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.

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.

Nutraceutical, Anti-Inflammatory, and Immune Modulatory Effects of ß-Glucan Isolated from Yeast.

ß-Glucan is a dietary fibre, found in many natural sources, and controls chronic metabolic diseases effectively. However, ß-glucan from the yeast has rarely been investigated. Objectively, conditions were optimized to isolate ß-glucan from the yeast (max. 66% yield); those optimized conditions included 1.0 M NaOH, pH 7.0, and 90°C. The purity and identity of the isolated ß-glucan were characterized through FT-IR, SEM, DSC, and physicofunctional properties. The obtained results from DSC revealed highly stable ß-glucan (m.p., 125°C) with antioxidant activity (TAC value 0.240 ± 0.0021 µg/mg, H2O2 scavenging 38%), which has promising bile acid binding 40.463% and glucose control (in vitro). In line with these results, we evaluated the in vivo anti-inflammatory potential, that is, myeloperoxidase activity and reduction in MDA and NO; protective effect on proteins and keeping viscosity within normal range exhibited improvement. Also, the in vivo cholesterol binding and reduction in the skin thickness by ß-glucan were highly encouraging. Finally, our results confirmed that yeast ß-glucan is effective against some of the inflammatory and oxidative stress markers studied in this investigation. In general, the effect of 4% ß-glucan was more noticeable versus 2% ß-glucan. Therefore, our results support the utilization of ß-glucan as a novel, economically cheap, and functional food ingredient.

Glutathione Depletion, Pentose Phosphate Pathway Activation, and Hemolysis in Erythrocytes Protecting Cancer Cells from Vitamin C-induced Oxidative Stress.

The discovery that oxidized vitamin C, dehydroascorbate (DHA), can induce oxidative stress and cell death in cancer cells has rekindled interest in the use of high dose vitamin C (VC) as a cancer therapy. However, high dose VC has shown limited efficacy in clinical trials, possibly due to the decreased bioavailability of oral VC. Because human erythrocytes express high levels of Glut1, take up DHA, and reduce it to VC, we tested how erythrocytes might impact high dose VC therapies. Cancer cells are protected from VC-mediated cell death when co-cultured with physiologically relevant numbers of erythrocytes. Pharmacological doses of VC induce oxidative stress, GSH depletion, and increased glucose flux through the oxidative pentose phosphate pathway (PPP) in erythrocytes. Incubation of erythrocytes with VC induced hemolysis, which was exacerbated in erythrocytes from glucose-6-phosphate dehydrogenase (G6PD) patients and rescued by antioxidants. Thus, erythrocytes protect cancer cells from VC-induced oxidative stress and undergo hemolysis in vitro, despite activation of the PPP. These results have implications on the use of high dose VC in ongoing clinical trials and highlight the importance of the PPP in the response to oxidative stress.

The Beneficial Effect of Cape Gooseberry Juice on Carbon Tetrachloride- Induced Neuronal Damage.

OBJECTIVE: Cape gooseberry (Physalis peruviana L.) belongs to the Solanaceae family. Physalis has many medicinal properties however, the beneficial effect of physalis in protecting against neurotoxins has not yet been evaluated. This experimental study investigated the protective effect of physalis juice against the oxidative damage induced by carbon tetrachloride (CCl4) in the rat brain. METHODS: The degrees of protection by physalis in brain tissues were evaluated by determining the brain levels of lipid peroxidation, nitric oxide, glutathione content and antioxidant enzyme activities (superoxide dismutase, catalase, glutathione-S-transferase, glutathione peroxidase and glutathione reductase), after CCl4) induction in the presence or absence of physalis. Adult male albino Wistar rats were divided into 4 groups, Group I served as the control group, Group II was intraperitoneally treated with 2 ml CCl4)/kg bwt for 12 weeks, Group III was supplemented with physalis juice via the drinking water for 12 weeks, Group IV was supplemented with physalis juice and was intraperitoneally injected weekly with CCl4). RESULTS: Treatment with CCl4) was significantly associated with a disturbance in the oxidative status in the brain tissues; this was marked by a significant (p<0.05) elevation in the lipid peroxidation and nitric oxide levels with a concomitant reduction in glutathione content compared to the control, along with a remarkable reduction in antioxidant enzymes. The administration of physalis along with CCl4) juice significantly (p<0.05) alleviated the changes in enzymatic antioxidant activity when compared to the CCl4) treated group. Furthermore, physalis juice supplemention inhibited apoptosis, as indicated by the increase of Bcl-2 immunoreactivity in brain tissue. CONCLUSION: Our results suggest that physalis juice could be effective in preventing neurotoxicity and the neuroprotective effect of physalis might be mediated via antioxidant and anti-apoptosis activities.

