Glutathione-dependent Detoxification Research Articles

High-fat high-fructose diet and alpha-ketoglutarate affect mouse behavior that is accompanied by changes in oxidative stress response and energy metabolism in the cerebral cortex

BackgroundHigh caloric diets with high amounts of fats and sweeteners such as fructose may predispose organisms to neurodegenerative diseases. MethodsThis study aimed to examine the effects of a high-fat high-fructose diet (HFFD) on the behavior of mice, energy metabolism, and markers of oxidative stress in murine cerebral cortex. Dietary α-ketoglutarate (AKG) was chosen as a treatment which could modulate the putative effects of HFFD. ResultsWe found that HFFD stimulated locomotion and defecation in mice, whereas an AKG-supplemented diet had a proclivity to promote anxiety-like behavior. HFFD stimulated lipid peroxidation, and in turn, the AKG-supplemented diet led to a higher ratio of reduced to oxidized glutathione, higher activity of NAD(P)H:quinone oxidoreductase 1, and higher mRNA levels of UDP-glucose 6-dehydrogenase and transcription factor EB. Both diets separately, but not in combination, led to a decrease in the activities of glutathione peroxidase, glutathione S-transferase, and phosphofructokinase. All experimental diets resulted in lower levels of transcripts of genes encoding pyruvate dehydrogenase kinase 4 (PDK4), glycine N-methyl transferase, and peroxisome proliferator receptor γ co-activator 1. ConclusionsOur results show that diet supplemented with AKG resulted in effects similar to those of HFFD on the cerebral cortex, but elicited substantial differences between these two diets with respect to behavior, glutathione-dependent detoxification, and processes related to autophagy. General significanceOur study provides insight into the metabolic effects of HFFD alone and in combination with alpha-ketoglutarate in the mouse brain.

Melatonin-Induced Detoxification of Organic Pollutants and Alleviation of Phytotoxicity in Selected Horticultural Crops

Environmental pollution with organic pollutants has increased drastically in recent decades. Despite the importance of minimizing organic pollutant content such as pesticide residue in edible crops, our understanding of induced xenobiotic metabolism in plants is poor. Melatonin is a potent stress-relieving biomolecule, which exerts beneficial effects on xenobiotic metabolism in plants. Exogenous melatonin treatment not only improves photosynthesis, antioxidant defense, and plant growth but also reduces pollutant residue and xenobiotic uptake. The overexpression of melatonin biosynthetic genes enhances organic pollutant metabolism, while the suppression of endogenous melatonin biosynthesis increases organic pollutant residue in horticultural products. Studies have revealed that the glutathione-dependent detoxification pathway plays a critical role in the melatonin-induced enhanced detoxification of xenobiotics. Moreover, a role for RESPIRATORY BURST HOMOLOG (RBOH)-derived reactive oxygen species signaling has been revealed which potentially acts upstream of glutathione-dependent xenobiotic metabolism. Based on the literature, here, we reviewed the effects of organic pollutants on plants and how melatonin aids plants in enduring the effects of organic pollutant-induced stress. We also discussed the potential melatonin signaling mechanism in enhanced pesticide metabolism. Our assessment suggests that melatonin has positive impacts on plant tolerance to organic pollution, which can be used to improve the food safety of edible horticultural crops.

Astrocyte heterogeneity within white matter tracts and a unique subpopulation of optic nerve head astrocytes.

Much of what we know about astrocyte form and function is derived from the study of gray matter protoplasmic astrocytes, whereas white matter fibrous astrocytes remain relatively unexplored. Here, we used the ribotag approach to isolate ribosome-associated mRNA and investigated the transcriptome of uninjured fibrous astrocytes from three regions: unmyelinated optic nerve head, myelinated optic nerve proper, and corpus callosum. Astrocytes from each region were transcriptionally distinct and we identified region-specific astrocyte genes and pathways. Energy metabolism, particularly oxidative phosphorylation and mitochondrial protein translation emerged as key differentiators of astrocyte populations. Optic nerve astrocytes expressed higher levels of neuroinflammatory pathways than corpus callosum astrocytes and we further identified CARTPT as a new marker of optic nerve head astrocytes. These previously uncharacterized transcriptional profiles of white matter astrocyte types reveal their functional diversity and a greater heterogeneity than previously appreciated.

