Involvement of Nitric Oxide and Melatonin Enhances Cadmium Resistance of Tomato Seedlings through Regulation of the Ascorbate-Glutathione Cycle and ROS Metabolism.

Melatonin (MT) and nitric oxide (NO) act as signaling molecules that can enhance cadmium (Cd) stress resistance in plants. However, little information is available about the relationship between MT and NO during seedling growth under Cd stress. We hypothesize that NO may be involved in how MT responds to Cd stress during seedling growth. The aim of this study is to evaluate the relationship and mechanism of response. The results indicate that different concentrations of Cd inhibit the growth of tomato seedlings. Exogenous MT or NO promotes seedling growth under Cd stress, with a maximal biological response at 100 µM MT or NO. The promotive effects of MT-induced seedling growth under Cd stress are suppressed by NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO), suggesting that NO may be involved in MT-induced seedling growth under Cd stress. MT or NO decreases the content of hydrogen peroxide (H2O2), malonaldehyde (MDA), dehydroascorbic acid (DHA), and oxidized glutathione (GSSG); improves the content of ascorbic acid (AsA) and glutathione (GSH) and the ratios of AsA/DHA and GSH/GSSG; and enhances the activities of glutathione reductase (GR), monodehydroascorbic acid reductase (MDHAR), dehydroascorbic acid reductase (DHAR), ascorbic acid oxidase (AAO), and ascorbate peroxidase (APX) to alleviate oxidative damage. Moreover, the expression of genes associated with the ascorbate-glutathione (AsA-GSH) cycle and reactive oxygen species (ROS) are up-regulated by MT or NO under Cd conditions, including AAO, AAOH, APX1, APX6, DHAR1, DHAR2, MDHAR, and GR. However, NO scavenger cPTIO reverses the positive effects regulated by MT. The results indicate that MT-mediated NO enhances Cd tolerance by regulating AsA-GSH cycle and ROS metabolism.

Biochemical and structural characterization of a robust and thermostable ascorbate recycling monodehydroascorbate reductase (MDHAR) from stress adapted pearl millet.

Ascorbate (AsA) is a crucial antioxidant in plants, and its recycling is necessary for protecting cells from oxidative damage and imparting stress tolerance. The monodehydroascorbate reductase (MDHAR) enzyme of the ascorbate-glutathione pathway plays a vital role in recycling AsA from monodehydroascorbate (MDHA) radical. Pennisetum glaucum (Pg), also known as pearl millet, is known to be more tolerant to abiotic stress than other food crops, such as rice. However, the contribution of MDHAR from this sessile plant to its unique stress tolerance mechanism is not well understood. In this study, we isolated a gene encoding the MDHAR enzyme from heat stress-adapted pearl millet and characterized it using enzyme kinetics, thermal stability assays, and crystal structure determination. Our results indicate that PgMDHAR is a more robust enzyme than its rice counterpart (Oryza sativa; Os). We solved the crystal structure of PgMDHAR at 1.8 Å and found that the enzyme has a more compact structure and greater stability than OsMDHAR. Using hybrid quantum mechanics and molecular mechanics calculations, we demonstrate that the structure of PgMDHAR contributes to increased stability towards bound FAD. Overall, the higher structural stability and affinity for NADH demonstrated by PgMDHAR are expected to impart improved stress tolerance. Our findings suggest that transgenic food crops expressing MDHAR from stress-adapted pearl millet may exhibit better tolerance to oxidative stress in the unpredictable climatic conditions prevalent today.

Identification of Structural Determinants of the Transport of the Dehydroascorbic Acid Mediated by Glucose Transport GLUT1.

GLUT1 is a facilitative glucose transporter that can transport oxidized vitamin C (i.e., dehydroascorbic acid) and complements the action of reduced vitamin C transporters. To identify the residues involved in human GLUT1's transport of dehydroascorbic acid, we performed docking studies in the 5 Å grid of the glucose-binding cavity of GLUT1. The interactions of the bicyclic hemiacetal form of dehydroascorbic acid with GLUT1 through hydrogen bonds with the -OH group of C3 and C5 were less favorable than the interactions with the sugars transported by GLUT1. The eight most relevant residues in such interactions (i.e., F26, Q161, I164, Q282, Y292, and W412) were mutated to alanine to perform functional studies for dehydroascorbic acid and the glucose analog, 2-deoxiglucose, in Xenopus laevis oocytes. All the mutants decreased the uptake of both substrates to less than 50%. The partial effect of the N317A mutant in transporting dehydroascorbic acid was associated with a 30% decrease in the Vmax compared to the wildtype GLUT1. The results show that both substrates share the eight residues studied in GLUT1, albeit with a differential contribution of N317. Our work, combining docking with functional studies, marks the first to identify structural determinants of oxidized vitamin C's transport via GLUT1.

