No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Effects of S-acetylglutathione in cell and animal model of herpes simplex virus type 1 infection
Abstract
Intracellular glutathione (GSH) plays an important regulatory role in the host response to viral infections. Replenishment of intracellular GSH is a desirable yet challenging goal, since systemic GSH supplementation is rather inefficient due to a short half-life of GSH in blood plasma. Further, GSH is not taken up by cells directly, but needs to be broken down into amino acids and resynthesized to GSH intracellularly, this process often being impaired during viral infections. These obstacles may be overcome by a novel glutathione derivative S-acetylglutathione (S-GSH), which is more stable in plasma and taken up directly by cells with subsequent conversion to GSH. In the present study, in vitro effects of supplementation with S-GSH or GSH on intracellular GSH levels, cell survival and replication of human herpes simplex virus type 1 (HSV-1) were studied in human foreskin fibroblasts. In addition, in vivo effects of supplementation with S-GSH or GSH on HSV-1-induced mortality were studied in hr/hr mice. In cell culture, viral infection resulted in a significant decrease of intracellular GSH levels. S-GSH efficiently and dose-dependently (5 and 10 mM tested) restored intracellular GSH, and this replenishment was more efficient than with GSH supplementation. In mice, S-GSH, but not GSH, significantly decreased HSV-1-induced mortality ( P<0.05). The data suggest that S-GSH is a suitable antiviral agent against HSV-1 both in vitro and in vivo, indicating that this drug may be of benefit in the adjunctive therapy of HSV-1 infections.
... Current strategies attempting to improve GSH bioavailability are based on the prodrug approach, using GSH prodrugs or derivatives, like N-acetylcysteine (NAC), a well absorbed precursor of cysteine, the main limiting factor in GSH synthesis [2,[7][8]. Currently, NAC administration has been the only therapeutic strategy against oxidative stress-associated disorder in HIV infection, even if the efficacy of NAC to increase intracellular GSH levels is correlated to the ability to synthesize GSH intracellularly by its precursors, a process often impaired by aging process, viral infections and liver diseases [8][9][10]. A promising approach is represented by GSH esters prodrugs, that just after enter the cell are hydrolyzed to GSH thus by-passing the de novo synthesis of GSH [8,11]. ...
... Moreover, cysteinyl acetylation prevents the oxidation of the thiol group before its absorption. After absorption, SAG is hydrolyzed by cytoplasmic thioesterases [8][9], so releasing a GSH pool available for the cells. The addition of SAG to cultures of fibroblasts originating from individuals suffering from a genetic glutathione synthetase deficiency has proved able to replenish the intracellular level of GSH effectively [12]. ...
... The addition of SAG to cultures of fibroblasts originating from individuals suffering from a genetic glutathione synthetase deficiency has proved able to replenish the intracellular level of GSH effectively [12]. SAG has proven to be more stable in plasma and more effective than GSH in replenishing the cell levels of GSH depleted by viral infections [7,9]. Moreover, SAG exhibits an interesting non-GSH-dependent activity that induces apoptosis in some human tumor cell lines in vitro [13]. ...
... The relationship between HSV-1 and ROS has been recognized since 1995 when it was observed that GSH levels were decreased in cells immediately after infection and that exogenous GSH treatment was deleterious for viral growth (Palamara, Perno et al. 1995). These studies were confirmed in rabbits (Nucci, Palamara et al. 2000) and mice (Vogel, Cinatl et al. 2005) using derivatives of GSH, suggesting that HSV requires a pro-oxidant state in cell culture and in live animal models. This is consistent with the accumulation of ROS in HSV infected cells, as detected with redox-sensitive fluorescent dyes (Kavouras, Prandovszky et al. 2007, Aubert, Chen et al. 2008, Schachtele, Hu et al. 2010, Hu, Sheng et al. 2011, Gonzalez-Dosal, Horan et al. 2012. ...
... Our finding that in the absence of UL32, capsid proteins UL6, UL25 and VP19C exhibited disulfide bonds not seen in wild-type infection is of interest. We and others have found that the cellular environment becomes oxidized during HSV-1 infection (Palamara, Perno et al. 1995, Nucci, Palamara et al. 2000, Vogel, Cinatl et al. 2005, Mathew, Bryant et al. 2010, Gonzalez-Dosal, Horan et al. 2011) (Albright manuscript in preparation), and an oxidized environment may promote disulfide bond formation. Although some disulfide bonds may be beneficial for the virus, it is also possible that an oxidizing environment could lead to aberrant disulfide bond formation between exposed reactive thiols that would be deleterious to the virus. ...
... Many viruses including HSV have been shown to induce oxidative stress, and antioxidants have shown promise in the treatment of viral infection (Palamara, Perno et al. 1995, Garland and Fawzi 1999, Beck, Handy et al. 2000, Nucci, Palamara et al. 2000, Cai, Chen et al. 2003, Abdalla, Ahmad et al. 2005, Vogel, Cinatl et al. 2005, Tian, Jiang et al. 2010). Viral proteins that play roles in sensing and responding to changes in redox status may thus provide targets for therapeutic intervention. ...
This thesis describes three separate but related projects exploring how herpes simplex virus 1 (HSV-1) induces and copes with a pro-oxidant environment during infection. We found that the UL6 portal ring, the conduit responsible for the uptake and release of viral DNA, required disulfide bonds for ring formation and stability. Mutagenesis studies revealed that these disulfide bonds might be mediated by cysteines 166 and 254. We next examined the protein UL32 as a potential virally encoded oxidoreductase. UL32 contains three C-X-X-C motifs reminiscent of proteins that regulate disulfide bond formation. Mutation of two of these C-X-X-C motifs abrogated protein function. We used a thiol-alkylation assay to show that viral capsid proteins, including UL6, had altered disulfide bond profiles in the absence of UL32, suggesting that UL32 influences disulfide bond formation in capsid proteins during assembly and maturation. We then confirmed that HSV induces oxidation using a redox-sensitive GFP probe. We also found that treating HSV-infected cells with the antioxidant glutathione (GSH) inhibited viral growth. This growth defect was likely due to delayed viral protein expression and replication compartment formation. We further investigated the role of ROS generating NADPH-oxidases (NOXs), which are important for the growth of several viruses. Using a pan-NOX inhibitor, we found similar defects in viral growth, protein expression and replication compartment formation as observed with GSH treatment, suggesting that NOX-derived ROS might be important for HSV infection. Collectively, we propose that HSV has evolved to use and manipulate the intracellular redox-balance for its own benefit.
... Somewhat surprisingly in this study, the UCP2 KO mice, be they on a resistant (C57BL/6) or susceptible (129SV) background, did not differ from the WT counterparts in terms of both survival and inflammatory response to HSV-1. This was surprising since we ( Figure 1) and others [24,36] have observed that alterations in oxidative stress occur in response to HSV-1 infection. ...
... This could not be explained by cytokine TNFα mRNA induction in the brain of infected mice. However, a previous study has shown that increases in brain TNFα can be detected after intranasal infection of infiltrating immune cells found in blood vessels and cells lining the brain but not inside the brain during the early stages of infection (days 1-3) [36]. Another possible explanation for the decrease in GSH is that responsible factors do not originate in the brain. ...
... This could reduce GSH pools and therefore increase oxidative stress. GSH has been suggested to play an important role in HSV infection [24,36]. It must be remembered that GSH requires appropriate levels of NADPH and ATP for its synthesis. ...
The present results demonstrate resistance to HSV-1 in C57Bl6 mice is associated with differential temporal body weight and GSH changes, compared to susceptible 129Sv mouse strain. Strong brain viral load TK and TLR-2 induction in the brain precedes TNF-α, iNOS, IκBα and UCP2 expression in 129Sv mice. Interestingly, we observe that UCP2 brain expression differs: UCP2 is found in higher quantities in the mid and hind brain in susceptible mice and in the forebrain in resistant mice. In contrast with previous data, UCP2 KO mice did not show differences in terms of survival, inflammatory gene expression and neurodegeneration compared to their WT controls. UCP2 brain expression is therefore a marker of resistance/susceptibility to infection but does not play a role in viral load or survival. In conclusion, UCP2s role in host survival to infection may be pathogen specific and largely subordinate to the direct effect of the toxicity of changes in antioxidants on the infectious agent itself.
... An increase in intracellular GSH by I-152 has been described in human monocyte-derived macrophages (MDMs) (Oiry et al., 2004); moreover, the same cells infected with macrophage-tropic HIV- 1/BaL were used to explore anti-HIV effects of I-152 which significantly decreased viral replication, with a 50% effective dose (ED 50 ) equal to 3–50 M regarding the multiplicity of infection (Oiry et al., 2004). The other molecule studied, which is an acetylated GSH derivative (S-acetylglutathione, SAG), was shown to restore GSH levels that had been decreased following HSV-1 infection and to delay HSV-1-induced mortality in hr/hr mice (Vogel et al., 2005). Herein, the antiviral effects exerted by these two molecules (I-152 and SAG) are discussed in comparison with GSH which was previously demonstrated to be effective in reducing the typical signs characterizing murine AIDS (Palamara et al., 1996). ...
... 1. Based on the data already available regarding efficacy and LD 50 (lethal dose 50) in mice of the two molecules (Oiry et al., 2001Oiry et al., , 2004 Vogel et al., 2005) and the effects of GSHFig. 1. Chemical structure of reduced glutathione (GSH) (a) and pro-GSH molecules: I-152 (b); S-acetylglutathione (SAG) (c). ...
... It has been demonstrated that therapeutic intervention aimed at normalization of oxidative disturbances in HIV infection might be of interest in addition to HAART, the standard protocol followed to treat HIV-infected patients (Nakamura et al., 2002; Aukrust et al., 2003; Spada et al., 2002; De Rosa et al., 2000). Indeed, GSH and some pro-GSH molecules are able to interfere with different steps in the biological cycle of the virus (Stevens et al., 2006; Oiry et al., 2004; Vogel et al., 2005; Palamara et al., 2004). Effective GSH replenishment has been described in HIV infection by oral administration of NAC, an effective precursor of cysteine for tissue GSH synthesis. ...
Immobilization of proteins on intact red blood cells (RBCs) can be valuable for several different reasons. In vivo, RBCs carrying
specific ligands can be targeted to cells exposing specific antigens, eventually for the delivery of drugs encapsulated in
the same RBC (1). Alternatively, RBCs carrying antigens or enzymes on the extracellular surface of their membrane can perform as antigen
delivery systems (2) or as bioreactors for the degradation of toxic metabolites (3). In vitro, RBCs carrying proteins can be used as diagnostic reagents in agglutination or hemolytic assays (4).
... To overcome the problems linked with the use of GSH as a therapeutic agent, many researchers have proposed the use of novel molecules able to exert antiviral effects comparable to or higher than those obtained with GSH. These pro-GSH mol- (Buhl et al., 1989) FIV (Mortola et al., 1998) LP-BM5 (Chen et al., 1996) Influenza and parainfluenza (Nencioni et al., 2003;Garaci et al., 1992) Rhinovirus (Papi and Johnston, 1999;Papi et al., 2002) Hepatitis (Okuda et al., 2002) Herpes simplex virus-1 (Nucci et al., 2000;Vogel et al., 2005) ecules can be either GSH carrying a hydrophobic group to make cellular entry easier, or a source of thiol groups from which GSH is synthesized intracellularly (Fig. 2). ...
... In vitro and in vivo experimental evidence suggests that viral infection causes a significant decrease in GSH and that the impairment of intracellular redox status is necessary for virus replication (Palamara et al., 1995;Nucci et al., 2000;Vogel et al., 2005). In vitro experiments performed on different cellular models, showed that GSH supplementation produces a concentration-dependent inhibition of HSV replication (Vogel et al., 2005;Palamara et al., 1995). ...
