Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases

Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione–GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.     

Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases

Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione-GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.     

The role of oxidative processes in the cytotoxicity of substituted 1,4-naphthoquinones in isolated hepatocytes

In order to clarify the role of oxidative processes in cytotoxicity we have studied the metabolism and toxicity of 2-methyl-1,4-naphthoquinone (menadione) and its 2,3 dimethyl (DMNQ) and 2,3 diethyl (DENQ) analogs in isolated rat hepatocytes. The two analogs, unlike menadione, cannot alkylate nucleophiles directly and were considerably less toxic than menadione. This decreased toxicity was consistent with the inability of DMNQ and DENQ to alkylate but we also found them to undergo lower rates of redox cycling in hepatocytes and a higher ratio of two electron as opposed to one electron reduction relative to menadione. Thus, facile analysis of the respective roles of alkylation and oxidation in cytotoxicity was not possible using these compounds. In hepatocytes pretreated with bischloroethyl-nitrosourea (BCNU) to inhibit glutathione reductase, all three naphthoquinones caused a potentiation of reduced glutathione (GSH) removal/oxidized glutathione (GSSG) generation and cytotoxicity relative to that observed in control cells. These data show that inhibition of hepatocyte glutathione reductase by BCNU results in enhanced naphthoquinone-induced oxidative challenge and subsequent cellular toxicity. That DMNQ and DENQ are cytotoxic, albeit at high concentrations, and that this cytotoxicity is potentiated by BCNU pretreatment suggest that oxidative processes alone can be a determinant of cytotoxicity.     

1,4-Naphthoquinones as inducers of oxidative damage and stress signaling in HaCaT human keratinocytes

Selected biological effects of 1,4-naphthoquinone, menadione (2-methyl-1,4-naphthoquinone) and structurally related quinones from natural sources - the 5-hydroxy-naphthoquinones juglone, plumbagin and the 2-hydroxy-naphthoquinones lawsone and lapachol - were studied in human keratinocytes (HaCaT). 1,4-naphthoquinone and menadione as well as juglone and plumbagin were highly cytotoxic, strongly induced reactive oxygen species (ROS) formation and depleted cellular glutathione. Moreover, they induced oxidative DNA base damage and accumulation of DNA strand breaks, as demonstrated in an alkaline DNA unwinding assay. Neither lawsone nor lapachol (up to 100 μM) were active in any of these assays. Cytotoxic and oxidative action was paralleled by stimulation of stress signaling: all tested quinones except lawsone and lapachol strongly induced phosphorylation of the epidermal growth factor receptor (EGFR) and the related ErbB2 receptor tyrosine kinase. EGFR activation by plumbagin, juglone and menadione was attenuated by a superoxide dismutase mimetic, indicating that ROS-related mechanisms contribute to EGFR activation by these naphthoquinones.     

Genome-wide association analysis of oxidative stress resistance in Drosophila melanogaster

Background

Aerobic organisms are susceptible to damage by reactive oxygen species. Oxidative stress resistance is a quantitative trait with population variation attributable to the interplay between genetic and environmental factors. Drosophila melanogaster provides an ideal system to study the genetics of variation for resistance to oxidative stress.

Methods and Findings

We used 167 wild-derived inbred lines of the Drosophila Genetic Reference Panel for a genome-wide association study of acute oxidative stress resistance to two oxidizing agents, paraquat and menadione sodium bisulfite. We found significant genetic variation for both stressors. Single nucleotide polymorphisms (SNPs) associated with variation in oxidative stress resistance were often sex-specific and agent-dependent, with a small subset common for both sexes or treatments. Associated SNPs had moderately large effects, with an inverse relationship between effect size and allele frequency. Linear models with up to 12 SNPs explained 67–79% and 56–66% of the phenotypic variance for resistance to paraquat and menadione sodium bisulfite, respectively. Many genes implicated were novel with no known role in oxidative stress resistance. Bioinformatics analyses revealed a cellular network comprising DNA metabolism and neuronal development, consistent with targets of oxidative stress-inducing agents. We confirmed associations of seven candidate genes associated with natural variation in oxidative stress resistance through mutational analysis.

Conclusions

We identified novel candidate genes associated with variation in resistance to oxidative stress that have context-dependent effects. These results form the basis for future translational studies to identify oxidative stress susceptibility/resistance genes that are evolutionary conserved and might play a role in human disease.     