5-Aminolevulinic acid promotes low-light tolerance by regulating chloroplast ultrastructure, photosynthesis, and antioxidant capacity in tall fescue.

The vital signaling molecule 5-Aminolevulinic acid (ALA) plays critical roles in signal transduction and biological modulation under abiotic stresses. In this study, we explored the effects of exogenous ALA on low-light (LL) stress-induced photosynthesis and antioxidant system damage in tall fescue (Festuca arundinacea Schreb.) seedlings. LL stress decreased morphological index values and chlorophyll contents, while also reduced net photosynthetic rate (Pn) and the maximum quantum yield of photosystem II photochemistry (Fv/Fm). Notably, these restrictions were substantially alleviated by exogenous ALA. Moreover, the contents of chlorophyll and its synthetic precursors were significantly increased after ALA treatment. Meanwhile, ALA observably enhanced expression level of FaCHLG, FaHEMA, FaPOR, and FaCAO, which encode the chlorophyll precursors biosynthesis enzymes. Exogenous ALA repaired the damage to the chloroplast ultrastructure caused by LL stress and promoted the formation of ordered thylakoids and grana lamella. ALA also improved Rubisco activity and expression level of the photosynthetic enzyme genes FaRuBP, FaPRK, and FaGADPH. Additionally, application of exogenous ALA decreased relative electrolytic leakage and the accumulation of malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide radicals (O2∙-), and increased the gene expression levels and activity of antioxidant enzymes. The ratios of ascorbic acid (AsA) to dehydroascorbic acid (DHA) and reduced glutathione (GSH) to oxidized glutathione (GSSG) were also increased significantly by application of ALA. Furthermore, all responses could be reversed by treatment with levulinic acid (LA). Thus, these results indicated that ALA protects tall fescue from LL stress through scavenging ROS, improving photosynthetic enzyme activity levels, increasing photosynthetic pigments contents, repairing chloroplast damage, and enhancing the photosynthesis rate.

Glioblastoma Invasiveness and Collagen Secretion Are Enhanced by Vitamin C.

Aims: Glioblastoma (GB) is one of the most aggressive brain tumors. These tumors modify their metabolism, increasing the expression of glucose transporters, GLUTs, which incorporate glucose and the oxidized form of vitamin C, dehydroascorbic acid (DHA). We hypothesized that GB cells preferentially take up DHA, which is intracellularly reduced and compartmentalized into the endoplasmic reticulum (ER), promoting collagen biosynthesis and an aggressive phenotype. Results: Our results showed that GB cells take up DHA using GLUT1, while GLUT3 and sodium-dependent vitamin C transporter 2 (SVCT2) are preferably intracellular. Using a baculoviral system and reticulum-enriched extracts, we determined that SVCT2 is mainly located in the ER and corresponds to a short isoform. Ascorbic acid (AA) was compartmentalized, stimulating collagen IV secretion and increasing in vitro and in situ cell migration. Finally, orthotopic xenografts induced in immunocompetent guinea pigs showed that vitamin C deficiency retained collagen, reduced blood vessel invasion, and affected glomeruloid vasculature formation, all pathological conditions associated with malignancy. Innovation and Conclusion: We propose a functional role for vitamin C in GB development and progression. Vitamin C is incorporated into the ER of GB cells, where it favors the synthesis of collagen, thus impacting tumor development. Collagen secreted by tumor cells favors the formation of the glomeruloid vasculature and enhances perivascular invasion. Antioxid. Redox Signal. 37, 538-559.

Transport of Vitamin C in Cancer.