Glutaredoxin like protein (RtGRL1) regulates H2O2 and Na+ accumulation by maintaining the glutathione pool during abiotic stress

Reaumuria trigyna, an endangered recretohalophyte, is a small archaic wild shrub endemic to arid and semiarid plateau regions of Inner Mongolia, China. Based on salt-related transcriptomic data, we isolated a GRX family gene, glutaredoxin like protein (RtGRL1), from R. trigyna that is associated with the removal of active oxygen and regulation of redox status. RtGRL1 encodes a plasma membrane and chloroplast-localized protein induced by salt, cold, drought stress, ABA, and H2O2. In Arabidopsis thaliana, ectopically expressed RtGRL1 positively regulated biomass accumulation, chlorophyll content, germination rate, and primary root length under salt and drought stress. Overexpression of RtGRL1 induced expression of genes related to antioxidant enzymes and proline biosynthesis, thus increasing glutathione biosynthesis, glutathione-dependent detoxification of reactive oxygen species (ROS), and proline content under stress. Changes in RtGRL1 expression consistently affected glutathione/oxidizedglutathione and ascorbate/dehydroascorbate ratios and H2O2 concentrations. Furthermore, RtGRL1 promoted several GSH biosynthesis gene transcripts, decreased leaf Na+ content, and maintained lower Na+/K+ ratios in transgenic A. thaliana compared to wild type plants. These results suggest a critical link between RtGRL1 and ROS modulation, and contribute to a better understanding of the mechanisms governing plant responses to drought and salt stress.

Overexpression of CsHMGB Alleviates Phytotoxicity and Propamocarb Residues in Cucumber.

Cucumber (Cucumis sativus L.) is one of the most economically important fruits of the Cucurbitaceae family, therefore consideration of potential pesticide residues in the fruit in the context of cucumber breeding and production programs is important. Propamocarb (a pesticide commonly used to prevent downy mildew) is widely used in cucumber cultivation, but the molecular mechanism underlying the degradation and metabolism of propamocarb in cucumber is not well understood. We screened a candidate CsHMGB gene (CsaV3-5G28190) for response to propamocarb exposure using transcriptome data. The coding region of CsHMGB was 624 bp in length and encoded the conserved HMB-box region. CsHMGB expression differed significantly between the “D0351” genotype, which accumulated low levels of propamocarb, and the “D9320” genotype, which accumulated high levels of propamocarb. CsHMGB expression was positively correlated with propamocarb levels in the cucumber peel. CsHMGB expression was upregulated in the fruit peels of the “D0351” genotype following exposure to propamocarb stress for 3–120 h, but no difference was observed in expression between propamocarb treatment and control for the “D9320” genotype. For the “D0351” genotype, CsHMGB expression was higher in the fruit peels and leaves than that in female flowers; expression was moderate in the stems and fruit pulps, and weak in male flowers and roots. The CsHMGB protein was targeted to the nucleus in Arabidopsis protoplasts and in the epidermis of Nicotiana benthamiana leaves. We measured MDA, O2–, and H2O2 levels in cucumber plants and found that they were likely to accumulate reactive oxygen species (ROS) in response to propamocarb stress. Analysis of antioxidant enzyme activity (SOD, POD, CAT, APX, GPX, GST, and GR) and the ascorbate-glutathione (AsA-GSH) system showed that the resistance of the plants was reduced and the levels of propamocarb residue was increased in CsHMGB-silenced plants in response to propamocarb stress. Conversely, overexpression of CsHMGB promoted glutathione-dependent detoxification by AsA-GSH system and improved the antioxidant potential, reduced the accumulation of ROS. Ultimately, the metabolism of propamocarb in cucumber was increased via increase in the wax levels and the stomatal conductance.