... In vitro and in vivo experimental evidence suggests that viral infection causes a significant decrease in GSH and that the impairment of intracellular redox status is necessary for virus replication (Palamara et al., 1995;Nucci et al., 2000;Vogel et al., 2005). In vitro experiments performed on different cellular models, showed that GSH supplementation produces a concentration-dependent inhibition of HSV replication (Vogel et al., 2005;Palamara et al., 1995). More interestingly, in the same models, efficacy of GSH analogs was compared with that of GSH. ...
Reduced glutathione (GSH) is the most prevalent non-protein thiol in animal cells. Its de novo and salvage synthesis serves to maintain a reduced cellular environment. GSH is the most powerful intracellular antioxidant and plays a role in the detoxification of a variety of electrophilic compounds and peroxides via catalysis by glutathione-S-transferases (GST) and glutathione peroxidases (GPx). As a consequence, the ratio of reduced and oxidized glutathione (GSH:GSSG) serves as a representative marker of the antioxidative capacity of the cell. A deficiency in GSH puts the cell at risk for oxidative damage. An imbalance in GSH is observed in a wide range of pathologies, such as cancer, neurodegenerative diseases, cystic fibrosis (CF), several viral infections including HIV-1, as well as in aging. Several reports have provided evidence for the use of GSH and molecules able to replenish intracellular GSH levels in antiviral therapy. This non-conventional role of GSH and its analogs as antiviral drugs is discussed in this chapter.
... An increase in intracellular GSH by I-152 has been described in human monocyte-derived macrophages (MDMs) (Oiry et al., 2004); moreover, the same cells infected with macrophage-tropic HIV-1/BaL were used to explore anti-HIV effects of I-152 which significantly decreased viral replication, with a 50% effective dose (ED 50 ) equal to 3-50 M regarding the multiplicity of infection (Oiry et al., 2004). The other molecule studied, which is an acetylated GSH derivative (S-acetylglutathione, SAG), was shown to restore GSH levels that had been decreased following HSV-1 infection and to delay HSV-1-induced mortality in hr/hr mice (Vogel et al., 2005). Herein, the antiviral effects exerted by these two molecules (I-152 and SAG) are discussed in comparison with GSH which was previously demonstrated to be effective in reducing the typical signs characterizing murine AIDS (Palamara et al., 1996). ...
... Based on the data already available regarding efficacy and LD 50 (lethal dose 50) in mice of the two molecules (Oiry et al., 2001(Oiry et al., , 2004Vogel et al., 2005) and the effects of GSH (at a concentration of 325 mol/mouse (4.5 g/kg)) on murine AIDS (Palamara et al., 1996), the doses reported in Section 2 were selected for testing. In detail, the intraperitoneal LD 50 in a chronic treatment with I-152 (30 days) had been reported equal to 700 mg/kg (Oiry et al., 2001). ...
... It has been demonstrated that therapeutic intervention aimed at normalization of oxidative disturbances in HIV infection might be of interest in addition to HAART, the standard protocol followed to treat HIV-infected patients (Nakamura et al., 2002;Aukrust et al., 2003;Spada et al., 2002;De Rosa et al., 2000). Indeed, GSH and some pro-GSH molecules are able to interfere with different steps in the biological cycle of the virus (Stevens et al., 2006;Oiry et al., 2004;Vogel et al., 2005;Palamara et al., 2004). Effective GSH replenishment has been described in HIV infection by oral administration of NAC, an effective precursor of cysteine for tissue GSH synthesis. ...
Antioxidant molecules can be used both to replenish the depletion of reduced glutathione (GSH) occurring during HIV infection, and to inhibit HIV replication. The purpose of this work was to assess the efficacy of two pro-GSH molecules able to cross the cell membrane more easily than GSH. We used an experimental animal model consisting of C57BL/6 mice infected with the LP-BM5 viral complex; the treatments were based on the intramuscular administration of I-152, a pro-drug of N-acetylcysteine and S-acetyl-beta-mercaptoethylamine, and S-acetylglutathione, an acetylated GSH derivative. The results show that I-152, at a concentration of 10.7 times lower than GSH, caused a reduction in lymph node and spleen weights of about 55% when compared to infected animals and an inhibition of about 66% in spleen and lymph node virus content. S-acetylglutathione, at half the concentration of GSH, caused a reduction in lymph node weight of about 17% and in spleen and lymph node virus content of about 70% and 30%, respectively. These results show that the administration of pro-GSH molecules may favorably substitute for the use of GSH as such.
... Moreover, proteins modulating the glutamate cysteine-ligase (GCL) activity were enriched in the set downregulated proteins (Figures 4B,C). GCL regulates glutathione levels, which are critical for protective, anti-viral responses in microglia (47,48). ...
... Glutathione is critical for HSV-1 production. In fact, HSV-1 needs to lower cellular glutathione levels for an efficient viral replication and GCL supplementation protects mice from HSV-1 infections (48,60,61). In primary cortical cultures, the virus was mainly detected in neurons, astrocytes and oligodendrocytes. ...
Infections of the brain with herpes simplex virus type 1 (HSV-1) cause life-threatening Herpes simplex encephalitis (HSE) characterized by viral replication in neurons and neuro-inflammation including an infiltration of peripheral immune cells. HSV-1 reprograms host cells to foster its own replication and for immune evasion, but eventually the immune responses clear the infection in most patients. However, many survivors suffer from long-term neuronal damage and cannot regenerate all brain functions. HSV-1 influences the physiology of neurons, astrocytes, oligodendrocytes and microglia, and significantly changes their protein expression and secretion pattern. To characterize temporal changes upon HSV-1 infection in detail, we inoculated mixed primary cultures of the murine brain cortex, and performed quantitative mass spectrometry analyses of the cell-associated proteome and the secretome. We identified 28 differentially regulated host proteins influencing inflammasome formation and intracellular vesicle trafficking during endocytosis and secretion. The NIMA-related kinase 7 (NEK7), a critical component of the inflammasome, and ArfGap1, a regulator of endocytosis, were significantly up-regulated upon HSV-1 infection. In the secretome, we identified 71 proteins including guidance cues regulating axonal regeneration, such as semaphorin6D, which were enriched in the conditioned media of HSV-1 infected cells. Modulation of inflammasome activity and intracellular membrane traffic are critical for HSV-1 cell entry, virus assembly, and intracellular spread. Our proteome analysis provides first clues on host factors that might dampen the inflammasome response and modulate intracellular vesicle transport to promote HSV infection of the brain. Furthermore, our secretome analysis revealed a set of proteins involved in neuroregeneration that might foster neuronal repair processes to restore brain functions after clearance of an HSV-1 infection.
... Our second MA (ANTIOX-MA) supports our hypothesis that administration of different antioxidants, especially piperitenone oxide, vaticaffinol, ebselen and resveratrol, may reduce infection. This suggests that herpes viruses are favoured by oxidative conditions, and that a disruption of these conditions can reduce herpes infection, as suggested by previous studies (Docherty et al. 2005;Kavouras et al. 2007;Palamara et al. 1995;Vogel et al. 2005). In this line of thoughts, it would be interesting to examine whether individuals with higher level of pre-existing oxidative damage are more susceptible to herpes virus than individuals with lower levels of oxidative damage. ...
... Regarding the in vivo experiments, we could not compare results from different studies because there was high variation in the experimental conditions (administration of antioxidants or dermal application of cream-based antioxidants; days of administration and number of administrations per day; results showed as number of survivals or percentage of virus reduction). Despite this strong heterogeneity between experiments, these studies suggest that administration of antioxidants may also reduce the virus yield in the whole organism (Docherty et al. 1999;Nucci et al. 2000;Vogel et al. 2005), indicating that availability of antioxidants may constrain individual's capability of controlling viral infection. ...
Herpes viruses are responsible for a variety of pathological effects in humans and in both wild and domestic animals. One mechanism that has been proposed to facilitate replication and activity of herpes viruses is oxidative stress (OS). We used meta-analytical techniques to test the hypotheses that (1) herpes virus infection causes OS and (2) supplementation of antioxidants reduces virus load, indicating that replication is favoured by a state of OS. Results based on studies on mammals, including humans, and birds show that (1) OS is indeed increased by herpes virus infection across multiple tissues and species, (2) biomarkers of OS may change differently between tissues, and (3) the effect size does not differ among different virus strains. In addition, the increase of oxidative damage in blood (tissue commonly available in ecological studies) was similar to that in the tissues most sensitive to the herpes virus. Our results also show that administration of antioxidants reduces virus yield, indicating that a condition of OS is favorable for the viral replication. In addition, some antioxidants may be more efficient than others in reducing herpes virus yield. Our results point to a potential mechanism linking herpes virus infection to individual health status.
... Поскольку введение GSH способствует снижению активности вирусной репликации, считают, что нарушение функционирования глутатионовой системы может быть одной из причин тяжелого течения ОРВИ. Высокий уровень GSH ингибирует репликацию как вируса гриппа, так и вируса простого герпеса [45]. Известно, что риновирусная инфекция сопровождается усилением экспрессии ICAM-1, которая является рецептором для 90 % различных серологических типов риновирусов. ...
В огляді літератури викладені сучасні дані щодо специфічних особливостей оксидативного стресу та функціональної активності антиоксидантної системи при гострих респіраторних вірусних інфекціях і хронічних запальних захворюваннях органів дихання. Показана взаємопов’язаність оксидативного стресу та запалення. Поданий шлях розвитку фіброзу легеневої тканини, асоційований з активованими кисневмісними метаболітами.
... described the synthesis of a new class of glutathione derivatives in which the cysteine mojety of the tripeptide molecule is linked with long chain, polyunsaturated fatty acids (PUFA) via a thioester bond 21 , thus obtaining a series of GSH's thioesthers (S-Acyl-GSH). The presence of a fatty acid confers lipophilic character to the adduct, thus favouring the passage through phospholipidic membrane bilayer.Once inside cytoplasm, the cytoplasmic thioesterases catalyse the hydrolysis of thioester bond giving rise to both GSH and fatty acid by thioesterase release22,23 . Wright et al demonstrated that acyl-GSHs are able to pass easily through plasma membranes of many distinct cell lines, and that an ...
Background:
Reactive oxygen species have a major role in the UV-induced short and long term damage, thus the exogenous supplementation of antioxidant molecules may allow better skin protection. Despite glutathione has pivotal properties in the complex cytoplasmic antioxidant system, its supplementation is hampered by limited transmembrane absorption. Modification of glutathione pharmacokinetic properties via acetylation with long chain polyunsaturated fatty acid may improve its passage through phospholipidic membranes.
Methods:
This was a single center randomized double-blinded clinical trial, versus placebo, cross sectional pairwise at time 0. The partecipants were 10 healthy volunteers with skin phototypes II or III and age ≥18 years interested in performing MED evaluation performed by photoallergology unit of Dermatology division, P. Palagi hospital, Florence. Each volunteer underwent UVB phototesting, treating 4 distinct areas with increasing UVB doses in 4 distinct conditions. One arm was treated as standard procedure ("Naïve" arm), one applying linolenic-glutathione conjugate (Lin-GSH) cream 2% before irradiation ("Pre Lin-GSH" arm), one with placebo ("placebo" arm) and one applying Lin-GSH cream 2% after irradiation ("post Lin-GSH" arm). The aim was to evaluate efficacy of Lin-GSH cream 2% before UVB irradiation compared with placebo. Secondary objective is the evaluation of the same cream applied after irradiation compared to no treatment. Other Secondary objective is the evaluation of safety in both conditions. Mean MED was evaluated at distinct conditions. Safety was evaluated reporting all grade 3-4 adverse events up to 30 days after treatment. All volunteers were treated in all 4 experimental conditions. "Pre Lin-GSH" and "Placebo" arms were applied in a double blind condition in each volunteer. Neither the volunteer nor the investigator executing MED evaluation knew which arm contained Lin-GSH and which one contained placebo.