Significance: Vitamin C is a powerful antioxidant that has an intricate relationship with cancer and has been studied for more than 60 years. However, the specific mechanisms that allow malignant cells to uptake, metabolize, and compartmentalize vitamin C remain unclear. In normal human cells, two different transporter systems are responsible for its acquisition: glucose transporters (GLUTs) transport the oxidized form of vitamin C (dehydroascorbic acid) and sodium-coupled ascorbic acid transporters (SVCTs) transport the reduced form (ascorbic acid [AA]). In this study, we review the mechanisms described for vitamin C uptake and metabolization in cancer. Recent Advances: Several studies performed recently in vivo and in vitro have provided the scientific community a better understanding of the differential capacities of cancer cells to acquire vitamin C: tumors from different origins do not express SVCTs in the plasma membrane and are only able to acquire vitamin C in its oxidized form. Interestingly, cancer cells differentially express a mitochondrial form of SVCT2. Critical Issues: Why tumors have reduced AA uptake capacity at the plasma membrane, but develop the capacity of AA transport within mitochondria, remains a mystery. However, it shows that understanding vitamin C physiology in tumor survival might be key to decipher the controversies in its relationship with cancer. Future Directions: A comprehensive analysis of the mechanisms by which cancer cells acquire, compartmentalize, and use vitamin C will allow the design of new therapeutic approaches in human cancer. Antioxid. Redox Signal. 35, 61-74.

Vitamin C Transporters and Their Implications in Carcinogenesis.

Vitamin C is implicated in various bodily functions due to its unique properties in redox homeostasis. Moreover, vitamin C also plays a great role in restoring the activity of 2-oxoglutarate and Fe2+ dependent dioxygenases (2-OGDD), which are involved in active DNA demethylation (TET proteins), the demethylation of histones, and hypoxia processes. Therefore, vitamin C may be engaged in the regulation of gene expression or in a hypoxic state. Hence, vitamin C has acquired great interest for its plausible effects on cancer treatment. Since its conceptualization, the role of vitamin C in cancer therapy has been a controversial and disputed issue. Vitamin C is transferred to the cells with sodium dependent transporters (SVCTs) and glucose transporters (GLUT). However, it is unknown whether the impaired function of these transporters may lead to carcinogenesis and tumor progression. Notably, previous studies have identified SVCTs' polymorphisms or their altered expression in some types of cancer. This review discusses the potential effects of vitamin C and the impaired SVCT function in cancers. The variations in vitamin C transporter genes may regulate the active transport of vitamin C, and therefore have an impact on cancer risk, but further studies are needed to thoroughly elucidate their involvement in cancer biology.

Crop Enhancement of Cucumber Plants under Heat Stress by Shungite Carbon.

Heat stress negatively impacts plant growth and yield. The effects of carbon materials on plants in response to abiotic stress and antioxidant activity are poorly understood. In this study, we propose a new method for improving heat tolerance in cucumber (Cucumis sativus L.) using a natural carbon material, shungite, which can be easily mixed into any soil. We analyzed the phenotype and physiological changes in cucumber plants maintained at 35 °C or 40 °C for 1 week. Our results show that shungite-treated cucumber plants had a healthier phenotype, exhibiting dark green leaves, compared to the plants in the control soil group. Furthermore, in the shungite-treated plants, the monodehydroascorbate content (a marker of oxidative damage) of the leaf was 34% lower than that in the control group. In addition, scavengers against reactive oxygen species, such as superoxide dismutase, catalase, and peroxidase were significantly upregulated. These results indicate that the successive pre-treatment of soil with a low-cost natural carbon material can improve the tolerance of cucumber plants to heat stress, as well as improve the corresponding antioxidant activity.

Investigating protein thiol chemistry associated with dehydroascorbate, homocysteine and glutathione using mass spectrometry.