Donor sex, age and ethnicity impact stored red blood cell antioxidant metabolism through mechanisms in part explained by glucose 6-phosphate dehydrogenase levels and activity.

Red blood cell (RBC) storage in the blood bank promotes the progressive accumulation of metabolic alterations that may ultimately impact the erythrocyte capacity to cope with oxidant stressors. However, the metabolic underpinnings of the capacity of RBC to resist oxidant stress and the potential impact of donor biology on this phenotype are not known. Within the framework of the REDS-III RBC-Omics study, RBC from 8,502 healthy blood donors were stored for 42 days and tested for their propensity to hemolyse following oxidant stress. A subset of extreme hemolysers donated a second unit of blood, which was stored for 10, 23, and 42 days and profiled again for oxidative hemolysis and metabolomics (599 samples). Alterations of RBC energy and redox homeostasis were noted in donors with high oxidative hemolysis. RBC from females, donors over 60 years old, donors of Asian/South Asian race-ethnicity, and RBC stored in additive solution- 3 were each independently characterized by improved antioxidant metabolism compared to, respectively, males, donors under 30 years old, Hispanic and African American race ethnicity donors, and RBC stored in additive solution-1. Merging metabolomics data with results from an independent genome-wide association study on the same cohort, we identified metabolic markers of hemolysis and glucose 6-phosphate dehydrogenasedeficiency, which were associated with extremes in oxidative hemolysis and dysregulation in nicotinamide adenine dinucleotide phosphate and glutathione- dependent detoxification pathways of oxidized lipids. Donor sex, age, ethnicity, additive solution and glucose 6-phosphate dehydrogenase status impact the metabolism of the stored erythrocyte and its susceptibility to hemolysis following oxidative insults.

From methylglyoxal to pyruvate: a genome-wide study for the identification of glyoxalases and D-lactate dehydrogenases in Sorghum bicolor

BackgroundThe glyoxalase pathway is evolutionarily conserved and involved in the glutathione-dependent detoxification of methylglyoxal (MG), a cytotoxic by-product of glycolysis. It acts via two metallo-enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII), to convert MG into D-lactate, which is further metabolized to pyruvate by D-lactate dehydrogenases (D-LDH). Since D-lactate formation occurs solely by the action of glyoxalase enzymes, its metabolism may be considered as the ultimate step of MG detoxification. By maintaining steady state levels of MG and other reactive dicarbonyl compounds, the glyoxalase pathway serves as an important line of defence against glycation and oxidative stress in living organisms. Therefore, considering the general role of glyoxalases in stress adaptation and the ability of Sorghum bicolor to withstand prolonged drought, the sorghum glyoxalase pathway warrants an in-depth investigation with regard to the presence, regulation and distribution of glyoxalase and D-LDH genes.ResultThrough this study, we have identified 15 GLYI and 6 GLYII genes in sorghum. In addition, 4 D-LDH genes were also identified, forming the first ever report on genome-wide identification of any plant D-LDH family. Our in silico analysis indicates homology of putatively active SbGLYI, SbGLYII and SbDLDH proteins to several functionally characterised glyoxalases and D-LDHs from Arabidopsis and rice. Further, these three gene families exhibit development and tissue-specific variations in their expression patterns. Importantly, we could predict the distribution of putatively active SbGLYI, SbGLYII and SbDLDH proteins in at least four different sub-cellular compartments namely, cytoplasm, chloroplast, nucleus and mitochondria. Most of the members of the sorghum glyoxalase and D-LDH gene families are indeed found to be highly stress responsive.ConclusionThis study emphasizes the role of glyoxalases as well as that of D-LDH in the complete detoxification of MG in sorghum. In particular, we propose that D-LDH which metabolizes the specific end product of glyoxalases pathway is essential for complete MG detoxification. By proposing a cellular model for detoxification of MG via glyoxalase pathway in sorghum, we suggest that different sub-cellular organelles are actively involved in MG metabolism in plants.