Results:
Ten volunteers among 12 recruited for the study were correctly randomized and completed all study evaluations. Recruitment went from 01 April 2016 to 01 May 2016, up to adequate population number. Mean MED in "Pre Lin-GSH" arm was superior to mean MED in "placebo" arm (135 mJ/cm2 ± 5.53 vs 105 mJ/cm2 ± 7.64, p=0.0003. No difference was observed among mean MED in "Post Lin-GSH" and "Naïve" arms. No grade 3-4 Adverse Events were reported.
Conclusions:
Lin-GSH 2% cream seems a safe and effective molecule able to exert significant increase in MED compared with placebo thanks to its antioxidant properties.
... ¹ 5 (73) • 2016 Çäîðîâüå ðåáåíêà, p-ISSN 2224-0551, e-ISSN 2307-1168 Íà äîïîìîãó ïåä³àòðó / To Help the Pediatrician рует репликацию как вируса гриппа, так и вируса простого герпеса [23,44]. Было показано, что риновирусная инфекция приводит к снижению уровня внутриклеточной концентрации восстановленного глутатиона, в то время как повышение уровня восстановленного глутатиона сопровождается ингибированием экспрессии молекулы ICAM-1, которая является рецептором для 90 % различных серологических типов риновирусов, и снижением активности риновирусной инфекции [33,34]. ...
The comprehensive analytic review reflects the characteristics of the virus-associated oxidative stress, and shows the need for antioxidant drug support for sanogenesis at acute respiratory viral infections. There are described the data on the antiviral activity and antioxidant protective effect of bioflavonoids of wild grasses Calamagrostis epigeios L. and Deschampsia caespitosa L. as the components of medicinal product Immunоflazidum. There are demonstrated the effectiveness and safety of Immunоflazidum as a drug of choice for the treatment of acute respiratory viral infections.
... This results in higher levels of intracellular glutathione. [49] S-acetyl-glutathione is also known to have antiviral and immunomodulatory properties. [50] However, there is no human data available to prove the superiority of S-acetyl-glutathione over plain glutathione for skin-lightening effects. ...
Glutathione is a low molecular weight thiol-tripeptide that plays a prominent role in maintaining intracellular redox balance. In addition to its remarkable antioxidant properties, the discovery of its antimelanogenic properties has led to its promotion as a skin-lightening agent. It is widely used for this indication in some ethnic populations. However, there is a dichotomy between evidence to support its efficacy and safety. The hype around its depigmentary properties may be a marketing gimmick of pharma-cosmeceutical companies. This review focuses on the various aspects of glutathione: its metabolism, mechanism of action and the scientific evidence to evaluate its efficacy as a systemic skin-lightening agent. Glutathione is present intracellularly in its reduced form and plays an important role in various physiological functions. Its skin-lightening effects result from direct as well as indirect inhibition of the tyrosinase enzyme and switching from eumelanin to phaeomelanin production. It is available in oral, parenteral and topical forms. Although the use of intravenous glutathione injections is popular, there is no evidence to prove its efficacy. In fact, the adverse effects caused by intravenous glutathione have led the Food and Drug Administration of Philippines to issue a public warning condemning its use for off-label indications such as skin lightening. Currently, there are three randomized controlled trials that support the skin-lightening effect and good safety profile of topical and oral glutathione. However, key questions such as the duration of treatment, longevity of skin-lightening effect and maintenance protocols remain unanswered. More randomized, double-blind, placebo-controlled trials with larger sample size, long-term follow-up and well-defined efficacy outcomes are warranted to establish the relevance of this molecule in disorders of hyperpigmentation and skin lightening.
... The overproduction of RS levels caused by virus can disrupt the GSH system and affect the host cell pro-/antioxidant balance by inhibiting the synthesis of antioxidant enzymes, such as SOD and CAT. In vitro cell culture studies revealed that oxidative stress occur in the host cells after the infection with viruses, including HIV [Westendorp et al., 1995], hepatitis C [Abdalla et al., 2005], herpes simplex type 1 [Vogel et al., 2005], and influenza virus [Nencioni et al., 2003]. In the present study, HSV-2 infection reduced CAT, SOD and GPx activities and (PhSe) 2 attenuated this effect. ...
Diphenyl diselenide, (PhSe)2 , is an organoselenium compound with pharmacological actions mostly related to antioxidant and anti-inflammatory properties. The study investigated its antiviral and virucidal actions against herpes simplex virus 2 (HSV-2) infection in vitro and in a vaginal infection model in mice. The plaque reduction assay indicated that (PhSe)2 showed virucidal and antiviral actions reducing infectivity in 70.8 and 47%, respectively. The antiviral action of (PhSe)2 against HSV-2 vaginal infection was performed by infecting mice (10(5) PFU/ml(-1) ) at day 6. The treatment with (PhSe)2 (5 mg/kg/day, intragastric (i.g.) followed 5 days before and for more 5 days after infection. The extravaginal lesion score was evaluated from day 6 to 10. At day 11, animals were killed and histological evaluation, determination of viral load and TNF-α and IFN-γ levels were performed in supernatants of homogenized vaginal tissue. The levels of reactive species (RS); protein carbonyl, non-protein thiols (NPSH), nitrate/nitrite (NOx) and malondialdehyde (MDA) and the activities of myeloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) were determined. (PhSe)2 reduced the histological damage, extravaginal lesion scores, the viral load of vaginal tissue and the activity of MPO but increased the levels of TNF-α, IFN-γ. (PhSe)2 attenuated the increase of RS, MDA, NOx levels and the activity of GR caused by infection. (PhSe)2 also attenuated the reduction of NPSH content and the inhibition of CAT, SOD and GPx activities. The antiviral action of (PhSe)2 against HSV-2 infection was related to its immunomodulatory, antioxidant and anti-inflammatory properties. This article is protected by copyright. All rights reserved.
... It is becoming increasingly clear that oxidative stress and cellular responses to it play critical roles in modulating a large number of biological processes, including pathogenesis by bacterial and viral infections. Many viruses, including HSV, have been shown to induce oxidative stress, and antioxidants have shown promise in the treatment of viral infection (54)(55)(56)(59)(60)(61)(62)(63). Viral proteins that play roles in sensing and responding to changes in redox status may thus provide targets for therapeutic intervention. ...
Unlabelled:
During DNA encapsidation, herpes simplex virus 1 (HSV-1) procapsids are converted to DNA-containing capsids by a process involving activation of the viral protease, expulsion of the scaffold proteins, and the uptake of viral DNA. Encapsidation requires six minor capsid proteins (UL6, UL15, UL17, UL25, UL28, and UL33) and one viral protein, UL32, not found to be associated with capsids. Although functions have been assigned to each of the minor capsid proteins, the role of UL32 in encapsidation has remained a mystery. Using an HSV-1 variant containing a functional hemagglutinin-tagged UL32, we demonstrated that UL32 was synthesized with true late kinetics and that it exhibited a previously unrecognized localization pattern. At 6 to 9 h postinfection (hpi), UL32 accumulated in viral replication compartments in the nucleus of the host cell, while at 24 hpi, it was additionally found in the cytoplasm. A newly generated UL32-null mutant was used to confirm that although B capsids containing wild-type levels of capsid proteins were synthesized, these procapsids were unable to initiate the encapsidation process. Furthermore, we showed that UL32 is redox sensitive and identified two highly conserved oxidoreductase-like C-X-X-C motifs that are essential for protein function. In addition, the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, were altered in the absence of UL32, suggesting that UL32 may act to modulate disulfide bond formation during procapsid assembly and maturation.
Importance:
Although functions have been assigned to six of the seven required packaging proteins of HSV, the role of UL32 in encapsidation has remained a mystery. UL32 is a cysteine-rich viral protein that contains C-X-X-C motifs reminiscent of those in proteins that participate in the regulation of disulfide bond formation. We have previously demonstrated that disulfide bonds are required for the formation and stability of the viral capsids and are also important for the formation and stability of the UL6 portal ring. In this report, we demonstrate that the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, are altered in cells infected with a newly isolated UL32-null mutant virus, suggesting that UL32 acts as a chaperone capable of modulating disulfide bond formation. Furthermore, these results suggest that proper regulation of disulfide bonds is essential for initiating encapsidation.
... The zinc finger motif of Hsp33 is not found in eukaryotic proteins (70) but is similar to a cysteine motif in the regulatory domain of UL16 (6). Moreover, it is known that HSV infections induce and require oxidative stress for the production of infectious virions (71)(72)(73)(74)(75)(76)(77)(78). But unlike those of Hsp33, the binding activities of the N-terminal portion of UL16 seem to be specific for particular viral proteins: UL11 (6), gE (9), and VP22 (this study). ...
UL16 is a tegument protein of herpes simplex virus (HSV) that is conserved among all the Herpesviridae, but its function is poorly understood. Previous studies revealed that UL16 is associated with capsids in the cytoplasm and interacts with the UL11 membrane protein, which suggested a "bridging" function during cytoplasmic envelopment, but this conjecture has not been tested. To gain further insight, cells infected with UL16-null mutants were examined by electron microscopy. Defects in the transport of capsids to cytoplasmic membranes were not observed, but the wrapping of capsids with membranes was delayed. Moreover, clusters of cytoplasmic capsids were often observed, but only near membranes, where they were wrapped to produce multiple capsids within a single envelope. Normal virion production was restored when UL16 was expressed either by complementing cells or from a novel position in the HSV genome. In analyzing the composition of the UL16-null viruses, a reduction in the packaging of glycoprotein E (gE) was observed, which was not surprising since it has been reported that UL16 interacts with this glycoprotein. However, the levels of tegument protein VP22 were also dramatically reduced in virions, even though this gE-binding protein has been shown not to depend upon its membrane partner for packaging. Co-transfection experiments revealed that UL16 and VP22 can interact in the absence of other viral proteins. These results extend the UL16 interaction network beyond its previously-identified binding partners to include VP22 and provide evidence that it plays an important function at the membrane during virion production.
... We hypothesize that the C-terminal regulatory domain of UL16 may also contain a switch that is activated in response to oxidation. In fact, it has been shown that HSV-infected cells undergo oxidative stress (17,21,33,49,56,59,65,70). Hence, it is possible that UL16 utilizes this change to modulate its function. ...
It is well known that proteins in the tegument (located between the viral capsid and envelope proteins) play critical roles in the assembly and budding of herpesviruses. Tegument proteins UL16 and UL11 of herpes simplex virus (HSV) are conserved among all the Herpesviridae. Although these proteins directly interact in vitro, UL16 was found to colocalize poorly with UL11 in cotransfected cells. To explain this discrepancy, we hypothesized that UL16 is initially made in an inactive form and is artificially transformed to the binding-competent state when cells are disrupted. Consistent with a regulated interaction, UL16 was able to fully colocalize with UL11 when a large C-terminal segment of UL16 was removed, creating mutant UL16(1-155). Moreover, membrane flotation assays revealed a massive movement of this mutant to the top of sucrose gradients in the presence of UL11, whereas both the full-length UL16 and the C-terminal fragment (residues 156 to 373) remained at the bottom. Further evidence for the presence of a C-terminal regulatory domain was provided by single-amino-acid substitutions at conserved cysteines (C269S, C271S, and C357S), which enabled the efficient interaction of full-length UL16 with UL11. Lastly, the binding site for UL11 was further mapped to residues 81 to 155, and to our surprise, the 5 Cys residues within UL16(1-155) are not required, even though the modification of free cysteines in UL16 with N-ethylmaleimide does in fact prevent binding. Collectively, these results reveal a regulatory function within the C-terminal region of UL16 that controls an N-terminal UL11-binding activity.
... In vitro and in vivo experiments show that enhanced GSH levels also inhibit replication of both the influenza virus (Nencioni et al., 2003), and the herpes simplex virus (Vogel et al., 2005). Not only do GSH levels appear to affect the activity of invading viruses, but they also affect the activation of important cell signaling pathways associated with the host immune system response. ...