RATIONALE: Oxidative stress is an imbalance between reactive free radical oxygen species and antioxidant defenses. Its consequences can lead to numerous pathologies. Regulating oxidative stress is the complex interplay between antioxidant recycling and thiol-containing regulatory proteins. Understanding these regulatory mechanisms is important for preventing onset of oxidative stress. The aim of this study was to investigae S-thiol protein chemistry associated with oxidized vitamin C (dehydroascorbate, DHA), homocysteine (HcySH) and glutathione (GSH) using mass spectrometry. METHODS: Glutaredoxin-1 (Grx-1) was incubated with DHA, with and without GSH and HcySH. Disulfide formation was followed by electrospray ionization mass spectrometry (ESI-MS) of intact proteins and by LC/ESI-MS/MS of peptides from protein tryptic digestions. The mechanism of DHA-mediated S-thiolation was investigated using two synthetic peptides: AcFHACAAK and AcFHACE. Three proteins, i.e. human hemoglobin (HHb), recombinant peroxiredoxin 2 (Prdx2) and Grx-1, were S-homocysteinylated followed by S-transthiolyation with GSH and investigated by ESI-MS and ESI-MS/MS. RESULTS: ESI-MS analysis reveals that DHA mediates disulfide formation and S-thiolation by HcySH as well as GSH of Grx-1. LC/ESI-MS/MS analysis allows identification of Grx-1 S-thiolated cysteine adducts. The mechanism by which DHA mediates S-thiolation of heptapeptide AcFHACAAK is shown to be via initial formation of a thiohemiketal adduct. In addition, ESI-MS of intact proteins shows that GSH can S-transthiolate S-homocysteinylated Grx-1_ HHb and Prdx2. The GS-S-protein adducts over time dominate the ESI-MS spectrum profile. CONCLUSIONS: Mass spectrometry is a unique analytical technique for probing complex reaction mechanisms associated with oxidative stress. Using model proteins, ESI-MS reveals the mechanism of DHA-facilitated S-thiolation, which consists of thiohemiketal formation, disulfide formation or S-thiolation. Furthermore, protein S-thiolation by HcySH can be reversed by reversible GSH thiol exchange. The use of mass spectrometry with in vitro models of protein S-thiolation in oxidative stress may provide significant insight into possible mechanisms of action occurring in vivo.

Resveratrol potentiates intracellular ascorbic acid enrichment through dehydroascorbic acid transport and/or its intracellular reduction in HaCaT cells.

L-Ascorbic acid (AsA), a reduced vitamin C (VC), is an important antioxidant, and the internal accumulation and maintenance of AsA are thought to play a significant role in various physiological activities in humans. We focused on resveratrol (RSV), a natural polyphenolic compound, as a candidate for an AsA transport modulator and investigated whether RSV can affect the intracellular VC accumulation after either AsA or dehydroascorbic acid (DHA) addition in HaCaT keratinocytes. Our results demonstrate that RSV treatment could significantly enhance intracellular VC levels after either AsA or DHA supplementation, and intracellular VC accumulated mainly as AsA. Our results also indicate that most of the intracellular transported DHA was reduced to AsA and accumulated after uptake into cells. In addition, RSV could induce several AsA or DHA transport-related and intracellular DHA reduction-related genes including SVCT2, GLUT3, TXNRD2, and TXNRD3, necessary for AsA transport, DHA transport, and DHA reduction/regeneration, respectively. On the other hand, the both protein expression levels and the localizations of sodium-dependent vitamin C transporters 2 (SVCT2) and glucose transporter 3(GLUT3) were scarcely affected by RSV treatment. Furthermore, RSV-induced enrichment of intracellular AsA levels was completely suppressed by a GLUT inhibitor cytochalasin B. These results suggest that RSV can potentiate intracellular AsA accumulation via activation of the DHA transport and subsequent intracellular reduction from DHA to AsA. Thus, RSV might be useful for maintaining substantial AsA accumulation in the skin keratinocytes.

Longitudinal Metabolome-Wide Signals Prior to the Appearance of a First Islet Autoantibody in Children Participating in the TEDDY Study.