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH.

More than half of human colorectal cancers (CRCs) carry either KRAS or BRAF mutations and are often refractory to approved targeted therapies. We found that cultured human CRC cells harboring KRAS or BRAF mutations are selectively killed when exposed to high levels of vitamin C. This effect is due to increased uptake of the oxidized form of vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased DHA uptake causes oxidative stress as intracellular DHA is reduced to vitamin C, depleting glutathione. Thus, reactive oxygen species accumulate and inactivate glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Inhibition of GAPDH in highly glycolytic KRAS or BRAF mutant cells leads to an energetic crisis and cell death not seen in KRAS and BRAF wild-type cells. High-dose vitamin C impairs tumor growth in Apc/Kras(G12D) mutant mice. These results provide a mechanistic rationale for exploring the therapeutic use of vitamin C for CRCs with KRAS or BRAF mutations.

Vitamin C suppresses cell death in MCF-7 human breast cancer cells induced by tamoxifen.

Vitamin C is generally thought to enhance immunity and is widely taken as a supplement especially during cancer treatment. Tamoxifen (TAM) has both cytostatic and cytotoxic properties for breast cancer. TAM engaged mitochondrial oestrogen receptor beta in MCF-7 cells and induces apoptosis by activation of pro-caspase-8 followed by downstream events, including an increase in reactive oxygen species and the release of pro-apoptotic factors from the mitochondria. In addition to that, TAM binds with high affinity to the microsomal anti-oestrogen-binding site and inhibits cholesterol esterification at therapeutic doses. This study aimed to investigate the role of vitamin C in TAM-mediated apoptosis. Cells were loaded with vitamin C by exposure to dehydroascorbic acid, thereby circumventing in vitro artefacts associated with the poor transport and pro-oxidant effects of ascorbic acid. Pre-treatment with vitamin C caused a dose-dependent attenuation of cytotoxicity, as measured by acridine-orange/propidium iodide (AO/PI) and Annexin V assay after treatment with TAM. Vitamin C dose-dependently protected cancer cells against lipid peroxidation caused by TAM treatment. By real-time PCR analysis, an impressive increase in FasL and tumour necrosis factor-α (TNF-α) mRNA was detected after TAM treatment. In addition, a decrease in mitochondrial transmembrane potential was observed. These results support the hypothesis that vitamin C supplementation during cancer treatment may detrimentally affect therapeutic response.

[Cellular and intracellular transport of vitamin C. The physiologic aspects]. / A C-vitamin celluláris, intracelluláris transzportja. Fiziológiai vonatkozások.

Vitamin C requirement is satisfied by natural sources and vitamin C supplements in the ordinary human diet. The two major forms of vitamin C in the diet are L-ascorbic acid and L-dehydroascorbic acid. Both ascorbate and dehydroascorbate are absorbed along the entire length of the human intestine. The reduced form, L-ascorbic acid is imported by an active mechanism, requiring two sodium-dependent vitamin C transporters (SVCT1 and SVCT2). The transport of the oxidized form, dehydroascorbate is mediated by glucose transporters GLUT1, GLUT3 and possibly GLUT4. Initial rate of uptake of both ascorbate and dehydroascorbate is saturable with increasing external substrate concentration. Vitamin C plasma concentrations are tightly controlled when the vitamin is taken orally. It has two simple reasons, on the one hand, the capacity of the transporters is limited, on the other hand the two Na+-dependent transporters can be down-regulated by an elevated level of ascorbate.

Hyperpolarized [1-13C]dehydroascorbate MR spectroscopy in a murine model of prostate cancer: comparison with 18F-FDG PET.