Glutathione (GSH) plays an important role in a multitude of cellular processes, including cell differentiation, proliferation, and apoptosis, and as a result, disturbances in GSH homeostasis are implicated in the etiology and/or progression of a number of human diseases, including cancer, diseases of aging, cystic fibrosis, and cardiovascular, inflammatory, immune, metabolic, and neurodegenerative diseases. Owing to the pleiotropic effects of GSH on cell functions, it has been quite difficult to define the role of GSH in the onset and/or the expression of human diseases, although significant progress is being made. GSH levels, turnover rates, and/or oxidation state can be compromised by inherited or acquired defects in the enzymes, transporters, signaling molecules, or transcription factors that are involved in its homeostasis, or from exposure to reactive chemicals or metabolic intermediates. GSH deficiency or a decrease in the GSH/glutathione disulfide ratio manifests itself largely through an increased susceptibility to oxidative stress, and the resulting damage is thought to be involved in diseases, such as cancer, Parkinson's disease, and Alzheimer's disease. In addition, imbalances in GSH levels affect immune system function, and are thought to play a role in the aging process. Just as low intracellular GSH levels decrease cellular antioxidant capacity, elevated GSH levels generally increase antioxidant capacity and resistance to oxidative stress, and this is observed in many cancer cells. The higher GSH levels in some tumor cells are also typically associated with higher levels of GSH-related enzymes and transporters. Although neither the mechanism nor the implications of these changes are well defined, the high GSH content makes cancer cells chemoresistant, which is a major factor that limits drug treatment. The present report highlights and integrates the growing connections between imbalances in GSH homeostasis and a multitude of human diseases.
Liver fibrosis, depending on the stage of the disease, could lead to organ dysfunction and cirrhosis, and no effective treatment is actually available. Emergent proof supports a link between oxidative stress, liver fibrogenesis and mitochondrial dysfunction as molecular bases of the pathology. A valid approach to protect against the disease would be to replenish the endogenous antioxidants; thus, we investigated the protective mechanisms of the S-acetyl-glutathione (SAG), a glutathione (GSH) prodrug. Preliminary in vitro analyses were conducted on primary hepatic cells. SAG pre-treatment significantly protected against cytotoxicity induced by CCl4. Additionally, CCl4 induced a marked increase in AST and ALT levels, whereas SAG significantly reduced these levels, reaching values found in the control group. For the in vivo analyses, mice were administered twice a week with eight consecutive intraperitoneal injections of 1 mL/kg CCl4 (diluted at 1:10 in olive oil) to induce oxidative imbalance and liver inflammation. SAG (30 mg/kg) was administered orally for 8 weeks. SAG significantly restored SOD activity, GSH levels and GPx activity, while it strongly reduced GSSG levels, lipid peroxidation and H2O2 and ROS levels in the liver. Additionally, CCl4 induced a decrease in anti-oxidants, including Nrf2, HO-1 and NQO-1, which were restored by treatment with SAG. The increased oxidative stress characteristic on liver disfunction causes the impairment of mitophagy and accumulation of dysfunctional and damaged mitochondria. Our results showed the protective effect of SAG administration in restoring mitophagy, as shown by the increased PINK1 and Parkin expressions in livers exposed to CCl4 intoxication. Thus, the SAG administration showed anti-inflammatory effects decreasing pro-inflammatory cytokines TNF-α, IL-6, MCP-1 and IL-1β in both serum and liver, and suppressing the TLR4/NFkB pathway. SAG attenuated reduced fibrosis, collagen deposition, hepatocellular damage and organ dysfunction. In conclusion, our results suggest that SAG administration protects the liver from CCl4 intoxication by restoring the oxidative balance, ameliorating the impairment of mitophagy and leading to reduced inflammation.
Host-directed therapy using drugs that target cellular pathways required for virus lifecycle or its clearance might represent an effective approach for treating infectious diseases. Changes in redox homeostasis, including intracellular glutathione (GSH) depletion, are one of the key events that favor virus replication and contribute to the pathogenesis of virus-induced disease. Redox homeostasis has an important role in maintaining an appropriate Th1/Th2 balance, which is necessary to mount an effective immune response against viral infection and to avoid excessive inflammatory responses. It is known that excessive production of reactive oxygen species (ROS) induced by viral infection activates nuclear factor (NF)-kB, which orchestrates the expression of viral and host genes involved in the viral replication and inflammatory response. Moreover, redox-regulated protein disulfide isomerase (PDI) chaperones have an essential role in catalyzing formation of disulfide bonds in viral proteins. This review aims at describing the role of GSH in modulating redox sensitive pathways, in particular that mediated by NF-kB, and PDI activity. The second part of the review discusses the effectiveness of GSH-boosting molecules as broad-spectrum antivirals acting in a multifaceted way that includes the modulation of immune and inflammatory responses.
Glutathione synthetase deficiency is an autosomal recessive inherited metabolic defect in the γ‐glutamyl cycle. Decreased intracellular glutathione levels are one of the characteristic biochemical features. In this study we show that addition of S‐acetylglutathione to the medium raised intracellular glutathione content in cultured fibroblasts from patients with glutathione synthetase deficiency. This has implications for the treatment of patients with this inborn error of metabolism.
Norovirus is a major cause of foodborne disease and nonbacterial gastroenteritis globally. This study evaluated the antiviral effects of Magnolia officinalis extract and its honokiol and magnolol constituents against human norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV) in vitro, and in model food systems. Pretreatment or cotreatment of M. officinalis extract at 1 mg/mL reduced MNV and FCV titers by 0.6-1.8 log. Honokiol and magnolol, which are the major polyphenols in the extract, showed significant antiviral effects against MNV and FCV. The virus-infected cells that were treated with M. officinalis extract exhibited significantly increased glutathione levels (p < 0.05). The extract, honokiol, and magnolol revealed ferric ion-reducing and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities in a dose-dependent manner. Furthermore, MNV and FCV titers were reduced by >1.6 log or to undetectable levels in apple, orange, and plum juices and by 0.9 and 1.6 log in milk, respectively, when they were treated with the extract at 5 mg/mL. Therefore, the present study suggests that M. officinalis extract can be used as an antiviral food material to control norovirus foodborne diseases.
Reduced glutathione (GSH) is the most prevalent non-protein thiol in animal cells. Its de novo and salvage synthesis serves to maintain a reduced cellular environment, which is important for several cellular functions. Altered intracellular GSH levels are observed in a wide range of pathologies, including several viral infections, as well as in ageing, all of which are also characterized by an unbalanced Th1/Th2 immune response. A central role in influencing the immune response has been ascribed to GSH. Specifically, GSH depletion in antigen-presenting cells (APCs) correlates with altered antigen processing and reduced secretion of Th1 cytokines. On the contrary, an increase in intracellular GSH content stimulates IL-12 and/or IL-27, which in turn induce differentiation of naive CD4
Introduction:
Cellular oxidoreductases catalyze thiol/disulfide exchange reactions in susceptible proteins and contribute to the cellular defense against oxidative stress. Oxidoreductases and oxidative stress are also involved in viral infections. In this overview, different aspects of the role of cellular oxidoreductases and oxidative stress during viral infections are discussed from a chemotherapeutic viewpoint. Areas covered: Entry of enveloped viruses into their target cells is triggered by the interaction of viral envelope glycoproteins with cellular (co)receptor(s) and depends on obligatory conformational changes in these viral envelope glycoproteins and/or cellular receptors. For some viruses, these conformational changes are mediated by cell surface-associated cellular oxidoreductases, which mediate disulfide bridge reductions in viral envelope glycoprotein(s). Therefore, targeting these oxidoreductases using oxidoreductase inhibitors might yield an interesting strategy to block viral entry of these viruses. Furthermore, since viral infections are often associated with systemic oxidative stress, contributing to disease progression, the enhancement of the cellular antioxidant defense systems might have potential as an adjuvant antiviral strategy, slowing down disease progression. Expert opinion: Promising antiviral data were obtained for both strategies. However, potential pitfalls have also been identified for these strategies, indicating that it is important to carefully assess the benefits versus risks of these antiviral strategies.
An efficient one step synthesis of S-acetyl-L-glutathione has been developed in a DMF-TFA mixed solvent with CoCl2 as catalyst. This process not only has the advantages of selective acylation of the glutathione thiol group without involving the free amino but also highlights recycling of the relatively costly solvent TFA, which improves the yield to 91% and quality to 99.7%. The reaction is cost-effective, efficient, and easy to scale-up.
Unlabelled:
Whey proteins (WP) exert anti-inflammatory and antioxidant effects. Hyperbaric pressurisation of whey increases its digestibility and changes the spectrum of peptides released during digestion. We have shown that dietary supplementation with pressurised whey improves nutritional status and systemic inflammation in patients with cystic fibrosis (CF). Both clinical indices are largely affected by airway processes, to which respiratory epithelial cells actively contribute. Here, we tested whether peptides released from the digestion of pressurised whey can attenuate the inflammatory responses of CF respiratory epithelial cells. Hydrolysates of pressurised WP (pWP) and native WP (nWP, control) were generated in vitro and tested for anti-inflammatory properties judged by the suppression of IL-8 production in CF and non-CF respiratory epithelial cell lines (CFTE29o- and 1HAEo-, respectively). We observed that, in both cell lines, pWP hydrolysate suppressed IL-8 production stimulated by lipopolysaccharide (LPS) to a greater magnitude compared with nWP hydrolysate. Neither hydrolysate suppressed IL-8 production induced by TNF-α or IL-1β, suggesting an effect on the Toll-like receptor (TLR) 4 pathway, the cellular sensor for LPS. Further, neither hydrolysate affected TLR4 expression or neutralised LPS. Both pWP and nWP hydrolysates similarly reduced LPS binding to surface TLR4, while pWP tended to more potently increase extracellular antioxidant capacity.
In conclusion:
(1) anti-inflammatory properties of whey are enhanced by pressurisation; (2) suppression of IL-8 production may contribute to the clinical effects of pressurised whey supplementation on CF; (3) this effect may be partly explained by a combination of reduced LPS binding to TLR4 and enhanced extracellular antioxidant capacity.
Selective syntheses of S-acyl glutathiones are achieved in 79–98% yields using 1-acyl-1H-benzotriazoles in the presence of potassium bicarbonate in aqueous methanol at 20 °C. N-Acylation of S-(p-nitrobenzoyl) glutathione with 1-acyl-1H-benzotriazoles followed by deprotection of the p-nitrobenzoyl groups under mild conditions gave 63–78% yields of N-acyl glutathiones. These meth-odologies should be useful for the S-acylation and N-acylation of peptides and glycopeptides.
Disulfide bonds reportedly stabilize the capsids of several viruses, including papillomavirus, polyomavirus, and simian virus 40, and have been detected in herpes simplex virus (HSV) capsids. In this study, we show that in mature HSV-1 virions, capsid proteins VP5, VP23, VP19C, UL17, and UL25 participate in covalent cross-links, and that these are susceptible to dithiothreitol (DTT). In addition, several tegument proteins were found in high-molecular-weight complexes, including VP22, UL36, and UL37. Cross-linked capsid complexes can be detected in virions isolated in the presence and absence of N-ethylmaleimide (NEM), a chemical that reacts irreversibly with free cysteines to block disulfide formation. Intracellular capsids isolated in the absence of NEM contain disulfide cross-linked species; however, intracellular capsids isolated from cells pretreated with NEM did not. Thus, the free cysteines in intracellular capsids appear to be positioned such that disulfide bond formation can occur readily if they are exposed to an oxidizing environment. These results indicate that disulfide cross-links are normally present in extracellular virions but not in intracellular capsids. Interestingly, intracellular capsids isolated in the presence of NEM are unstable; B and C capsids are converted to a novel form that resembles A capsids, indicating that scaffold and DNA are lost. Furthermore, these capsids also have lost pentons and peripentonal triplexes as visualized by cryoelectron microscopy. These data indicate that capsid stability, and especially the retention of pentons, is regulated by the formation of disulfide bonds in the capsid.