Diesel Exhaust Particles Induce Cysteine Oxidation and S-Glutathionylation in House Dust Mite Induced Murine Asthma

Background

Diesel exhaust particle (DEP) exposure enhances allergic inflammation and has been linked to the incidence of asthma. Oxidative stress on the thiol molecules cysteine (Cys) and glutathione (GSH) can promote inflammatory host responses. The effect of DEP on the thiol oxidation/reduction (redox) state in the asthmatic lung is unknown.

Objective

To determine if DEP exposure alters the Cys or GSH redox state in the asthmatic airway.

Methods

Bronchoalveolar lavage fluid was obtained from a house dust mite (HDM) induced murine asthma model exposed to DEP. GSH, glutathione disulfide (GSSG), Cys, cystine (CySS), and s-glutathionylated cysteine (CySSG) were determined by high pressure liquid chromatography.

Results

DEP co-administered with HDM, but not DEP or HDM alone, decreased total Cys, increased CySS, and increased CySSG without significantly altering GSH or GSSG.

Conclusions

DEP exposure promotes oxidation and S-glutathionylation of cysteine amino acids in the asthmatic airway, suggesting a novel mechanism by which DEP may enhance allergic inflammatory responses.     

Interaction of menadione (2-methyl-1,4-naphthoquinone) with glutathione

The interaction of menadione with reduced glutathione (GSH) led to a removal of menadione and formation of menadione-GSH conjugate and glutathione disulfide (GSSG). The changes in thiol level were essentially biphasic with an initial rapid decrease in GSH and appearance of GSSG (less than 1 min) followed by secondary less pronounced changes. The interaction of menadione and GSH caused an oxygen uptake and both superoxide anion radical and hydrogen peroxide were produced during the reaction, the amount dependent on the GSH/menadione ratio. Catalase did not protect against the initial decrease in GSH level but markedly inhibited the secondary changes while superoxide dismutase had little effect. These results suggest that the initial changes in thiol level are the result in part of a redox reaction between menadione and GSH as well as conjugate formation, whilst the secondary changes reflect conjugate formation and the activity of other oxidants such as hydrogen peroxide. The potential biological significance of this reaction was investigated using hepatocytes depleted of reduced pyridine nucleotides and thus not able to perform enzyme-catalyzed reduction of menadione. In these cells menadione induced GSSG formation at a rate similar to that observed in control cells. This suggests that quinone-induced oxidative challenge caused by the chemical interactions of a quinone and glutathione may have biological relevance.     

Inhibition of AMPK expression in skeletal muscle by systemic inflammation in COPD rats

Background

Analysis of exhaled breath condensates (EBC) is a non-invasive technique to evaluate biomarkers such as antioxidants in the pediatric population, but limited data exists of its use in intubated patients, particularly newborns. Currently, tracheal aspirate (TA) serves as the gold standard collection modality in critically ill newborns, but this method remains invasive. We tested the hypothesis that glutathione status would positively correlate between EBC and TA collections in intubated newborns in the Newborn Intensive Care Unit (NICU). We also hypothesized that these measurements would be associated with alveolar macrophage (AM) glutathione status in the newborn lung.

Methods

Reduced glutathione (rGSH), glutathione disulfide (GSSG), and total GSH (rGSH + (2 X GSSG)) were measured in sequential EBC and TA samples from 26 intubated newborns via high performance liquid chromatography (HPLC). Additionally, AM glutathione was evaluated via immunofluorescence. Pearson’s correlation coefficient and associated 95% confidence intervals were used to quantify the associations between raw and urea-corrected concentrations in EBC and TA samples and AM staining. Statistical significance was defined as p ≤ 0.05 using two-tailed tests. The sample size was projected to allow for a correlation coefficient of 0.5, with 0.8 power and alpha of 0.05.

Results

EBC was obtainable from intubated newborns without adverse clinical events. EBC samples demonstrated moderate to strong positive correlations with TA samples in terms of rGSH, GSSG and total GSH. Positive correlations between the two sampling sites were observed in both raw and urea-corrected concentrations of rGSH, GSSG and total GSH. AM glutathione staining moderately correlated with GSSG and total GSH status in both the TA and EBC.