Children at increased genetic risk for type 1 diabetes (T1D) after environmental exposures may develop pancreatic islet autoantibodies (IA) at a very young age. Metabolic profile changes over time may imply responses to exposures and signal development of the first IA. Our present research in The Environmental Determinants of Diabetes in the Young (TEDDY) study aimed to identify metabolome-wide signals preceding the first IA against GAD (GADA-first) or against insulin (IAA-first). We profiled metabolomes by mass spectrometry from children's plasma at 3-month intervals after birth until appearance of the first IA. A trajectory analysis discovered each first IA preceded by reduced amino acid proline and branched-chain amino acids (BCAAs), respectively. With independent time point analysis following birth, we discovered dehydroascorbic acid (DHAA) contributing to the risk of each first IA, and γ-aminobutyric acid (GABAs) associated with the first autoantibody against insulin (IAA-first). Methionine and alanine, compounds produced in BCAA metabolism and fatty acids, also preceded IA at different time points. Unsaturated triglycerides and phosphatidylethanolamines decreased in abundance before appearance of either autoantibody. Our findings suggest that IAA-first and GADA-first are heralded by different patterns of DHAA, GABA, multiple amino acids, and fatty acids, which may be important to primary prevention of T1D.

Characterisation of the non-oxidative degradation pathway of dehydroascorbic acid in slightly acidic aqueous solution.

Although l-ascorbate (vitamin C) is an important biological antioxidant, its degradation pathways in vivo remain incompletely characterised. Ascorbate is oxidised to dehydroascorbic acid, which can be either hydrolysed to diketogulonate (DKG) or further oxidised. DKG can be further degraded, oxidatively or non-oxidatively. Here we characterise DKG products formed non-enzymically and non-oxidatively at 20 °C and at a slightly acidic pH typical of the plant apoplast. High-voltage electrophoresis revealed at least five products, including two novel CPLs (epimers of 2-carboxy-l-threo-pentonolactone), which slowly interconverted with CPA (2-carboxy-l-threo-pentonate). One of the two CPLs has an exceptionally low pKa. The CPL structures were supported by MS [(C6H7O7)-] and by 1H and 13C NMR spectroscopy. Xylonate and its lactone also appeared. Experiments with [1-14C]DKG showed that all five products (including the 5-carbon xylonate and its lactone) retained DKG's carbon-1; therefore, most xylonate arose by decarboxylation of CPLs or CPA, one of whose -COOH groups originates from C-2 or C-3 of DKG after a 'benzilic acid rearrangement'. Since CPLs appeared before CPA, a DKG lactone is probably the main species undergoing this rearrangement. CPA and CPL also form non-enzymically in vivo, where they may be useful to researchers as 'fingerprints', or to organisms as 'signals', indicating a non-oxidative, slightly acidic biological compartment.

Procyanidins in rice cooked with adzuki bean and their contribution to the reduction of nitrite to nitric oxide (•NO) in artificial gastric juice.

In Japan, adzuki bean is cooked with rice. During the cooking, the colour of rice becomes pale red. It is postulated that the red pigment is produced from procyanidins and that the ingestion of red rice causes the production of nitric oxide (•NO) in the stomach by reacting with salivary nitrite. The increase in colour intensity accompanied the decrease in the amounts of procyanidins, suggesting the conversion of procyanidins into the red pigment during the cooking. In addition, the red pigment combined with rice strongly. The red-coloured rice produced •NO by reacting with nitrite in artificial gastric juice, and the amounts were dependent on the contents of procyanidins and the equivalents. It is suggested that although adzuki procyanidins were oxidised during cooking with rice, procyanidins and the equivalents bound to rice still have the ability to produce bioactive •NO in the stomach using nitrite in mixed whole saliva.

The sour side of vitamin C might mediate neuroprotective, anticonvulsive and antidepressant-like effects.

In animal experiments, neuroprotective, anticonvulsive and antidepressant-like properties have been increasingly attributed to administrations of ascorbic acid (AA, vitamin C) in at least medium (low millimolar) doses, which however await validation in well controlled clinical studies. In mammalian cortical and subcortical neurons, small to modest acidification (<0.4-0.5 pH-units) is belonging to the key strategies for controlling local excitability and is associated with neuroprotection, e.g. by limiting excitotoxicity. Such acidifications are furthermore involved in the mechanisms of some anticonvulsants and antidepressants. As AA-transport and regulation of intracellular pH (pHi) are closely interwoven on the level of special transmembrane solute carriers, I suppose that the aforementioned beneficial AA-effects might be based upon a discrete "hormetic" acidification of cortical and or subcortical neurons via an AA-mediated weakening of their pHi-regulation. This assumption is supported by findings in non-neuronal cells suggesting both, intracellular acidification and inhibition of a core-element of the pHi-regulation apparatus by millimolar AA. In mammalian subcortical neurons, there is already first evidence of a modest acidification after adding low millimolar AA.