Reduced glutathione (GSH) is the most abundant non-protein thiol in mammalian cells and the preferred substrate for several enzymes in xenobiotic metabolism and antioxidant defense. It plays an important role in many cellular processes, such as cell differentiation, proliferation and apoptosis. GSH deficiency has been observed in aging and in a wide range of pathologies, including neurodegenerative disorders and cystic fibrosis (CF), as well as in several viral infections. Use of GSH as a therapeutic agent is limited because of its unfavorable biochemical and pharmacokinetic properties. Several reports have provided evidence for the use of GSH prodrugs able to replenish intracellular GSH levels. This review discusses different strategies for increasing GSH levels by supplying reversible bioconjugates able to cross the cellular membrane more easily than GSH and to provide a source of thiols for GSH synthesis.
Cystic Fibrosis (CF), the most common fatal genetic disorder, is due to mutations of the cystic fibrosis transmembrane regulator protein (CFTR). Loss of functional CFTR leads to mucus thickening with the consequences of chronic pulmonary infection and inflammation, the most common fatal complication of this disorder. This thesis aimed to examine one aspect of the pathophysiology of CF, the potential role of glutathione (GSH) and oxidant stress. CFTR is permeable to GSH, at least in vitro, where evidence of the ability of CFTR to transport GSH is strong. Furthermore, extracellular GSH is diminished in CF airway lining fluid and it has been hypothesized that deficiency of CFTR leads to higher intracellular GSH coupled with lower extracellular GSH. This would be consistent with the observation of low extracellular GSH in CF patients and increased resistance to apoptosis of CF cell lines, where falling intracellular GSH concentrations ([GSH]) are required to initiate apoptosis. In addition to CFTR, other channels transport GSH, including Multi-drug Resistance Protein 1 (MRP1) which has structural and functional homology to CFTR. Higher expression of MRP1 has been associated with fewer symptoms in CF patients, suggesting that MRP1 may contribute to CF pathophysiology. We therefore set out to study GSH at the cellular level, with or without functional CFTR, in an airway epithelial cell model. Given the importance of oxidant stress in CF, we tested whether cells were more susceptible to stress in the absence of CFTR function. We hypothesized that lack of CFTR function would increase intracellular and decrease extracellular [GSH]. We also hypothesized that MRP1 function would influence intracellular and extracellular [GSH]. Blockade of CFTR function in Calu-3 cells failed to alter either intra- or extracellular [GSH], independent of oxidant stress conditions, suggesting the channel was not a determinant of [GSH]. However, inhibition of other anion channels did increase intracellular [GSH], suggesting an alternate mechanism for regulating cellular [GSH]. In contrast to CFTR, inhibition of MRP1 increased intracellular [GSH], suggesting a role for this channel in regulating intracellular [GSH]. Paradoxically however, extracellular [GSH] was higher after administration of the MRP1 antagonist MK-571. In addition, we also observed higher CFTR activity with use of MK-571. These findings suggest that CFTR could be indirectly responsible for the changes in [GSH] through interaction with MRP1. CFTR blockade in the presence of MK-571 administration returned extracellular [GSH] to normal, which reinforces a role for CFTR in the extracellular [GSH] increases. Administration of either MK-571 or Montelukast increased both wild-type and deltaF508 CFTR expression and function in the BHK cell model. Montelukast may have beneficial effects in restoring CFTR expression and function, which if confirmed in other models and in vivo, may pave the way for future therapeutic treatments for CF patients. La fibrose kystique (FK) est causée par des mutations dans la protéine CFTR (pour « Cystic fibrosis transmembrane conductance regulator »). La perte de fonction de CFTR mène à l’épaississement des muqueuses des voies respiratoires et l’avènement d’infections pulmonaires chroniques avec réponse inflammatoire, la cause primaire de mortalité chez les personnes affligées de FK. Cette thèse va examiner une partie de la pathophysiologie de la FK, celle du rôle du glutathion (GSH) et du stress oxydatif. Le CFTR est perméable au GSH et les données expérimentales in vitro suggèrent que le CFTR puisse transporter le GSH. De plus, le GSH extracellulaire est diminué dans le liquide de surface recouvrant les voies respiratoires des patients avec la FK et de cela découle l’hypothèse que la déficience en CFTR mène à une augmentation du GSH intracellulaire, avec une diminution concomitante du GSH extracellulaire. Cette hypothèse s’alignerait bien avec le bas taux de GSH extracellulaire observé chez les patients affectés par la FK et la résistance accrue contre l’apoptose vue dans les lignées cellulaires de la FK. Cette résistance découle du fait que la cellule a besoin d’une diminution de concentration de GSH ([GSH]) intracellulaire pour entamer le processus d’apoptose. À part du CFTR, il existe d’autres canaux membranaires qui transportent le GSH, tels que le Multi-drug Resistance Protein 1 (MRP1). Ce canal a une forte homologie structurelle et fonctionnelle avec le CFTR et son expression se trouve liée avec moins de symptômes cliniques chez les patients atteints de FK. Ces observations suggèrent que le MRP1 pourrait jouer un rôle important dans l’évolution de la physiopathologie de la maladie. Notre but est d’étudier l’état du GSH du point de vue de la cellule, avec ou sans stress oxydatif, dans une lignée de cellules épithéliales des voies respiratoires. Considérant l’importance du stress oxydatif dans la FK, il a fallu déterminer si les cellules étaient plus susceptibles au stress en absence du fonctionnement du CFTR. On a mis devant l’hypothèse que le manque de fonctionnement du CFTR amène à l’augmentation de [GSH] interne avec diminution de [GSH] externe. De plus, nous croyions que le fonctionnement du MRP1 aurait des effets sur le [GSH] intracellulaire ou le [GSH] extracellulaire.En utilisant un bloqueur de canal visant le CFTR, nous n’avons pas observé de changement dans le [GSH] intracellulaire ou extracellulaire, chez les cellules Calu-3, avec ou sans stress oxydatif. Ces observations suggéraient que le CFTR n’était pas impliqué dans la régulation du [GSH]. Cependant, nous avons observé une augmentation du [GSH] intracellulaire avec les traitements utilisant les bloqueurs de canaux anioniques, ce qui amenait la possibilité qu’un autre mécanisme soit impliqué dans la régulation du [GSH]. Contrairement à l’inhibition de la fonction du CFTR, le blocage de MRP1 causait une augmentation du [GSH] intracellulaire, apportant au canal un rôle possible dans la régulation du GSH. De façon paradoxale, le [GSH] extracellulaire augmentait avec l’utilisation de MK-571, bloqueur de MRP1. De plus, nous avons constaté une augmentation de l’activité de CFTR avec l’utilisation de MK-571. Ces observations prises ensemble suggéraient que CFTR pouvait être impliqué de façon indirecte dans les changements de [GSH], à travers une interaction avec MRP1. En bloquant CFTR et MRP1 ensemble nous avons constaté que le [GSH] extracellulaire était revenu au niveau de base, ce qui venait appuyer un rôle pour le CFTR dans l’augmentation du [GSH] extracellulaire. Par après, nous avons constaté qu’un traitement de MK-571 ou de Montelukast, utilisant le modèle cellulaire BHK, causait une augmentation de l’expression et de la fonction du CFTR normal ainsi que du CFTR deltaF508. Le Montelukast pourrait alors avoir des effets bénéfiques sur le rétablissement de l’expression et de fonction de CFTR ce qui pourra ouvrir la porte à de nouvelles thérapies.
A series of glutathione (GSH) derivatives with aliphatic chains of different lengths, coupled by peptides bound to the alpha-NH2 group of Glu, were synthesized. When added to several cell lines, the C6 (n-hexanoyl), C8 (n-octanoyl) and C12 (n-dodecanoyl) derivatives were toxic while the C2 (nethanoyl) and C4 (n-butanoyl) derivatives were not. Preliminary experiments were performed to investigate the potential antiviral activity of the C2 and C4 derivatives compared to GSH. The C4 derivative was the most potent and fully characterized. GSH-C4 is a poor substrate of GSH metabolizing enzymes; once oxidized by disulphide-bound formation, C4 is slowly reduced by GSH-reductase. GSH-C4 completely abrogated Sendai virus replication at 7.5 mM with an EC50 of 3.6 mM, compared to 7.5 mM for GSH. GSH-C4 completely inhibited herpes simplex virus (HSV-1) virus production in Vero cells at 10 mM, while the same dose of GSH caused only a 2.5 log10 reduction. Furthermore, the GSH-C4 treatment (7.5 mM) was able to markedly reduce the cytopathic effect of HSV-1 in Vero cells. Thus, GSH derivatives with increased hydrophobic properties are more effective antiviral agents against Sendai and HSV-1 viruses than GSH, suggesting their usefulness in antiviral therapy.
Glutathione synthetase deficiency is an autosomal recessive inherited metabolic defect in the gamma-glutamyl cycle. Decreased intracellular glutathione levels are one of the characteristic biochemical features. In this study we show that addition of S-acetylglutathione to the medium raised intracellular glutathione content in cultured fibroblasts from patients with glutathione synthetase deficiency. This has implications for the treatment of patients with this inborn error of metabolism.
Reduced glutathione (GSH) is present in millimolar concentrations in mammalian cells. It is involved in many cellular functions such as detoxification, amino acid transport, production of coenzymes, and the recycling of vitamins E and C. GSH acts as a redox buffer to preserve the reduced intracellular environment. Decreased glutathione levels have been found in numerous diseases such as cancer, viral infections, and immune dysfunctions. Many antioxidant molecules, such as GSH and N-acetylcysteine (NAC), have been demonstrated to inhibit in vitro and in vivo viral replication through different mechanisms of action. Accumulating evidence suggests that intracellular GSH levels in antigen-presenting cells such as macrophages, influence the Th1/Th2 cytokine response pattern, and more precisely, GSH depletion inhibits Th1-associated cytokine production and/or favours Th2 associated responses. It is known that GSH is not transported to most cells and tissues in a free form. Therefore, a number of different approaches have been developed in the last years to circumvent this problem. This review discusses the capacity of some new molecules with potent pro-GSH effects either to exert significant antiviral activity or to augment GSH intracellular content in macrophages to generate and maintain the appropriate Th1/Th2 balance. The observations reported herein show that pro-GSH molecules represent new therapeutic agents to treat antiviral infections and Th2-mediated diseases such as allergic disorders and AIDS.
Herpes simplex virus (HSV) can enter the central nervous system and cause encephalitis (HSV-1) or meningitis (HSV-2). Microglia, the immunocompetent cells of the central nervous system, are potentially able to detect viral infections. Microglia have been shown to express the glutamate transporter GLT-1 during pathological events, leading to increased microglial glutamate uptake and glutathione synthesis. This study aims to address the role of GLT-1 and glutathione, a major antioxidant with antiviral properties, during HSV infections. Using neuron-enriched mixed primary cultures from rat, it was found that microglia have higher resistance to HSV infections than neurons or astrocytes after 24 h incubation with HSV. Purified microglia in culture were used to further address this. It was found that microglia were able to detect HSV and responded by releasing tumor necrosis factor-alpha (TNF-alpha) and upregulating GLT-1 after 24 h incubation with 1 PFU/cell HSV-1 or HSV-2. Furthermore, the microglial glutathione levels were not significantly diminished after 24 h. Inhibition of the microglial glutathione synthesis with 200 microM buthionine sulfoximide (BSO) led to significantly more infected cells after 24 h incubation with 1 PFU/cell HSV-1 or HSV-2. These data indicate that the higher resistance in microglia against HSV infections may be due to the expression of GLT-1, which can maintain the glutathione levels and provide a mechanism for microglial self-defense against HSV.