Dopamine alleviates bisphenol A-induced phytotoxicity by enhancing antioxidant and detoxification potential in cucumber

Bisphenol A (BPA) is an emerging organic pollutant, widely distributed in environment. Plants can uptake and metabolize BPA, but BPA accumulation induces phytotoxicity. In this study, we administered dopamine, a kind of catecholamines with strong antioxidative potential, to unveil its role in cucumber tolerance to BPA stress. The results showed that exposure to BPA (20 mg L−1) for 21 days significantly reduced growth and biomass accumulation in cucumber seedlings as revealed by decreased lengths and dry weights of shoots and roots. While BPA exposure decreased the chlorophyll content, cell viability and root activity, it remarkably increased reactive oxygen species (ROS) accumulation, electrolyte leakage and malondialdehyde (MDA) content, suggesting that BPA induced oxidative stress in cucumber. However, exogenous dopamine application significantly improved the photosynthetic pigment content, root cell viability, growth and biomass accumulation, and decreased the ROS and MDA levels by increasing the activity of antioxidant enzymes under BPA stress. Further analysis revealed that dopamine application significantly increased the glutathione content and the transcripts and activity of glutathione S-transferase under co-administration of dopamine and BPA compared with only BPA treatment. Moreover, dopamine decreased the BPA content in both leaves and roots, suggesting that dopamine promoted BPA metabolism by enhancing the glutathione-dependent detoxification. Our results show that dopamine has a positive role against BPA phytotoxicity and it may reduce the risks-associated with the dietary intake of BPA through consumption of vegetables.

Regulation of boron toxicity responses via glutathione-dependent detoxification pathways at biochemical and molecular levels in Arabidopsisthaliana

The fine-tuned regulation of the Halliwell-Asada cycle (ascorbate-glutathione pathway) in Arabidopsis thaliana under boron (B) toxicity was shown in our previous report. In this study, we investigated the expression levels of some members of the glutathione S-transferase (GST) superfamily, such as phi (GSTF2, GSTF6, GSTF7, and GSTF8), tau (GSTU19), and zeta (GSTZ1) classes in Arabidopsis thaliana that were exposed to 1 mM boric acid (1B) and 3 mM boric acid (3B). Additionally, the expression levels of genes for glutathione (GSH) and phytochelatin biosynthesis as well as miR169 and miR156 were evaluated in Arabidopsis thaliana exposed to 1B and 3B. Moreover, changes in the levels of total GST activity; GSH; and total, protein-bound, and nonprotein thiols were spectrophotometrically determined. GSH levels and nonprotein thiol content did not change significantly following both B-toxicity conditions. Expression levels of GSH1 and GSH2 stayed stable under 1B toxicity; however, GSH1 expression increased significantly under 3B conditions in Arabidopsis thaliana. The expression levels of four genes from phi class members of GST were not dramatically changed under B-toxicity conditions. However, the transcript levels of miR169, ATGSTU19, and ATGSTZ1 were significantly increased after 1B and 3B exposure. These GST genes may have a role in the dramatic increase of total GST activity under toxic B. To the best of our knowledge, this is the first report displaying an integrative view of high-B-induced regulation of GSH-dependent enzymatic machinery at different biological organization levels in Arabidopsis thaliana.

Melatonin promotes metabolism of bisphenol A by enhancing glutathione-dependent detoxification in Solanum lycopersicum L