The Role of Physiological Vitamin C Concentrations on Key Functions of Neutrophils Isolated from Healthy Individuals.

Vitamin C (ascorbate) is important for neutrophil function and immune health. Studies showing improved immune function have primarily used cells from scorbutic animals or from individuals with infectious conditions or immune cell disorders. Few studies have focused on the requirements of neutrophils from healthy adults. Therefore, we have investigated the role of vitamin C, at concentrations equivalent to those obtained in plasma from oral intakes (i.e., 50-200 µmol/L), on key functions of neutrophils isolated from healthy individuals. Cells were either pre-loaded with dehydroascorbic acid, which is rapidly reduced intracellularly to ascorbate, or the cells were activated in the presence of extracellular ascorbate. We measured the effects of enhanced ascorbate uptake on the essential functions of chemotaxis, oxidant production, programmed cell death and neutrophil extracellular trap (NET) formation. We found that neutrophils isolated from healthy individuals already had replete ascorbate status (0.35 nmol/106 cells), therefore they did not uptake additional ascorbate. However, they readily took up dehydroascorbic acid, thus significantly increasing their intracellular ascorbate concentrations, although this was found to have no additional effect on superoxide production or chemotaxis. Interestingly, extracellular ascorbate appeared to enhance directional mobilityin the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). Stimulation of the cells in the presence of ascorbate significantly increased intracellular ascorbate concentrations and, although this exhibited a non-significant increase in phosphatidylserine exposure, NET formation was significantly attenuated. Our findings demonstrate the ability of neutrophils to regulate their uptake of ascorbate from the plasma of healthy humans to maintain an optimal level within the cell for proper functioning. Higher oral intakes, however, may help reduce tissue damage and inflammatory pathologies associated with NET formation.

Metabolic control by dehydroascorbic acid: Questions and controversies in cancer cells.

For a long time, the effect of vitamin C on cancer cells has been a controversial concept. From Linus Pauling's studies in 1976, it was proposed that ascorbic acid (AA) could selectively kill tumor cells. However, further research suggested that vitamin C has no effect on tumor survival. In the last decade, new and emerging functions for vitamin C have been discovered using the reduced form, AA, and the oxidized form, dehydroascorbic acid (DHA), independently. In this review, we summarized the latest findings related to the effects of DHA on the survival and metabolism of tumor cells. At the same time, we put special emphasis on the bystander effect and the recycling capacity of vitamin C in various cellular models, and how these concepts can affect the experimentation with vitamin C and its therapeutic application in the treatment against cancer.

Determination of cellular vitamin C dynamics by HPLC-DAD.

A redox-sensitive inter-conversion between ascorbic acid (ASC) and its oxidized form dehydroascorbic acid (DHA) in the intracellular environment has been of exceptional interest to recent metabolomics and pharmaceutical research. We developed a chromatographic protocol to instantly determine these vitamers with each identity from cellular extracts, without any labeling and pretreatments. Owing to its simplicity, one can readily continue the assay for hours, an otherwise difficult to cover timescale at which the intracellular DHA-ASC conversion comes into play. The method was validated for the analysis of pancreatic cancer cells, to our knowledge the first-ever study on a nucleated cell type, to trace in detail their kinetics of glucose transporter-dependent DHA uptake and, simultaneously, that for the intracellular ASC conversion. The simplest of all the relevant techniques and yet with the unique ability to provide each vitamer identity on a high-throughput basis, this method should offer the most practical option for VC-involved physiological and pharmaceutical studies including high-dose VC cancer therapy.