Current thinking attributes the balance between T helper 1 (Th1) and Th2 cytokine response patterns in immune responses to the nature of the antigen, the genetic composition of the host, and the cytokines involved in the early interaction between T cells and antigen-presenting cells. Here we introduce glutathione, a tripeptide that regulates intracellular redox and other aspects of cell physiology, as a key regulatory element in this process. By using three different methods to deplete glutathione from T cell receptor transgenic and conventional mice and studying in vivo and/or in vitro responses to three distinct antigens, we show that glutathione levels in antigen-presenting cells determine whether Th1 or Th2 response patterns predominate. These findings present new insights into immune response alterations in HIV and other diseases. Further, they potentially offer an explanation for the well known differences in immune responses in "Th1" and "Th2" mouse strains.
The activation of nuclear factor kappa B (NF-kappa B) has been implicated in the regulation of transcription of a variety of genes and has been shown to be essential for the expression of genes controlled by the long terminal repeat of human immunodeficiency virus (HIV LTR). We show here that intracellular thiol levels play a key role in regulating this process. That is, stimulation with tumor necrosis factor alpha and/or phorbol 12-myristate 13-acetate activates NF-kappa B and markedly decreases intracellular thiols; N-acetyl-L-cysteine, an efficient thiol source, prevents this thiol decrease and blocks the activation of NF-kappa B; and the lack of activated NF-kappa B prevents the activation of the HIV LTR and the transcription of genes under its control. These findings reveal a previously unrecognized genetic regulatory mechanism in which cytokine-induced shifts in intracellular thiol levels are crucial in the control of NF-kappa B activity and thereby influence the spectrum of genes expressed by cytokine-stimulated cells.
Recent studies have shown that there is an interorgan cycle of glutathione metabolism in which glutathione is translocated from certain cells into the blood plasma, and that plasma glutathione is utilized by cells that have gamma-glutamyl transpeptidase. The present studies indicate that there is a significant intravascular phase of glutathione metabolism. The level of total glutathione (GSH + GSSG) in rat blood plasma was found to be 22 to 27 microM GSH equivalents, as determined by the glutathione reductase recycling method. About 85% of the total is in the form of GSH. These findings contrast with previous reports of total levels of 3 to 6 microM and 50 to 75% GSSG. We found that plasma allowed to stand at 23 degrees C for 30 to 60 min has total glutathione levels of 4 to 7 microM, most (95%) of which is GSSG; after treatment of this plasma (following deproteinization) with KBH4, levels of 21 to 24 microM were found. GSH disappears rapidly from plasma, whereas GSSG disappears very slowly. gamma-Glutamyl transpeptidase does not account for the loss of plasma GSH, nor does binding to proteins account for more than a small fraction of the GSH that disappears. Most of the GSH that disappears can be found in the deproteinized samples after treatment with KBH4. The findings are in accord with the view that glutathione is translocated to plasma in the form of GSH and that such GSH constitutes the major source of plasma thiol. The intravascular phase of GSH metabolism seems to involve reduction of disulfide bonds of plasma constituents and mobilization of compounds bound by disulfide linkage to plasma proteins to form GSSG and low molecular weight derivatives of glutathione such as disulfides.
Even a moderate increase in the cellular cysteine supply elevates the intracellular glutathione (GSH) and glutathione disulfide (GSSG) levels and potentiates immunological functions of lymphocytes in vitro. At low GSSG levels, T cells cannot optimally activate the immunologically important transcription factor NF kappa B, whereas high GSSG levels inhibit the DNA binding activity of NF kappa B. The effects of GSSG are antagonized by reduced thioredoxin (TRX). As the protein tyrosine kinase activities p56lck and p59fyn are activated in intact cells by hydrogen peroxide, they are likely targets for GSSG action. These redox-regulated enzymes trigger signal cascades for NF kappa B activation and transduce signals from the T cell antigen receptor, from CD4 and CD8 molecules, and from the IL-2 receptor beta-chain. The effector phase of cytotoxic T cell responses and IL-2-dependent functions are inhibited even by a partial depletion of the intracellular GSH pool. As signal transduction is facilitated by prooxidant conditions, we propose that the well-known immunological consequences of GSH depletion ultimately may be results of the accompanying GSSG deficiency. As HIV-infected patients and SIV-infected rhesus macaques have, on the average, significantly decreased plasma cyst(e)ine and intracellular GSH levels, we also hypothesize that AIDS may be the consequence of a GSSG deficiency as well.
Antiviral activity of L-ascorbic acid-2-phosphate (ASC-2P), a long-acting derivative of L-ascorbic acid, against several human cytomegalovirus (CMV) strains was examined in cultures of human foreskin fibroblasts (HFF) and endothelial cells (EC). ASC-2P at concentrations ranging from 0.2 to 2 mM had no effect on the number of cells expressing 72 kDa CMV immediate early antigen (IEA) while it inhibited expression of 68 kDa late antigen (LA) in infected cultures of both cell types (30% and 55% reduction for EC and HFF, respectively). In HFF cells, virus yield was reduced up to 4-fold, when ASC-2P was added after CMV infection. Antiviral effects were significantly increased in cultures pretreated with ASC-2P. In HFF and EC pretreated for three subcultures (18 days) with 0.2 mM ASC-2P, a significant reduction of cells expressing IEA (75% and 80% reduction in EC and HFF, respectively) and LA (92% and 90% reduction for EC and HFF, respectively) was observed. Pretreatment for three subcultures with ASC-2P inhibited virus yield 50- to 100-fold in EC and 100- to 1000-fold in HFF. The continuous presence of ASC-2P was not required for its antiviral activity. A significantly higher reduction of virus replication with ganciclovir and foscarnet was obtained in ASC-2P pretreated cells than in untreated controls. The results showed that ASC-2P provides L-ascorbic acid with long-lasting antiviral activity against CMV. ASC-2P may be of benefit for the adjunctive treatment of CMV infection.
In a double-blind placebo-controlled trial, human immunodeficiency virus (HIV)-seropositive patients with a CD4 lymphocyte cell count of more than 200 x 10(6) . l-1 were randomised to receive either 800 mg N-acetylcysteine (NAC) or placebo for 4 months. Before treatment low plasma cysteine levels, high free radical activity in neutrophils in the presence of autologous plasma-measured by the nitroblue tetrazolium (NBT) test- and increased tumor necrosis factor (TNF)-alpha levels were found in the HIV positive patients.
After treatment the low plasma cysteine level in the NAC group increased to normal, and the decline of the CD4+ lymphocyte count before the study start, was less steep in the NAC group than in the placebo group after treatment. There was also a reduction in TNF-alpha level. However, NAC had no effect on the radical production by neutrophils, and although it did not increase the CD4+ cell count, it may have decreased the decline in CD4+ cells.
Further controlled trials with NAC are needed to determine whether it has a beneficial effect in the treatment of asymptomatic HIV-infected individuals.
In vitro HIV-1 infection induced a significant decrease in intracellular reduced glutathione (GSH) in human macrophages. Such a decrease was observed at the time of infection corresponding to maximum release of virus from infected cells and was not related to cell cytotoxicity. GSH los was not related to its oxidation or leakage through the cell membrane. Inhibition of intracellular GSH synthesis by buthionine sulfoximine (BSO) did not further decrease GSH levels with respect to the decrease caused by HIV alone. However, treatment of macrophages with BSO significantly increased the HIV yield in the supernatant. Exogenous GSH strongly suppressed the production of p24 gag protein as well as the virus infectivity. Previous observations with other RNA and DNA viruses consistently showed that GSH antiviral effect occurred at late stages of virus replication and was related to the selective decrease of specific glycoproteins, such as gp120, which are particularly rich in disulfide bonds.
Dysregulated apoptosis may underlie the etiology of T cell depletion by human immunodeficiency virus type 1 (HIV-1). We show that HIV-induced apoptosis is preceded by an exponential increase in reactive oxygen intermediates (ROIs) produced in mitochondria. This leads to caspase-3 activation, phosphatidylserine (PS) externalization, and GSH depletion. Since mitochondrial ROI levels are regulated by the supply of NADPH from the pentose phosphate pathway (PPP), the effect of transaldolase (TAL), a key enzyme of PPP, was investigated. Jurkat and H9 human CD4+ T cells were transfected with TAL expression vectors oriented in the sense or antisense direction. TAL overexpression down-regulated glucose-6-phosphate dehydrogenase activities and GSH levels. Alternatively, decreased TAL expression up-regulated glucose-6-phosphate dehydrogenase activities and GSH levels. HIV-induced 1) mitochondrial ROI production, 2) caspase-3 activation, 3) proteolysis of poly(ADP-ribose) polymerase, and 4) PS externalization were accelerated in cells overexpressing TAL. In contrast, suppression of TAL abrogated these four activities. Thus, susceptibility to HIV-induced apoptosis can be regulated by TAL through controlling the balance between mitochondrial ROI production and the metabolic supply of reducing equivalents by the PPP. The dominant effect of TAL expression on oxidative stress, caspase activation, PS externalization, and cell death suggests that this balance plays a pivotal role in HIV-induced apoptosis.
Glutathione (GSH) is an important intracellular peptide with multiple functions ranging from antioxidant defense to modulation of cell proliferation. GSH is synthesized in the cytosol of all mammalian cells in a tightly regulated manner. The major determinants of GSH synthesis are the availability of cysteine, the sulfur amino acid precursor, and the activity of the rate-limiting enzyme, gamma-glutamylcysteine synthetase (GCS). In the liver, major factors that determine the availability of cysteine are diet, membrane transport activities of the three sulfur amino acids cysteine, cystine and methionine, and the conversion of methionine to cysteine via the trans-sulfuration pathway. Many conditions alter GSH level via changes in GCS activity and GCS gene expression. These include oxidative stress, activators of Phase II detoxifying enzymes, antioxidants, drug-resistant tumor cell lines, hormones, cell proliferation, and diabetes mellitus. Since the molecular cloning of GCS, much has been learned about the regulation of this enzyme. Both transcriptional and post-transcriptional mechanisms modulate the activity of this critical cellular enzyme.--Lu, S. C. Regulation of hepatic glutathione synthesis: current concepts and controversies.
Glutathione is central to the antioxidant defences of the lung. The aim of this study was to determine whether sputum induction can be used for the measurement of glutathione in the respiratory tract.
Saliva and induced sputum (3% NaCl, 20 minutes) samples were collected from 10 healthy individuals and 10 patients with stable asthma receiving treatment with inhaled corticosteroids. Samples were chilled on ice and dispersed by dilution with ice cold phosphate buffered saline and pipetting. Cell-free supernatants were obtained by centrifugation of samples and filtration of supernatants and analysed for total glutathione, glutathione disulfide, and albumin content. The cells were treated with dithiothreitol and cell numbers, cell viability, and differential cell patterns were determined.
As judged by cell viability and percentage of non-squamous cells, adequate sputum samples were obtained from nine healthy and nine asthmatic subjects. The salivary total glutathione content was low (median concentration 1.2 microM (range 0.8-1.5) in healthy subjects and 0.9 microM (0.7-1. 2) in asthmatic subjects). The sputum total glutathione content of both healthy and asthmatic subjects was within the same range (3.9 (1.0-12.3) microM and 6.4 (1.3-19.2) microM, respectively; p=0.35). Surprisingly, and in marked contrast to results obtained with bronchoalveolar lavage, sputum levels of glutathione disulfide represented more than 50% of the total glutathione in both groups (50.9% (range 24.6-83.1) and 72.3% (range 36.5-97.4), respectively; p=0.2).
The results of this study indicate that sputum induction can be used to measure the glutathione content of bronchial secretions. Sputum glutathione levels of stable asthmatic patients did not differ significantly from healthy controls.