Conclusions

GSH status in EBC samples of intubated newborns significantly correlated with the GSH status of the TA sample and was reflective of cellular GSH status in this cohort of neonatal patients. Non-invasive EBC sampling of intubated newborns holds promise for monitoring antioxidant status such as GSH in the premature lung. Further studies are necessary to evaluate the potential relationships between EBC biomarkers in the intubated premature newborn and respiratory morbidities.     

Changes in Non-Enzymatic Antioxidants in the Blood Following Anaerobic Exercise in Men and Women

Purpose

The aim of this study was to compare changes in total oxidative status (TOS), total antioxidative capacity (TAC) and the concentration of VitA, VitE, VitC, uric acid (UA), reduced (GSH) and oxidized glutathione (GSSG) in blood within 24 hours following anaerobic exercise (AnEx) among men and women.

Methods

10 women and 10 men performed a 20-second bicycle sprint (AnEx). Concentrations of oxidative stress indicators were measured before AnEx and 3, 15 and 30 minutes and 1 hour afterwards. UA, GSH and GSSH were also measured 24 hours after AnEx. Lactate and H⁺ concentrations were measured before and 3 minutes after AnEx.

Results

The increase in lactate and H⁺ concentrations following AnEx was similar in both sexes. Changes in the concentrations of all oxidative stress indicators were significant and did not differ between men and women. In both sexes, TOS, TAC, TOS/TAC and VitA and VitE concentrations were the highest 3 minutes, VitC concentration was the highest 30 minutes, and UA concentration was the highest 1 hour after AnEx. GSH concentration was significantly lower than the initial concentration from 15 minutes to 24 hour after AnEx. GSSG concentration was significantly higher, while the GSH/GSSG ratio was significantly lower than the initial values 1 hour and 24 hour after AnEx.

Conclusions

With similar changes in lactate and H⁺ concentrations, AnEx induces the same changes in TAC, TOS, TOS/TAC and non-enzymatic antioxidants of low molecular weight in men and women. Oxidative stress lasted at least 24 hours after AnEx.     

即时浸酸显著降低滞育家蚕卵的还原型与氧化型谷胱甘肽比值

即时浸酸在阻止家蚕Bombyx mori卵滞育发动的同时, 显著提高了家蚕卵H₂O₂含量。还原型谷胱甘肽（reduced glutathione, GSH）与氧化型谷胱甘肽（oxidized glutathione, GSSG）的比值是一种氧化胁迫状态的动态指标。为了调查即时浸酸是否造成滞育家蚕卵氧化胁迫, 本研究利用分光光度法分别测定了滞育家蚕卵和5 min即时浸酸滞育家蚕卵中GSH和GSSG含量以及谷胱甘肽转移酶（glutathione-S-transferase, GST）活性。结果表明: 处理后24 h, 即时浸酸处理家蚕卵的总谷胱甘肽（GSH+2GSSG）含量、 GSH含量、 GSSG含量、 GSH/GSSG比值和GST活性分别相当于同期滞育家蚕卵的204%, 78%, 550%, 14%和97%。据此推测, 即时浸酸在阻止滞育发动的同时, 可能通过促进GSH氧化为GSSG, 而显著降低了GSH/GSSG比值, 使家蚕卵处于过氧化状态。     

Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells

AimsMenadione, a redox-cycling quinone known to cause oxidative stress, binds to reduced glutathione (GSH) to form glutathione S-conjugate. Glutathione S-conjugates efflux is often mediated by multidrug-resistance-associated protein (MRP). We investigated the effect of a transporter inhibitor, MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), on menadione-induced oxidative stress in bovine aortic endothelial cells (BAECs).Main methodsBAECs were treated with menadione and MK571, and cell viability was measured. Modulation of intracellular GSH levels was performed with buthionine sulfoximine and GSH ethyl ester treatments. Intracellular superoxide was estimated by dihydroethidium oxidation using fluorescence microscopy or flow cytometry. Expression of MRP was determined by flow cytometry using phycoerythrin-conjugated anti-MRP monoclonal antibody.Key findingsIntracellular GSH depletion by buthionine sulfoximine promoted the loss of viability of BAECs exposed to menadione. Exogenous GSH, which does not permeate the cell membrane, or GSH ethyl ester protected BAECs against the loss of viability induced by menadione. The results suggest that GSH binds to menadione outside the cells as well as inside. Pretreatment of BAECs with MK571 dramatically increased intracellular levels of superoxide generated from menadione, indicating that menadione may accumulate in the intracellular milieu. Finally, we found that MK571 aggravated menadione-induced toxicity in BAECs and that MRP levels were increased in menadione-treated cells.SignificanceWe conclude that MRP plays a vital role in protecting BAECs against menadione-induced oxidative stress, presumably due to its ability to transport glutathione S-conjugate.     