Bisphenol A (BPA), a widely distributed organic compound, is toxic to animals and plants. Here we show the mechanism of BPA detoxification by melatonin (MEL) in tomato, which is otherwise poorly understood in plants. BPA treatment decreased the quantum yield of photosystem II (Fv/Fm) and increased the membrane lipid peroxidation and reactive oxygen species (ROS) accumulation dose-dependently, whereas exogenous MEL alleviated the BPA effects on Fv/Fm, lipid peroxidation, ROS accumulation and BPA uptake. Furthermore, BPA elevated the glutathione (GSH) content, activities of glutathione S-transferase (GST), and glutathione reductase (GR), and the transcript levels of GSH1, GR1, GST1 and MEL biosynthesis genes (COMT, T5H, and SNAT), whereas BPA + MEL showed even a more profound induction. Silencing GSH1, GR1 and GST1 genes compromised the BPA detoxification potential of tomato plants as revealed by an increased level of ROS, lipid peroxidation and BPA uptake, and a decreased Fv/Fm and GST activity; these changes were alleviated by MEL application. Under in vitro conditions, BPA was glutathionylated by GSH, which was further catalyzed by GST to cysteine and N-acetylcysteine conjugates. These findings suggest a crucial role for MEL in BPA detoxification via GSH and GST, and can be useful to reduce BPA residue for food safety.

Overexpression of GmNAC085 enhances drought tolerance in Arabidopsis by regulating glutathione biosynthesis, redox balance and glutathione-dependent detoxification of reactive oxygen species and methylglyoxal

Glutathione (GSH)-dependent detoxification mechanisms are crucial for protecting plants from the adverse effects of abiotic stresses, including drought. Plant tolerance to drought is regulated by a set of transcription factors that are considered as an integral part of signaling networks in plants. Recently, there have been excellent appreciations on the drought tolerance roles of NAC (NAM, ATAF1/2, and CUC2) transcription factors in plants. In the current study, we sought the mechanisms on how the soybean GmNAC085 functions in the regulation of GSH biosynthesis and GSH-dependent detoxifications of reactive oxygen species (ROS) and methylglyoxal (MG) in order to promote drought tolerance in Arabidopsis thaliana. Our results showed that overexpression of GmNAC085 led to a marked increase in the level of reduced GSH, while decreasing oxidized GSH (GSSG) with a concomitant enhancement of the level of GSH/GSSG ratio in the leaves of Arabidopsis 35S:GmNAC085 transgenic plants. This GSH-based redox balance in transgenic lines was likely due to the increased expression of GSH-biosynthesis genes GSH1 and GSH2, as well as enhanced recycling of GSSG to GSH by glutathione reductase. The significant inductions of the activities of GSH-dependent enzymes glutathione peroxidase and glutathione-S-transferase were also evident in the 35S:GmNAC085 plants, revealing higher protection of transgenic lines against oxidative stress. We also observed that 35S:GmNAC085 plants efficiently removed toxic aldehyde MG by enhancing the activities of glyoxalase (Gly) I and Gly II, which is coincided with the increased transcript levels of Gly I and Gly II genes in the leaves. These results demonstrate a previously unknown function of GmNAC085-guided GSH-dependent detoxifications of ROS and MG, thereby providing a new insight into the mechanisms associated with GmNAC085-mediated drought tolerance. Additionally, expression of GmNAC085 under drought-inducible Arabidopsis RD29A promoter also enhanced drought tolerance without growth retardation, suggesting GmNAC085 as a potential genetic tool for developing plants with improved drought tolerance capacity.

Pathogenetic Justification for the Use of Folates for the Prevention of Congenital Malformations