Glutathione (GSH; gamma-glutamylcysteinylglycine) is ubiquitous in mammalian and other living cells. It has several important functions, including protection against oxidative stress. It is synthesized from its constituent amino acids by the consecutive actions of gamma-glutamylcysteine synthetase and GSH synthetase. gamma-Glutamylcysteine synthetase activity is modulated by its light subunit and by feedback inhibition of the end product, GSH. Treatment with an inhibitor, buthionine sulfoximine (BSO), of gamma-glutamylcysteine synthetase leads to decreased cellular GSH levels, and its application can provide a useful experimental model of GSH deficiency. Cellular levels of GSH may be increased by supplying substrates and GSH delivery compounds. Increasing cellular GSH may be therapeutically useful.
The role of glutathione (GSH) in the in vitro infection and replication of human herpes simplex virus type 1 (HSV-1) was investigated. Intracellular endogenous GSH levels dramatically decreased in the first 24 h after virus adsorption, starting immediately after virus challenge. The addition of exogenous GSH was not only able to restore its intracellular levels almost up to those found in uninfected cells, but also to inhibit > 99% the replication of HSV-1. This inhibition was concentration-dependent, not related to toxic effects on host cells and also maintained if the exogenous GSH was added as late as 24 h after virus challenge, i.e. when virus infection was fully established. Electron microscopic examination of HSV-1-infected cells showed that GSH dramatically reduced the number of extracellular and intracytoplasmic virus particles, whereas some complete nucleocapsids were still detected within the nuclei of GSH-treated cells. Consistent with this observation, immunoblot analysis showed that the expression of HSV-1-glycoprotein B, crucial for the release and the infectivity of virus particles, was significantly decreased. Data suggest that exogenous GSH inhibits the replication of HSV-1 by interfering with very late stages of the virus life cycle, without affecting cellular metabolism.
Human carcinoma of the cervix cell line HeLa, adapted to continuous growth in a protein-free chemically defined medium, was used as substrate for the replication of several human pathogenic viruses. Growth characteristics of the cells designated as HeLa-PF in protein-free 1:1 nutrient mixture of Dulbecco's modified MEM and Ham's F-12 supplemented with L-ascorbic acid 2-phosphate were similar to those of the cells grown in a serum-supplemented medium. After 30 months (135 subcultures) in the protein-free medium, HeLa-PF cells were infected with poliovirus types 1, 2 and 3; adenovirus types 2 and 5 and herpes simplex virus type 1. Both adenoviruses and polioviruses developed in HeLa-PF cells titers and showed cytopathic effects comparable to those obtained in conventionally grown and maintained cells; in contrast, significantly lower herpes simplex virus type 1 titers and changed characteristics of the cytopathic effects were observed in HeLa-PF cells. The results show that HeLa-PF cells grown continuously in protein-free medium provide a unique system for the study of virus replication.
Addition of reduced glutathione inhibited the production of Sendai virus in African green monkey kidney (AGMK) cells. This result could be accounted for by a direct action of GSH on viral replication. The inhibitory action was associated to an increase of the GSH intracellular level, while the host cell metabolism was unaffected. The antiviral effect was related to decrease and inactivation of the hemagglutinin-neuraminidase (HN) virus glycoprotein.
Markedly decreased plasma cystine and cysteine concentrations have been found in HIV-infected patients at all stages of the disease and in SIV-infected rhesus macaques. The elevated glutamate levels found in the same patients aggravate the cysteine deficiency by inhibiting the membrane transport activity for cystine. The intact immune system appears to require a delicate balance between pro-oxidant and antioxidant conditions, maintained by a limited and well-regulated supply of cysteine. This balance is obviously disturbed in HIV infection and may contribute to the pathogenesis of AIDS.
The effects of glutathione (GSH), glutathione ester (GSE), and N-acetyl-L-cysteine (NAC) on the induction of human immunodeficiency virus (HIV) expression were investigated in the chronically infected monocytic U1 cell line, a previously described cellular model for HIV latency. U1 cells constitutively express low levels of virus, which can be increased by phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and other inducers. GSH, GSE, and NAC suppressed in a dose-dependent fashion the induction of HIV expression mediated by PMA, TNF-alpha, and IL-6, in the absence of cytotoxic or cytostatic effects. Reverse transcriptase activity, inducible by PMA, TNF-alpha, or IL-6, was decreased by 80-90% after pretreatment with GSH, GSE, or NAC. The induction of total HIV protein synthesis was also decreased appreciably after pretreatment with GSH, GSE, or NAC. The accumulation of HIV mRNA was substantially suppressed after pretreatment with NAC but to a lesser extent after pretreatment with GSH or GSE. Although PMA induces the expression of TNF-alpha in U1 cells, the suppressive effect of GSH, GSE, and NAC on PMA-induced HIV expression in U1 cells was not associated with the inhibition of TNF-alpha expression. The present findings, which elucidate relationships between cellular GSH and HIV expression, suggest that therapy with thiols may be of value in the treatment of HIV infection.
Glutathione, which is synthesized within cells, is a component of a pathway that uses NADPH to provide cells with their reducing milieu. This is essential for (a) maintenance of the thiols of proteins (and other compounds) and of antioxidants (e.g. ascorbate, α-tocopherol), (b) reduction of ribonucleotides to form the deoxyribonucleic precursors of DNA, and (c) protection against oxidative damage, free radical damage, and other types of toxicity. Glutathione interacts with a wide variety of drugs. Despite its many and varied cellular functions, it is possible to achieve therapeutically useful modulations of glutathione metabolism.
The plasma concentration and appearance rate of norepinephrine are increased in the elderly. A hypothesis to explain this observation is that the elderly have a diminished response of the alpha 2-adrenoreceptor in the brainstem that modulates peripheral sympathetic tone. To evaluate the effect of age on alpha 2-adrenoreceptor function, we studied 12 healthy elderly subjects and 12 healthy young volunteers and compared the decrease in plasma norepinephrine and blood pressure in response to increasing doses of orally administered clonidine. We found that, for the same plasma clonidine concentration, the blood pressure and plasma norepinephrine concentration fell equivalently in both groups. These data imply that the increased plasma norepinephrine and the elevated blood pressure in the healthy elderly population do not appear to be secondary to a decrease in alpha 2-adrenergic response to an agonist in the central nervous system.
Functions of GSH in detoxication during radical-induced injury in specific pathological and toxicological conditions are discussed. GSH protects against oxidative damage in systems that scavenge radicals, eliminate lipid peroxidation products, preserve thiol-disulfide status of proteins, and repair oxidant damage. Several factors which affect cellular GSH homeostasis can affect these functions, including nutritional status, hypoxia and pharmacological intervention. Evidence from a variety of pathological and toxicological conditions, e.g. ischemia-reperfusion injury, chemically induced oxidative injury, radiation damage, aging, and degenerative diseases, indicate that GSH is a primary component of physiological systems to protect against oxidant and free-radical-mediated cell injury.
To find out whether systemic glutathione deficiency is associated with human immunodeficiency virus (HIV) infection, thus contributing to the immunodeficiency state, glutathione concentrations in venous plasma and lung epithelial lining fluid (ELF) of symptom-free HIV-seropositive and normal individuals were measured. Total and reduced glutathione concentrations in the plasma of the HIV-infected subjects were about 30% of those in the normal individuals. Concentrations of these substances in the ELF of HIV-infected subjects were about 60% of those in the controls. There was no correlation between ELF and plasma concentrations of total or reduced glutathione. Since glutathione enhances immune function, glutathione deficiency may contribute to the progressive immune dysfunction of HIV infection.
In a single in vivo experiment we determined the apparent half-life of glutathione in human plasma to be 1.6 min. The mean total glutathione content in the plasma of 11 male and 9 female adults was found to be 0.34±0.11 μmol/l. GSSG was ≤0.1 μmol/l in 12 subjects and 0.1-0.28 μmol/l in 8 subjects. No sex-related differences were observed. We also determined total plasma glutathione of a bilaterally nephrectomized female patient and found a level of 0.53 μmol/l.
S-acetyl- and S-phenylacetyl-glutathione derivatives were synthesized by using a new procedure. The derivatives were incubated with rat plasma and red blood cells, and also with cytosol from rat liver, kidney and heart, or tissue slices from rat heart, kidney and liver. A limited hydrolysis of the compounds occurs in plasma, whereas hydrolysis occurs to a larger extent in tissue cytosols. Both purified and crude gamma-glutamyl-transpeptidase from different sources recognized the S-acetyl- and S-phenylacetyl derivatives as substrates. Intracellular glutathione increases after incubating the derivatives with red blood cells. A potential role of S-acetyl- and S-phenylacetyl-glutathione in replenishing cells with exogenous glutathione is envisaged.
Because glutathione (GSH) in plasma and lymphocytes of HIV-infected patients is low, adjunct therapy with N-acetylcysteine (NAC) to restore GSH homeostasis has been proposed. To investigate the effect of NAC on the GSH status we treated six patients with AIDS with 1.8 g/day of NAC for 2 weeks. During treatment the plasma concentration of cysteine, a precursor for GSH synthesis, increased significantly. Nevertheless, there was no significant increase in GSH in plasma and peripheral blood mononuclear cells. The failure of sulfhydryl supplementation to increase GSH suggests that the low concentrations of the tripeptide are not the result of an increased consumption secondary to an oxidant stress, but rather the consequence of a decreased rate of synthesis of GSH in HIV infection.
This chapter discusses the preparation and properties of glutathione diethyl ester and related derivatives. Glutathione diethyl ester, like glutathione mono(glycyl) esters, is an effective glutathione delivery compound. When mice are injected intraperitoneally with glutathione diethyl ester, the tissue levels of glutathione increase to about the same extent as found after injection of glutathione monoethyl ester. Essentially equivalent effects are found after administration of the monoester and the diester is explained by the finding that mouse blood plasma contains appreciable amounts of glutathione diester α-esterase, which rapidly converts the diester to the monoester. On the other hand, human plasma does not have this esterase; for this reason, glutathione diethyl ester is stable in human blood and serves as a cellular glutathione delivery agent for human cells. Studies indicate that glutathione diethyl ester is effectively transported into human cells. It is evident that neither rats nor mice are good experimental models for studies on glutathione diester. However, studies on hamsters, guinea pigs, rabbits, and sheep have shown that plasma from these species lacks glutathione diethyl ester α-esterase; thus, these animals are potentially useful as experimental models for studies on the in vivo disposition of glutathione diethyl ester.
This chapter discusses the preparation and use of glutathione monoesters. Glutathione monoesters are useful glutathione delivery agents because they are readily transported into many types of cells and split intracellularly to form glutathione. Treatment of cells (in suspension or culture) and of intact animals with glutathione itself may lead to some increase in cellular glutathione levels; such increases are usually caused by extracellular breakdown of glutathione into its constituent amino acids and into dipeptides and the transport of these into cells followed by intracellular glutathione synthesis. Studies in which glutathione synthesis is completely inhibited, for example, by treatment with buthionine sulfoximine, show that administration of glutathione leads to little increase in cellular glutathione levels. Glutathione monoesters are transported into many rodent tissues, including kidney, liver, pancreas, spleen, skeletal muscle, heart, lung, and, in neonatal animals, the lens and the brain. Monoesters of glutathione are also transported into human red blood cells, lymphocytes, and fibroblasts.