Dimethylthiourea protects against chlorine induced changes in airway function in a murine model of irritant induced asthma

Background

Exposure to chlorine (Cl₂) causes airway injury, characterized by oxidative damage, an influx of inflammatory cells and airway hyperresponsiveness. We hypothesized that Cl₂-induced airway injury may be attenuated by antioxidant treatment, even after the initial injury.

Methods

Balb/C mice were exposed to Cl₂ gas (100 ppm) for 5 mins, an exposure that was established to alter airway function with minimal histological disruption of the epithelium. Twenty-four hours after exposure to Cl₂, airway responsiveness to aerosolized methacholine (MCh) was measured. Bronchoalveolar lavage (BAL) was performed to determine inflammatory cell profiles, total protein, and glutathione levels. Dimethylthiourea (DMTU;100 mg/kg) was administered one hour before or one hour following Cl₂ exposure.

Results

Mice exposed to Cl₂ had airway hyperresponsiveness to MCh compared to control animals pre-treated and post-treated with DMTU. Total cell counts in BAL fluid were elevated by Cl₂ exposure and were not affected by DMTU treatment. However, DMTU-treated mice had lower protein levels in the BAL than the Cl₂-only treated animals. 4-Hydroxynonenal analysis showed that DMTU given pre- or post-Cl₂ prevented lipid peroxidation in the lung. Following Cl₂ exposure glutathione (GSH) was elevated immediately following exposure both in BAL cells and in fluid and this change was prevented by DMTU. GSSG was depleted in Cl₂ exposed mice at later time points. However, the GSH/GSSG ratio remained high in chlorine exposed mice, an effect attenuated by DMTU.

Conclusion

Our data show that the anti-oxidant DMTU is effective in attenuating Cl₂ induced increase in airway responsiveness, inflammation and biomarkers of oxidative stress.     

Is Nitric Oxide Decrease Observed with Naphthoquinones in LPS Stimulated RAW 264.7 Macrophages a Beneficial Property?

The search of new anti-inflammatory drugs has been a current preoccupation, due to the need of effective drugs, with less adverse reactions than those used nowadays. Several naphthoquinones (plumbagin, naphthazarin, juglone, menadione, diosquinone and 1,4-naphthoquinone), plus p-hydroquinone and p-benzoquinone were evaluated for their ability to cause a reduction of nitric oxide (NO) production, when RAW 264.7 macrophages were stimulated with lipopolysaccharide (LPS). Dexamethasone was used as positive control. Among the tested compounds, diosquinone was the only one that caused a NO reduction with statistical importance and without cytotoxicity: an IC(25) of 1.09±0.24 μM was found, with 38.25±6.50% (p<0.001) NO reduction at 1.5 μM. In order to elucidate if this NO decrease resulted from the interference of diosquinone with cellular defence mechanisms against LPS or to its conversion into peroxynitrite, by reaction with superoxide radical formed by naphthoquinones redox cycling, 3-nitrotyrosine and superoxide determination was also performed. None of these parameters showed significant changes relative to control. Furthermore, diosquinone caused a decrease in the pro-inflammatory cytokines: tumour necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6). Therefore, according to the results obtained, diosquinone, studied for its anti-inflammatory potential for the first time herein, has beneficial effects in inflammation control. This study enlightens the mechanisms of action of naphthoquinones in inflammatory models, by checking for the first time the contribution of oxidative stress generated by naphthoquinones to NO reduction.     

Impaired Glutathione Redox System Paradoxically Suppresses Angiotensin II-Induced Vascular Remodeling

Background

Angiotensin II (AII) plays a central role in vascular remodeling via oxidative stress. However, the interaction between AII and reduced glutathione (GSH) redox status in cardiovascular remodeling remains unknown.

