinants of Nrf2 activity, and therefore may contribute to the maintenance of cellular homeostasis. Here, we dissect evidence supporting these Keap1-dependent and -independent mechanisms of Nrf2 regulation. Furthermore, we highlight key knowledge gaps in this important field of biology, and suggest how these may be addressed experimentally.
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Dysregulation of glutathione homeostasis and alterations in glutathione-dependent enzyme activities are increasingly implicated in the induction and progression of neurodegenerative diseases, including Alzheimer's, Parkinson's and Huntington's diseases, amyotrophic lateral sclerosis, and Friedreich's ataxia. In this review background is provided on the steady-state synthesis, regulation, and transport of glutathione, with primary focus on the brain. A brief overview is presented on the distinct but vital roles of glutathione in cellular maintenance and survival, and on the functions of key glutathione-dependent enzymes. Major contributors to initiation and progression of neurodegenerative diseases are considered, including oxidative stress, protein misfolding, and protein aggregation. In each case examples of key regulatory mechanisms are identified that are sensitive to changes in glutathione redox status and/or in the activities of glutathione-dependent enzymes. Mechanisms of dysregulation of glutathione and/or glutathione-dependent enzymes are discussed that are implicated in pathogenesis of each neurodegenerative disease. Limitations in information or interpretation are identified, and possible avenues for further research are described with an aim to elucidating novel targets for therapeutic interventions. The pros and cons of administration of N-acetylcysteine or glutathione as therapeutic agents for neurodegenerative diseases, as well as the potential utility of serum glutathione as a biomarker, are critically evaluated.
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Reactive oxygen species regulate redox-signaling processes, but in excess they can cause cell damage, hence underlying the aetiology of several neurological diseases. Through its ability to down modulate reactive oxygen species, glutathione is considered an essential thiol-antioxidant derivative, yet under certain circumstances it is dispensable for cell growth and redox control. Here we show, by directing the biosynthesis of γ-glutamylcysteine-the immediate glutathione precursor-to mitochondria, that it efficiently detoxifies hydrogen peroxide and superoxide anion, regardless of cellular glutathione concentrations. Knocking down glutathione peroxidase-1 drastically increases superoxide anion in cells synthesizing mitochondrial γ-glutamylcysteine. In vitro, γ-glutamylcysteine is as efficient as glutathione in disposing of hydrogen peroxide by glutathione peroxidase-1. In primary neurons, endogenously synthesized γ-glutamylcysteine fully prevents apoptotic death in several neurotoxic paradigms and, in an in vivo mouse model of neurodegeneration, γ-glutamylcysteine protects against neuronal loss and motor impairment. Thus, γ-glutamylcysteine takes over the antioxidant and neuroprotective functions of glutathione by acting as glutathione peroxidase-1 cofactor.
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The tripeptide glutathione (GSH) is the most abundant free radical scavenger synthesized endogenously in humans. Increasing mechanistic, clinical, and epidemiological evidence demonstrates that GSH status is significant in acute and chronic diseases. Despite ease of delivery, little controlled clinical research data exist evaluating the effects of oral GSH supplementation. The study objectives were to determine the effect of oral GSH supplementation on biomarkers of systemic oxidative stress in human volunteers. This was a randomized, double-blind, placebo-controlled clinical trial. The study was conducted at Bastyr University Research Institute, Kenmore, WA and the Bastyr Center for Natural Health, Seattle, WA. Forty (40) adult volunteers without acute or chronic disease participated in this study. Intervention: Oral GSH supplementation (500 mg twice daily) was given to the volunteers for 4 weeks. Primary outcome measures included change in creatinine-standardized, urinary F2-isoprostanes (F2-isoP) and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG). Changes in erythrocyte GSH concentrations, including total reduced glutathione (GSH), oxidized glutathione (GSSG), and their ratio (GSH:GSSG) were also measured by tandem liquid chromatography/mass spectrometry. Analysis of variance was used to evaluate differences between groups. There were no differences in oxidative stress biomarkers between treatment groups at baseline. Thirty-nine (39) participants completed the study per protocol. Changes in creatinine standardized F2-isoP (ng/mg creatinine) (0.0±0.1 versus 0.0±0.1, p=0.38) and 8-OHdG (μg/g creatinine) (-0.2±3.3 versus 1.0±3.2, p=0.27) were nonsignificant between groups at week 4. Total reduced, oxidized, and ratio measures of GSH status were also unchanged. No significant changes were observed in biomarkers of oxidative stress, including glutathione status, in this clinical trial of oral glutathione supplementation in healthy adults.