N-Acetylcysteine (NAC) is highly nontoxic for peripheral blood T cells and immunostimulatory enhancing T cell functions such as mitogenesis, interleukin-2 (IL-2) production, and growth in culture. NAC has been proposed for the treatment of AIDS based on its inhibition of human immunodeficiency virus (HIV) replication in cultured cells. Therefore its effect on normal T cells from 10 young donors and one elderly donor has been investigated as a prelude to clinical consideration. T cell function was evaluated in the presence and absence of accessory cells. With concanavalin A and anti-CD3 activation, NAC enhanced mitogenesis by approximately 2- to 2.5-fold at 5-10 mM. Mitogenesis of purified T cells with anti-CD2 was not affected by NAC; in the presence of accessory cells, NAC enhanced mitogenesis by approximately 2-fold at 1-10 mM. Importantly, NAC levels above 10 mM completely inhibited activation of peripheral blood mononuclear cells by anti-CD2. IL-2 secreted by T cells was also enhanced by NAC, approximately 1.5-fold, but IL-2 secreted by cells from old donors was enhanced by 3-fold. In cultures of peripheral blood T cells, NAC (10 mM) stimulated growth by at least 4- to 6-fold after two passages. These results show that NAC, nontoxic even at 20 mM, is an effective enhancer of T cell function and a remarkable enhancer of growth. Results from other laboratories show that NAC, which increases glutathione levels, suppresses HIV replication presumably via suppression of the activation of transcriptional factor NF-kappa B.(ABSTRACT TRUNCATED AT 250 WORDS)
In studies presented here, we demonstrate that antioxidants regulate NF-kappa B activation and signal transduction pathways leading to HIV expression. We show (1) that N-acetyl-L-cysteine (NAC), an antioxidant and an efficient glutathione (GSH) precursor, inhibits NF-kappa B activation and HIV expression under conditions in which GSH is depleted and NAC cannot be converted to GSH, (2) that the D-stereoisomer of NAC and a wide variety of chemically unrelated antioxidants also inhibit NF-kappa B activation and/or transcription directed by the HIV LTR, and (3) that depletion of GSH, the principal intracellular antioxidant, augments HIV production in an acute infection model. Taken together, these findings suggest direct antioxidant action as the mechanism for inhibition of HIV transcription by NAC. They also confirm that GSH, acting in its capacity as an antioxidant, regulates HIV expression and that exogenous antioxidants can potentiate this regulation.
Cocaine was found to increase parainfluenza-1 Sendai virus (SV) replication in Madin Darby canine kidney (MDCK) cells. Its effect was maximal when it was added before SV infection, while practically no effect was observed when cocaine was added at the time of or after infection. Enhanced SV replication was associated with increased viral protein expression. Cocaine also greatly reduced the intracellular level of glutathione (GSH), namely the most abundant cell thiol with antioxidant functions, recently proposed as an important factor influencing viral infection. Support for this view was provided in the present study by the reversal of cocaine-induced enhancement of SV replication when the intracellular content of GSH was restored by addition of exogenous GSH.
Reduced glutathione (GSH) is decreased in patients affected by chronic hepatitis C (CHC) as well as in patients who are HIV positive. Because the liver is the most important source of plasmatic GSH, we measured the concentrations of GSH in the liver (H-GSH) of patients with CHC who were either HIV positive or negative, correlating it to the concentrations of GSH in plasma (P-GSH) and in peripheral blood mononuclear cells (PBMCs) (L-GSH), to the replication activity of hepatitis C virus (HCV) in PBMCs, to the activity of the liver disease, and to the state of immunodeficiency in patients who were HIV positive.
One hundred, five patients with serologically and histologically demonstrated CHC (55 HIV positive and 50 HIV negative) entered the trial. Fifty-one healthy individuals made up a control group for P-GSH and L-GSH concentrations. H-GSH concentration was determined by high performance liquid chromatography on liver specimens obtained by ultrasound-guided biopsy according to the method described by Reed et al. The concentrations of P-GSH and L-GSH were determined according to the method described by Suarez et al. The detection of HCV RNA strands in PBMCs was performed according to the method described by Qian et al. Histological findings and degree of fibrosis were scored according to the numerical scoring system proposed by Scheuer and by Knodell et al.
H-GSH, P-GSH, and L-GSH were significantly reduced in patients affected by CHC compared with healthy controls (p < 0.001). H-GSH and particularly L-GSH were more significantly reduced in patients who were HIV positive compared with those who were HIV negative (p < 0.001), without significant correlation with the values of the T cell subset CD4+. The reductions in H-GSH, P-GSH, and L-GSH were significantly correlated to the replication activity of HCV in PBMCs (p < 0.001) and to the grade of activity of the liver disease assessed by the values of ALT (p < 0.001) and by histological and fibrosis scores of CHC (p < 0.001). In both groups of patients with CHC, H-GSH, P-GSH, and L-GSH were more reduced in patients addicted to drugs than in patients who were not addicted.
In patients with CHC, particularly those who are HIV positive, a systemic depletion of GSH is present. This depletion may be a factor underlying the resistance to interferon therapy and, in patients who are HIV positive, to antiretroviral drugs, fostering HCV and/or HIV replication. This may represent the biological basis for GSH replacement therapy.
An L-glutamine antagonist, 6-diazo-5-oxo-L-norleucin (L-DON), inhibits replication of vesicular stomatitis virus, poliovirus and paramyxoviruses in cultured cells. We tested the antiviral activity of L-DON against different strains of herpes simplex virus type 1 (HSV-1) in Vero cells. In the presence of a physiological plasma concentration of L-glutamine (0.5mM) L-Don inhibited 50% production of virus plaques at concentrations ranging from 7.9 to 16 microM. At concentrations of 40 microM L-Don inhibited infectious virus yield by 99%. The antiviral activity of L-DON decreased with increasing L-glutamine concentrations. A concentration of 5000 microM of L-Don had no significant effects on the viability of Vero cells. Transmission electron microscopical investigations showed that L-DON prevented mainly envelopment of viral nucleocapsids in the cytoplasm. The immunoprecipitation experiments demonstrated selective inhibition of synthesis of HSV-1 glycoproteins in L-DON treated cells. The results showed that L-DON inhibits HSV-1 replication at a late stage in the virus replication cycle, probably the cytoplasmic maturation of virions and subsequent virion egress from the cells.
Glutathione (GSH), a cysteine-containing tripeptide, is essential for the viability and function of virtually all cells. In vitro studies showing that low GSH levels both promote HIV expression and impair T cell function suggested a link between GSH depletion and HIV disease progression. Clinical studies presented here directly demonstrate that low GSH levels predict poor survival in otherwise indistinguishable HIV-infected subjects. Specifically, we show that GSH deficiency in CD4 T cells from such subjects is associated with markedly decreased survival 2-3 years after baseline data collection (Kaplan-Meier and logistic regression analyses, P < 0.0001 for both analyses). This finding, supported by evidence demonstrating that oral administration of the GSH prodrug N-acetylcysteine replenishes GSH in these subjects and suggesting that N-acetylcysteine administration can improve their survival, establishes GSH deficiency as a key determinant of survival in HIV disease. Further, it argues strongly that the unnecessary or excessive use of acetaminophen, alcohol, or other drugs known to deplete GSH should be avoided by HIV-infected individuals.
Oxidative stress could play a role in the pathogenesis of hepatitis C virus infection. We investigated the oxidant/antioxidant status in peripheral blood mononuclear cells from patients with chronic hepatitis C and controls.
Lipid peroxidation products and superoxide dismutase activity in peripheral blood mononuclear cells were higher in chronic hepatitis C patients than in healthy subjects while glutathione S-transferase activity was reduced in patients as compared to controls. Catalase, glutathione peroxidase and glutathione reductase were similar in chronic hepatitis C and normal individuals. No statistically significant differences were found between patients and controls with regard to glutathione levels in peripheral blood mononuclear cells, but 35% of patients with chronic hepatitis C showed values of glutathione and oxidized glutathione which were below and above, respectively, the limits of normal controls. Finally, the glutathione synthetic capacity of the cytosol of peripheral blood mononuclear cells was significantly higher in patients than in controls, indicating increased glutathione turnover in lymphocytes from patients with chronic hepatitis C.
Oxidative stress is observed in peripheral blood mononuclear cells from chronic hepatitis C patients. This process might alter lymphocyte function and facilitate the chronicity of the infection.
A significant decrease in the antioxidant glutathione (GSH) was found in the corneal tissue of rabbits with Herpes Simplex 1 (HSV-1)-induced keratitis. Such a decrease was due to a loss of the reduced species, since no increase in its oxidized form was observed. Topical administration of purified GSH was able to reduce the virus titre in corneal tissue and, at the same time, was effective in reducing the severity and progression of keratitis and conjunctivitis. This effect was paralleled by a partial recovery in the corneal GSH content. In vitro experiments performed on HSV-1 infected corneal-derived rabbit cells showed that exogenous GSH reduced virus titre in the supernatant of infected cells. These results are in agreement with our previous findings that an oxidative environment, due to GSH depletion, is necessary for virus replication and suggest that topical GSH treatment could be considered as complementary therapy in HSV-1-induced keratitis.
The tripeptide glutathione is the thiol compound present in the highest concentration in cells of all organs. Glutathione has many physiological functions including its involvement in the defense against reactive oxygen species. The cells of the human brain consume about 20% of the oxygen utilized by the body but constitute only 2% of the body weight. Consequently, reactive oxygen species which are continuously generated during oxidative metabolism will be generated in high rates within the brain. Therefore, the detoxification of reactive oxygen species is an essential task within the brain and the involvement of the antioxidant glutathione in such processes is very important. The main focus of this review article will be recent results on glutathione metabolism of different brain cell types in culture. The glutathione content of brain cells depends strongly on the availability of precursors for glutathione. Different types of brain cells prefer different extracellular glutathione precursors. Glutathione is involved in the disposal of peroxides by brain cells and in the protection against reactive oxygen species. In coculture astroglial cells protect other neural cell types against the toxicity of various compounds. One mechanism for this interaction is the supply by astroglial cells of glutathione precursors to neighboring cells. Recent results confirm the prominent role of astrocytes in glutathione metabolism and the defense against reactive oxygen species in brain. These results also suggest an involvement of a compromised astroglial glutathione system in the oxidative stress reported for neurological disorders.
Glutathione (GSH) deficiency is common in HIV-infected individuals and is associated with impaired T cell function and impaired survival. N-acetylcysteine (NAC) is used to replenish GSH that has been depleted by acetaminophen overdose. Studies here test oral administration of NAC for safe and effective GSH replenishment in HIV infection.
Oral NAC administration in a randomized, 8-week double-blind, placebo-controlled trial followed by optional open-label drug for up to 24 weeks.
HIV-infected, low GSH, CD4 T cells < 500 micro L(-1), no active opportunistic infections or other debilitation; n = 81. Study conducted prior to introduction of protease inhibitors.
Whole blood GSH levels in NAC arm subjects significantly increased from 0.88 mM to 0.98 mM, bringing GSH levels in NAC-treated subjects to 89% of uninfected controls (P = 0.03). Baseline GSH levels in the placebo group (0.91) remained essentially the same during the 8 week placebo-controlled trial. T cell GSH, adjusted for CD4 T cell count and beta2-microglobulin levels, also increased in the NAC-treated subjects (P = 0.04). Adverse effects were minimal and not significantly associated with NAC ingestion.
NAC treatment for 8 weeks safely replenishes whole blood GSH and T cell GSH in HIV-infected individuals. Thus, NAC offers useful adjunct therapy to increase protection against oxidative stress, improve immune system function and increase detoxification of acetaminophen and other drugs. These findings suggest that NAC therapy could be valuable in other clinical situations in which GSH deficiency or oxidative stress plays a role in disease pathology, e.g. rheumatoid arthritis, Parkinson's disease, hepatitis, liver cirrhosis, septic shock and diabetes.
Glutathione in the brain: disorders of glutathione metabolism The molecular and genetic basis of neurological disease
- Cooper
- Rn Rosenberg
- Sb Prusiner
- S Dimauro
- Rl Barchi
- Lm Kunk
Decreased synthesis of glutathione in patients with AIDS
- B Helbling
- Von Overbeck
- J Lauterburg
- Helbling