Methods

In vivo: The cuff-induced vascular injury model was applied to Sprague Dawley rats. Then we administered saline or a GSH inhibitor, buthionine sulfoximine (BSO, 30 mmol/L in drinking water) for a week, subsequently administered 4 more weeks by osmotic pump with saline or AII (200 ng/kg/minute) to the rats. In vitro: Incorporation of bromodeoxyuridine (BrdU) was measured to determine DNA synthesis in cultured rat vascular smooth muscle cells (VSMCs).

Results

BSO reduced whole blood GSH levels. Systolic blood pressure was increased up to 215±4 mmHg by AII at 4 weeks (p<0.01), which was not affected by BSO. Superoxide production in vascular wall was increased by AII and BSO alone, and was markedly enhanced by AII+BSO. The left ventricular weight to body weight ratio was significantly increased in AII and AII+BSO as compared to controls (2.52±0.08, 2.50±0.09 and 2.10±0.07 mg/g respectively, p<0.05). Surprisingly, the co-treatment of BSO totally abolished these morphological changes. Although the vascular circumferential wall stress was well compensated in AII, significantly increased in AII+BSO. The anti-single-stranded DNA staining revealed increasing apoptotic cells in the neointima of injured arteries in BSO groups. BrdU incorporation in cultured VSMCs with AII was increased dose-dependently. Furthermore it was totally abolished by BSO and was reversed by GSH monoethyl ester.

Conclusions

We demonstrated that a vast oxidative stress in impaired GSH redox system totally abolished AII-induced vascular, not cardiac remodeling via enhancement of apoptosis in the neointima and suppression of cell growth in the media. The drastic suppression of remodeling may result in fragile vasculature intolerable to mechanical stress by AII.     

Inhibition of biliary taurocholate excretion during menadione metabolism in perfused rat liver

In perfused rat liver menadione elicits substantial oxidation in both the NADPH and GSH redox systems. Biliary excretion of GSSG is increased several-fold. Menadione derivatives appear in the bile predominantly as the menadione-S-glutathione conjugate, thiodione (60%), or as conjugates derived therefrom (17%). About 10% appear as menadione glucuronides. The excretion of taurocholate into bile is strongly inhibited upon menadione infusion. The inhibition of taurocholate excretion is small in livers with a low content of Se-GSH-peroxidase and in glutathione-depleted livers. In these livers intracellular GSSG and biliary GSSG release remain at low values, although menadione still imposes oxidative stress as indicated by an oxidation of intracellular NADPH. Under anoxic conditions menadione has little influence on both the NADPH and GSH redox systems and also on biliary taurocholate excretion. The amount of thiodione released into bile is similar to that found under normoxia, whereas the amount of glucuronidated products almost doubled. We conclude (a) that intracellular formation of GSSG by menadione occurs via the generation of hydrogen peroxide; (b) that the inhibition of biliary taurocholate excretion by menadione is related to the increased formation of glutathione disulfide; and (c) that menadione derivatives show little, if any, contribution to the inhibition of taurocholate excretion.     

A tale of two toxicities: malformed selenoproteins and oxidative stress both contribute to selenium stress in plants

Background

Despite selenium''s toxicity in plants at higher levels, crops supply most of the essential dietary selenium in humans. In plants, inorganic selenium can be assimilated into selenocysteine, which can replace cysteine in proteins. Selenium toxicity in plants has been attributed to the formation of non-specific selenoproteins. However, this paradigm can be challenged now that there is increasingly abundant evidence suggesting that selenium-induced oxidative stress also contributes to toxicity in plants.

Scope

This Botanical Briefing summarizes the evidence indicating that selenium toxicity in plants is attributable to both the accumulation of non-specific selenoproteins and selenium-induced oxidative stress. Evidence is also presented to substantiate the claim that inadvertent selenocysteine replacement probably impairs or misfolds proteins, which supports the malformed selenoprotein hypothesis. The possible physiological ramifications of selenoproteins and selenium-induced oxidative stress are discussed.

Conclusions

Malformed selenoproteins and oxidative stress are two distinct types of stress that drive selenium toxicity in plants and could impact cellular processes in plants that have yet to be thoroughly explored. Although challenging, deciphering whether the extent of selenium toxicity in plants is imparted by selenoproteins or oxidative stress could be helpful in the development of crops with fortified levels of selenium.