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Aging is associated with oxidative stress, but underlying mechanisms remain poorly understood. We tested whether glutathione deficiency occurs because of diminished synthesis and contributes to oxidative stress in aging and whether stimulating glutathione synthesis with its precursors cysteine and glycine could alleviate oxidative stress. Eight elderly and 8 younger subjects received stable-isotope infusions of [2H(2)]glycine, after which red blood cell (RBC) glutathione synthesis and concentrations, plasma oxidative stress, and markers of oxidant damage (eg, F(2)-isoprostanes) were measured. Elderly subjects were restudied after 2 wk of glutathione precursor supplementation. Compared with younger control subjects, elderly subjects had markedly lower RBC concentrations of glycine (486.7 ± 28.3 compared with 218.0 ± 23.7 μmol/L; P < 0.01), cysteine (26.2 ± 1.4 compared with 19.8 ± 1.3 μmol/L; P < 0.05), and glutathione (2.08 ± 0.12 compared with 1.12 ± 0.18 mmol/L RBCs; P < 0.05); lower glutathione fractional (83.14 ± 6.43% compared with 45.80 ± 5.69%/d; P < 0.01) and absolute (1.73 ± 0.16 compared with 0.55 ± 0.12 mmol/L RBCs per day; P < 0.01) synthesis rates; and higher plasma oxidative stress (304 ± 16 compared with 346 ± 20 Carratelli units; P < 0.05) and plasma F(2)-isoprostanes (97.7 ± 8.3 compared with 136.3 ± 11.3 pg/mL; P < 0.05). Precursor supplementation in elderly subjects led to a 94.6% higher glutathione concentration, a 78.8% higher fractional synthesis rate, a 230.9% higher absolute synthesis rate, and significantly lower plasma oxidative stress and F(2)-isoprostanes. No differences in these measures were observed between younger subjects and supplemented elderly subjects. Glutathione deficiency in elderly humans occurs because of a marked reduction in synthesis. Dietary supplementation with the glutathione precursors cysteine and glycine fully restores glutathione synthesis and concentrations and lowers levels of oxidative stress and oxidant damages. These findings suggest a practical and effective approach to decreasing oxidative stress in aging.
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Modulation of monocyte function is a critical factor in the resolution of inflammatory responses. This role is mediated mainly by the production of TNF-α. Investigations of the actions of TNF have mostly focused on acute activation of other cell types such as fibroblasts and endothelial cells. Less is known about the effects of TNF on monocytes themselves, and little is known about the regulation of cell responses to TNF beyond the activation of NF-κB. In this study, we investigated the regulation of NF-E2-related factor 2 (Nrf2) cyctoprotective responses to TNF in human monocytes. We found that in monocytes TNF induces sustained Nrf2 activation and Nrf2 cytoprotective gene induction in a TNFR1-dependent manner. Under TNF activation, monocytes increased their expression of Nrf2-dependent genes, including NAD(P)H:quinone oxidoreductase 1 and glutamyl cysteine ligase modulatory, but not heme oxygenase-1. We also showed that autocrine TNF secretion was responsible for this sustained Nrf2 response and that Nrf2 activation by TNF was mediated by the generation of reactive oxygen species. Moreover, we showed that Nrf2-mediated gene induction can modulate TNF-induced NF-κB activation. These results show for the first time, to our knowledge, that TNF modulates prolonged Nrf2-induced gene expression, which in turn regulates TNF-induced inflammatory responses.