The frequency and severity of congenital malformations (CMF) do not tend to decline in modern society. CMF for etiologic factors are referred to the pathologies of a multifactorial nature. Among the many causative factors of CMF there is a hereditary predisposition. The objective of the study was to increase the effectiveness of complex individualized prophylaxis of congenital malformations in women with polymorphic alleles of genes of folate cycle enzymes, the second phase of the detoxification system through the use of diagnostic, preventive, and therapeutic measures. Materials and methods. 120 women of reproductive age who live in the city of Odessa and the Odessa region were examined. The alleles of the genes of the folate cycle enzymes of methylenetetrahydrofolate reductase (MTHFR), 5-methyltetrahydrofolate homocysteine methyltransferase reductase (MTRR), glutathione-S-transferase M1 (GSTM1), folate acid, cyanocobalamin were determined. Results of the study and their discussion. The determination of the polymorphic alleles of the genes of the folate cycle enzymes of methylenetetrahydrofolate reductase (MTHFR), 5-methyltetrahydrofolate homocysteine methyltransferase reductase (MTRR), glutathione-S-transferase M1 (GSTM1), low folate, cyanocobalamin indicates the presence of a hereditary predisposition to the emergence of CMF, before and during pregnancy. Polymorphism of genes that control the synthesis of GSTM1 may alter the activity of detoxification enzymes. Expression of genes of GSTM1 enzymes begins in the embryonic period. Glutathione-dependent detoxification plays a key role in disinfecting of DNA peroxides. Mistakes of metabolism, functions of the corresponding enzymes are realized by chromosomal abnormalities and the risk of the occurrence of CMF, which requires the use of antioxidant therapy before and during pregnancy. Conclusions. It was found that a high (about 55%) frequency of the polymorphic alleles of the genes of folate cycle enzymes MTHFR (homozygous – 10.0%, heterozygous – 16.7%), 5 MTRR (homozygous 12.5%, heterozygous – 15.5%) , the second phase of the GSTM1 detoxification system (homozygous – 13.3%, heterozygous – 15.8%), the content below the reference values of folic acid in 26.7%, cyanocobalamin – in 63.4% of observations indicates a hereditary predisposition and may contribute the emergence of CMF, which explains the expediency of timely use of preventive measures including folates, antioxidants.

Cellular mechanisms of action and resistance of Plasmodium falciparum to artemisinin.

The recent reports of high failure rates and decline in in vitro sensitivity of Plasmodium falciparum to artemisinin-based combination therapies (ACTs) suggest the possibility of clinical artemisinin resistance along the Thai-Cambodian and Thai-Myanmar borders. The study investigated cellular mechanisms of action and resistance of P. falciparum to artesunate (stage specific activity, interaction with hemozoin, and anti-oxidant levels) in the two paired P. falciparum isolates (MSF046 and MSF060) collected before treatment with a 3-day artesunate-mefloquine and at the time of recrudescence. In addition, the link of these cellular mechanisms to the polymorphisms of the candidate artemisinin-resistant genes (pfatp6, pfcrt, pfmdr1, pfmrp1, and K13 propeller) was also investigated. Morphological change was observed in both pairs of the primary and recrudesced P. falciparum isolates during 12-48h of exposure to artesunate (at IC90). A marked decrease in parasite viability was found in the recrudesced isolates of both MSF046 and MSD060. The extent of the reduction (% change of baseline) in total glutathione concentrations was significantly lower in recrudesced (32.1 and 1.7%) compared with primary (45.5 and 53.7%) isolates of both MSF046 and MSF060. The extent of reduction of hemozoin content in MSF046 was significantly higher in the recrudesced (76.8%) isolate compared with the primary isolate (99.5%). For MSF060 on the other hand, increase in hemozoin content was found in the recrudesced isolate and the extent of such increase was significantly higher in recrudesced (93.1%) than the primary isolate (87.5%). Polymorphism of K13 (N458Y) together with pfmdr1 copy number correlated well with sensitivity of both isolates to artesunate. Results of this preliminary study suggests possible role of glutathione-dependent detoxification system as well as heme degradation as cellular mechanisms of action and resistance of artemisinins.

Generation and characterization of mouse knockout for glyoxalase 1

Glyoxalase 1 (Glo1) is the first enzyme involved in glutathione-dependent detoxification of methylglyoxal, eventually generating d-lactate by the second enzyme glyoxalase 2 (Glo2). An accumulation of intracellular glyoxal and methylglyoxal leads to protein malfunction and mutation via formation of the advanced glycation end products (AGEs). Studies on mouse behavior suggest that methylglyoxal has anxiolytic properties. In this report, we generated and characterized a mouse knockout for Glo1. The knockout mice were viable without a pronounced phenotypic defect. Increased level of AGEs in Glo1 knockout mice was detected by immunoblotting with anti-MGH1 in liver homogenate, but not in brain. Alterations in behavior were observed in open field, light-dark transition, and tail suspension test. Open field data indicate increased exploration for novel environment and entry/stay in center zone in Glo1 knockout mice. In addition, increased light-dark transition and immobility was observed in the knockout mice. These data indicate that Glo1 knockout reduces anxiety-like behavior, but increases depression-like behavior.