The oxidation of dehydroascorbic acid and 2,3-diketogulonate by distinct reactive oxygen species.

l-Ascorbate, dehydro-l-ascorbic acid (DHA), and 2,3-diketo-l-gulonate (DKG) can all quench reactive oxygen species (ROS) in plants and animals. The vitamin C oxidation products thereby formed are investigated here. DHA and DKG were incubated aerobically at pH 4.7 with peroxide (H2O2), 'superoxide' (a ∼50 : 50 mixture of [Formula: see text] and [Formula: see text]), hydroxyl radicals (•OH, formed in Fenton mixtures), and illuminated riboflavin (generating singlet oxygen, 1O2). Products were monitored electrophoretically. DHA quenched H2O2 far more effectively than superoxide, but the main products in both cases were 4-O-oxalyl-l-threonate (4-OxT) and smaller amounts of 3-OxT and OxA + threonate. H2O2, but not superoxide, also yielded cyclic-OxT. Dilute Fenton mixture almost completely oxidised a 50-fold excess of DHA, indicating that it generated oxidant(s) greatly exceeding the theoretical •OH yield; it yielded oxalate, threonate, and OxT. 1O2 had no effect on DHA. DKG was oxidatively decarboxylated by H2O2, Fenton mixture, and 1O2, forming a newly characterised product, 2-oxo-l-threo-pentonate (OTP; '2-keto-l-xylonate'). Superoxide yielded negligible OTP. Prolonged H2O2 treatment oxidatively decarboxylated OTP to threonate. Oxidation of DKG by H2O2, Fenton mixture, or 1O2 also gave traces of 4-OxT but no detectable 3-OxT or cyclic-OxT. In conclusion, DHA and DKG yield different oxidation products when attacked by different ROS. DHA is more readily oxidised by H2O2 and superoxide; DKG more readily by 1O2 The diverse products are potential signals, enabling organisms to respond appropriately to diverse stresses. Also, the reaction-product 'fingerprints' are analytically useful, indicating which ROS are acting in vivo.

Imaging glutathione depletion in the rat brain using ascorbate-derived hyperpolarized MR and PET probes.

Oxidative stress is a critical feature of several common neurologic disorders. The brain is well adapted to neutralize oxidative injury by maintaining a high steady-state concentration of small-molecule intracellular antioxidants including glutathione in astrocytes and ascorbic acid in neurons. Ascorbate-derived imaging probes for hyperpolarized 13C magnetic resonance spectroscopy and positron emission tomography have been used to study redox changes (antioxidant depletion and reactive oxygen species accumulation) in vivo. In this study, we applied these imaging probes to the normal rat brain and a rat model of glutathione depletion. We first studied hyperpolarized [1-13C]dehydroascorbate in the normal rat brain, demonstrating its robust conversion to [1-13C]vitamin C, consistent with rapid transport of the oxidized form across the blood-brain barrier. We next showed that the kinetic rate of this conversion decreased by nearly 50% after glutathione depletion by diethyl maleate treatment. Finally, we showed that dehydroascorbate labeled for positron emission tomography, namely [1-11C]dehydroascorbate, showed no change in brain signal accumulation after diethyl maleate treatment. These results suggest that hyperpolarized [1-13C]dehydroascorbate may be used to non-invasively detect oxidative stress in common disorders of the brain.

Effects of nicosulfuron on growth, oxidative damage, and the ascorbate-glutathione pathway in paired nearly isogenic lines of waxy maize (Zea mays L.).

Nicosulfuron is a postemergence herbicide used for weed control in maize fields (Zea mays L.). We used the pair of nearly isogenic inbred lines, SN509-R (nicosulfuron resistant) and SN509-S (nicosulfuron sensitive), to study the effect of nicosulfuron on growth, oxidative stress, and the ascorbate-glutathione (AA-GSH) cycle in waxy maize seedlings. Nicosulfuron treatment was applied when the fourth leaves were fully developed and the obtained effects were compared to water treatment as control. After nicosulfuron treatment, compared to SN509-R, the death of SN509-S might be associated with increased oxidative stress, since higher O2- and H2O2 accumulations were observed in SN509-S. This in turn might have caused severe damage to lipids and proteins, thus reducing membrane stability. These effects were exacerbated with increasing exposure time. After nicosulfuron treatment, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, and guaiacol peroxidase of SN509-S were significantly lower than those of SN509-R. Compared to SN509-R, dehydroascorbate content, glutathione (GSH) content, and GSH to glutathione disulphide ratios significantly declined with increasing exposure time in SN509-S. Our results suggest that the rapid degradation of nicosulfuron in SN509-R results in only a small and transient increase in reactive oxygen species (ROS). In contrast, in SN509-S, reduced nicosulfuron degradation leads to increase ROS, while at the same time, the AA-GSH pathway is not activated.