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Lymphocytes are an important immunological cell and have been played a significant role in acquired immune system; hence, may play in pivotal role in immunosenescence. Oxidative stress has been reported to increase in elderly subjects, possibly arising from an uncontrolled production of free radicals with aging and decreased antioxidant defenses. This study was aimed to evaluate the level of lipid-protein damage, and antioxidant status in lymphocytes of healthy individuals to correlate between oxidative damage with the aging process. Twenty healthy individuals of each age group (11 - 20; 21 - 30; 31 - 40; 41 - 50; and 51 - 60 years) were selected randomly. Blood samples were drawn by medical practitioner and lymphocytes were isolated from blood samples. Malondialdehyde (MDA), protein carbonyls (PC) level were evaluated to determine the lipid, and protein damage in lymphocytes. Superoxide dismutase (SOD), catalase (CAT), glutathione and glutathione dependent enzymes were estimated to evaluate the antioxidant status in the lymphocytes. Increased MDA and PC levels strongly support the increased oxidative damage in elderly subject than young subjects. The results indicated that, balance of oxidant and antioxidant systems in lymphocytes shifts in favor of accelerated oxidative damage during aging. Thus oxidative stress in lymphocytes may particular interest in aging, and may play important role in immunosenescence.
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Rushworth, S. A., S. Shah, and D. J. MacEwan. 2011. TNF mediates the sustained activation of Nrf2 in human monocytes. J. Immunol. 187: [702–707][1]. In [Fig. 4 D ][2], the incorrect immunoblot for the loading control was mistakenly used. The correct immunoblot has now been used in the updated [
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Glutamate-cysteine ligase (EC 6.3.2.2, GLCL), formerly called γ-glutamylcysteine synthetase (GCS), is the rate-limiting enzyme in the de novo synthesis of the antioxidant tripeptide glutathione. GLCL consists of a heavy subunit, which possesses catalytic activity and is the site of glutathione feedback inhibition, and a light subunit, which has a regulatory function. Glutathione is ubiquitous in mammalian tissues and performs a variety of functions, including protection from reactive oxygen species through antioxidant properties; detoxification of xenobiotics, organic peroxides, and heavy metals; and maintenance of sulfhydryl groups of other molecules. Increased intracellular levels of glutathione have also been found in tumor cells resistant to chemotherapeutic agents. Increased expression of GLCL in melphalan-resistant myeloma and prostate carcinoma cells and cisplatinum-resistant ovarian carcinoma cells suggests that this enzyme may be involved in glutathione-associated drug resistance. Moreover, GLCL has been shown to be induced by phenolic antioxidants and heavy metals. Recently, Mulcahy and Gipp have shown that the GLCL catalytic subunit gene (GLCLC) contains a putative antioxidant regulatory element, which may explain the responsiveness of this gene to agents that induce oxidative stress. To further our understanding of GLCL, which is linked to such a wide variety of metabolic and physiological functions through its role in glutathione synthesis, we have mapped both the catalytic and regulatory subunit genes (GLCLC and GLCLR) to human and mouse chromosomes by fluorescence in situ hybridization (FISH). 16 refs., 1 fig.
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Erythrocytes endure constant exposure to oxidative stress. The major oxidative stress scavenger in erythrocytes is glutathione. The rate-limiting enzyme for glutathione synthesis is glutamate-cysteine ligase, which consists of a catalytic subunit (GCLC) and a modifier subunit (GCLM). Here, we examined erythrocyte survival in GCLM-deficient (gclm -/-) mice. Erythrocytes from gclm -/- mice showed greatly reduced intracellular glutathione. Prolonged incubation resulted in complete lysis of gclm -/- erythrocytes, which could be reversed by exogenous delivery of the antioxidant Trolox. To test the importance of GCLM in vivo, mice were treated with phenylhydrazine (PHZ; 0.07 mg/g b.w.) to induce oxidative stress. Gclm -/- mice showed dramatically increased hemolysis compared with gclm +/+ controls. In addition, PHZ-treated gclm -/- mice displayed markedly larger accumulations of injured erythrocytes in the spleen than gclm +/+ mice within 24 h of treatment. Iron staining indicated precipitations of the erythrocyte-derived pigment hemosiderin in kidney tubules of gclm -/- mice and none in gclm +/+ controls. In fact, 24 h after treatment, kidney function began to diminish in gclm -/- mice as evident from increased serum creatinine and urea. Consequently, while all PHZ-treated gclm +/+ mice survived, 90% of PHZ-treated gclm -/- mice died within 5 days of treatment. In vitro, upon incubation in the absence or presence of additional oxidative stress, gclm -/- erythrocytes exposed significantly more phosphatidylserine, a cell death marker, than gclm +/+ erythrocytes, an effect at least partially due to increased cytosolic Ca 2+ concentration. Under resting conditions, gclm -/- mice exhibited reticulocytosis, indicating that the enhanced erythrocyte death was offset by accelerated erythrocyte generation. GCLM is thus indispensable for erythrocyte survival, in vitro and in vivo, during oxidative stress.