Glutathione-Dependent Detoxification Processes in Astrocytes.

Astrocytes have a pivotal role in brain as partners of neurons in homeostatic and metabolic processes. Astrocytes also protect other types of brain cells against the toxicity of reactive oxygen species and are considered as first line of defence against the toxic potential of xenobiotics. A key component in many of the astrocytic detoxification processes is the tripeptide glutathione (GSH) which serves as electron donor in the GSH peroxidase-catalyzed reduction of peroxides. In addition, GSH is substrate in the detoxification of xenobiotics and endogenous compounds by GSH-S-transferases which generate GSH conjugates that are efficiently exported from the cells by multidrug resistance proteins. Moreover, GSH reacts with the reactive endogenous carbonyls methylglyoxal and formaldehyde to intermediates which are substrates of detoxifying enzymes. In this article we will review the current knowledge on the GSH metabolism of astrocytes with a special emphasis on GSH-dependent detoxification processes.

Degradation of Glutathione S-Conjugates in Physcomitrella patens is Initiated by Cleavage of Glycine

Glutathione-dependent detoxification is a key pathway that allows plants to efficiently remove toxic compounds like heavy metals or electrophilic xenobiotics. Under persistent exposure to toxins plants need to respond to continuous demand with efficient synthesis of glutathione (GSH) and ideally fast and efficient removal of potentially toxic glutathione S-conjugates. With the aim of studying the respective degradation pathway in Physcomitrella patens we initially characterized fluorescence labeling of protonema cultures with GSH-specific xenobiotic monochlorobimane (MCB). Incubation of protonema with 200 μM MCB for 24 h resulted in a steady increase of total bimane label, which was not confined to glutathione S-bimane (GS-B), but predominantly present in γ-glutamylcysteine S-bimane (γ-EC-B) and cysteine S-bimane (Cys-B). Pulse-chase experiments identified γ-EC-B and Cys-B as degradation products of GS-B, suggesting initial cleavage of the C-terminal glycine, followed by cleavage of the γ-glutamyl bond. The amount of GS-B formed, increased linearly at 90 nmol GSH g fw⁻¹ h⁻¹ for 24 h and after ∼1.5 h already surpassed the amount of GSH present in control protonema. This demand-driven biosynthesis of GSH depends on sufficient supply of sulfate in the incubation medium.

γ-Tocopherol abolishes postprandial increases in plasma methylglyoxal following an oral dose of glucose in healthy, college-aged men

Postprandial hyperglycemia contributes to the risk of cardiovascular disease in part by increasing concentrations of the reactive dicarbonyl methylglyoxal (MGO), a byproduct of glucose metabolism. Oxidative stress increases MGO formation from glucose in vitro and decreases its glutathione-dependent detoxification to lactate. We hypothesized that the antioxidant γ-tocopherol, a form of vitamin E, would decrease hyperglycemia-mediated postprandial increases in plasma MGO in healthy, normoglycemic, college-aged men. Participants (n=12 men; 22.3±1.0 years; 29.3±2.4 kg/m2) received an oral dose of glucose (75 g) in the fasted state prior to and following 5-day ingestion of a vitamin E supplement enriched in γ-tocopherol (500 mg/day). γ-Tocopherol supplementation increased (P<.0001) plasma γ-tocopherol from 2.22±0.32 to 7.06±0.71 μmol/l. Baseline MGO concentrations and postprandial hyperglycemic responses were unaffected by γ-tocopherol supplementation (P>.05). Postprandial MGO concentrations increased in the absence of supplemental γ-tocopherol (P<.05), but not following γ-tocopherol supplementation (P>.05). Area under the curve for plasma MGO was significantly (P<.05) smaller with the supplementation of γ-tocopherol than without (area under the curve 0–180 min, −778±1010 vs. 2277±705). Plasma concentrations of γ-carboxyethyl-hydroxychroman, reduced glutathione and markers of total antioxidant capacity increased after supplementation, and these markers and plasma γ-tocopherol were inversely correlated with plasma MGO (r=−0.48 to −0.67, P<.05). These data suggest that short-term supplementation of γ-tocopherol abolishes the oral glucose-mediated increases in postprandial MGO through its direct and indirect antioxidant properties and may reduce hyperglycemia-mediated cardiovascular disease risk.