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Alzheimer's disease (AD) is characterized by progressive loss of memory and cognition. Our laboratory and many others have shown that the AD brain is under extensive oxidative stress. Numerous therapeutic approaches to AD therapy have been hypothesized. Among these are exogenous antioxidants. We suggest that the body's own endogenous antioxidant systems should be mobilized against the oxidative stress inherent in AD brain. One of the most versatile antioxidants in the brain is glutathione. Glutathione is capable of protecting the cell against reactive oxygen species, redox metal ions, and reactive lipid peroxidation products and other electrophiles associated with AD. There are many ways to increase glutathione levels, and one or more of these ways to increase glutathione in the brain may be a promising therapeutic strategy for AD. Drug Dev. Res. 56:428–437, 2002. © 2002 Wiley-Liss, Inc.
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In our prior studies (Orr et al., 2005) we achieved a 30-50% increase in the life span of Drosophila by manipulating glutathione (GSH) production in neuronal tissues, through over-expression of glutamate-cysteine ligase (GCL), a key enzyme in glutathione biosynthesis. In the present study, we identified gene response patterns from which plausible mechanisms responsible for the observed effects on life span might be inferred. Functional clustering analysis of the transcriptome data revealed that biological processes affected by GCLc in young flies (10 days) were generally related to cell morphogenesis and differentiation, while those in older flies were associated with nucleosome organization and detoxification processes. Notably, in older flies there was considerable reduction in the expression of genes related to humoral immunity in the GCLc over-expressors and this was observed in flies of the same chronological age (∼40 days old flies) and in flies of equivalent physiological age (10% dead for both experimentals and controls). Our study demonstrates that most of the GSH-mediated processes and targets are relatively distinct in young and old flies. Nevertheless there exists a restricted number of related processes affected by GCLc in both young and old flies and prominent among them are those associated with proteolysis and metabolism.
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Individually, γ-glutamylcysteine (GGC), a dipeptide and precursor of glutathione (GSH), and conjugated linoleic acid (CLA), a trans-fatty acid, exhibit antioxidant properties. The objective of this study was to compare effects of co-administration of GGC and CLA to treatment with GGC alone on oxidative stress and GSH synthesis in human endothelial cells. Changes in levels of 8-epi-PGF2α, thiobarbituric acid reactive substances (TBARS), GSH, total antioxidants, GSH synthetase (GSS) expression, and transcription factor DNA binding were assessed in human umbilical vein endothelial cells (HUVEC) treated with GGC alone (100 μmol/L) or combined with CLA isomer mixture (10, 50, 100 μmol/L) for 24h. Significantly higher levels of TBARS, 8-epi-PGF2α, GSH, and GSS protein were found in cells treated with GGC and 10 μmol/L CLA, compared to cells treated with GGC alone, indicative of prooxidant effects of CLA. Approximately 40% cell death was microscopically observed in cells incubated with GGC and 100 μmol/L CLA. Despite lower levels of GSH, treatment with GGC and 50 μmol/L CLA appeared to be protective from oxidative stress similar to treatment with GGC alone, as indicated by lower levels of TBARS, compared to control cells not treated with GGC and CLA.