Cellular Response to Cigarette Smoke and Oxidants: Adapting to Survive

The gaseous and soluble phases of cigarette smoke are sources of oxidants that contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD). Chronic oxidative stress of cigarette smoking induces mucus secretion and inhibits cystic fibrosis transmembrane conductance regulator function. The increased mucus viscosity renders the airways susceptible to bacterial infections, a hallmark of chronic bronchitis. Furthermore, lungs chronically exposed to the toxic mixture of oxidants in cigarette smoke show signs of endoplasmic reticulum stress, unfolded protein response, altered ceramide metabolism, and apoptosis. Fortunately, the respiratory tract has developed effective adaptive cellular mechanisms to limit oxidant damage. Numerous antioxidant enzymes and glutathione-dependent detoxification systems are increased in healthy smokers. The regulation of the antioxidant response is largely dependent on the nuclear factor erythroid 2-related factor-2 (Nrf2) pathway. However, patients with COPD have defective Nrf2 responses. Novel therapies such as 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) to correct defective Nrf2-dependent cellular response may hold promise for patients with COPD.

The different responses of glutathione-dependent detoxification pathway to fungicide chlorothalonil and carbendazim in tomato leaves

Chlorothalonil (CHT) and carbendazim (CAR) are two widely used fungicides in agriculture. Despite their agronomic importance in pest control, little is known about their detoxification in the plant. In this study, we investigated the effects of these fungicides on glutathione (GSH) content, GSH-dependent enzyme activities and gene expression in tomato leaves. Results showed that exposure to CHT resulted in increases in GSH content, activities of glutathione S-transferases (GSTs) and glutathione reductase (GR), as well as the transcriptional levels of glutathione S-transferase genes ( GST1, GST2 and GST3), glutathione synthetase gene ( GSH), glutathione reductase gene ( GR) and glutathione peroxidase gene ( GPX) in tomato leaves, but such increases were not observed in leaves exposed to CAR. In addition, GSTs, GR, peroxidase (POD) activities and most of GSH-dependent gene expression were induced by CHT in a concentration- and time-dependent manner. These results suggest that GSH-dependent pathway plays an important role in the CHT detoxification but not in the CAR detoxification in tomato leaves.

In vivo subcellular targeting analysis validates a novel peroxisome targeting signal type 2 and the peroxisomal localization of two proteins with putative functions in defense in Arabidopsis

Plant peroxisomes are essential organelles that house diverse metabolic activities. To understand the full spectrum of peroxisomal functions, we recently performed a proteomic analysis of Arabidopsis leaf peroxisomes followed by in vivo subcellular targeting validation of some novel proteins. Here, we continue with the targeting analysis and demonstrate that the nonapeptide RVx5HF, which is present near the N terminus of the newly identified peroxisomal protein, HIT3 (histidine triad family protein 3), is a functional peroxisome targeting signal type 2 (PTS2). In addition, we have confirmed the peroxisomal localization of UP6 (unknown protein 6) and GLX1 (glyoxalase 1 homolog), two proteins with possible roles in stress responses and glutathione-dependent detoxification, respectively. These data, together with results from our previous analysis of the peroxisomal proteome, reinforce the notion that peroxisomes are involved in various stress responses and suggest glutathione as a major antioxidant in plant peroxisomes.