UNLABELLED: Reduction and oxidation (redox) chemistry is increasingly implicated in cancer pathogenesis. To interrogate the redox status of prostate tumors noninvasively, we developed hyperpolarized [1-(13)C]dehydroascorbate ((13)C-DHA), the oxidized form of vitamin C, as an MR probe. In a model of transgenic adenocarcinoma of the mouse prostate (TRAMP), increased reduction of hyperpolarized (13)C-DHA to vitamin C was observed in tumor, as compared with normal prostate and surrounding benign tissue. We hypothesized that this difference was due to higher concentrations of glutathione and increased transport of hyperpolarized (13)C-DHA via the glucose transporters (GLUT1, GLUT3, and GLUT4) in TRAMP tumor. To test these hypotheses, hyperpolarized (13)C-DHA MR spectroscopy (MRS) and (18)F-FDG PET were applied as complementary technologies in the TRAMP model. METHODS: Late-stage TRAMP tumors (>4 cm(3)) were studied at similar time points (MR studies conducted < 24 h after PET) in fasting mice by (18)F-FDG PET and hyperpolarized (13)C-DHA MR imaging on a small-animal PET/CT scanner and a (1)H/(3)C 3-T MR scanner. PET data were processed using open-source AMIDE software to compare the standardized uptake values of tumor with those of surrounding muscle, and (13)C-DHA MRS data were processed using custom software to compare the metabolite ratios (vitamin C/[vitamin C + (13)C-DHA]). After in vivo studies, the tumor glutathione concentrations were determined using a spectrophotometric assay, and thiol staining was performed using mercury orange. Real-time polymerase chain reaction was used to evaluate the relevant transporters GLUT1, GLUT3, and GLUT4 and vitamin C transporters SVCT1 and SVCT2. GLUT1 was also evaluated by immunohistochemistry. RESULTS: The average metabolite ratio was 0.28 ± 0.02 in TRAMP tumor, versus 0.11 ± 0.02 in surrounding benign tissue (n = 4), representing a 2.5-fold difference. The corresponding tumor-to-nontumor (18)F-FDG uptake ratio was 3.0. The total glutathione was 5.1 ± 0.4 mM in tumor and 1.0 ± 0.2 mM in normal prostate, whereas reduced glutathione was 2.0 ± 0.3 mM and 0.8 ± 0.3 mM, respectively, corresponding to a 2.5-fold difference. In TRAMP tumor, mercury orange staining demonstrated increased thiols. Real-time polymerase chain reaction showed no significant difference in GLUT1 messenger RNA between TRAMP tumor and normal prostate, with immunohistochemistry (anti-GLUT1) also showing comparable staining. CONCLUSION: Both hyperpolarized (13)C-DHA and (18)F-FDG provide similar tumor contrast in the TRAMP model. Our findings suggest that the mechanism of in vivo hyperpolarized (13)C-DHA reduction and the resulting tumor contrast correlates most strongly with glutathione concentration. In the TRAMP model, GLUT1 is not significantly upregulated and is unlikely to account for the contrast obtained using hyperpolarized (13)C-DHA or (18)F-FDG.

New insights on dimethylaminoethanol (DMAE) features as a free radical scavenger.

Recently, a number of synthetic drugs used in a variety of therapeutic indications have been reported to have antiaging effects. Among them, Dimethylaminoethanol (DMAE), an anologue of dietylaminoethanol, is a precursor of choline, which in turn allows the brain to optimize the production of acetylcholine that is a primary neurotransmitter involved in learning and memory. The data presented here includes new information on the ability of the compound to scavenge specific free radicals, assessed by Electron Spectroscopic Resonance (EPR), to further analyze the role of DMAE as an antioxidant. DMAE ability to directly react with hydroxyl, ascorbyl and lipid radicals was tested employing in vitro assays, and related to the supplemented dose of the compound.

Soybean ferritin: isolation, characterization, and free radical generation.

The main aim of this work was to assess the multi-task role of ferritin (Ft) in the oxidative metabolism of soybean (Glycine max). Soybean seeds incubated for 24 h yielded 41 ± 5 µg Ft/g fresh weight. The rate of in vitro incorporation of iron (Fe) into Ft was tested by supplementing the reaction medium with physiological Fe chelators. The control rate, observed in the presence of 100 µM Fe, was not significantly different from the values observed in the presence of 100 µM Fe-his. However, it was significantly higher in the presence of 100 µM Fe-citrate (approximately 4.5-fold) or of 100 µM Fe-ATP (approximately 14-fold). Moreover, a substantial decrease in the Trp-dependent fluorescence of the Ft protein was determined during Fe uptake from Fe-citrate, as compared with the control. On the other hand, Ft addition to homogenates from soybean embryonic axes reduced endogenously generated ascorbyl radical, according to its capacity for Fe uptake. The data presented here suggest that Ft could be involved in the generation of free radicals, such as hydroxyl radical, by Fe-catalyzed reactions. Moreover, the scavenging of these radicals by Ft itself could then lead to protein damage. However, Ft could also prevent cellular damage by the uptake of catalytically active Fe.

Ascorbic acid contents in transgenic potato plants overexpressing two dehydroascorbate reductase genes.

Ascorbic acid (AsA, vitamin C) is one of the most important nutritional quality factors in many horticultural crops and has many biological activities in the human body. Dehydroascorbate reductase (EC 1.8.5.1; DHAR) plays an important role in maintaining the normal level of ascorbic acid (AsA) by recycling oxidized ascorbic acid. To increase AsA content of potato, we isolated and characterized the cDNAs encoding two isoform DHARs localized in cytosol and chloroplast from potato, and developed two types of transgenic potato plants overexpressing cytosolic DHAR gene and chloroplastic DHAR, respectively. Incorporation of the transgene in the genome of potato was confirmed by PCR and real time RT-PCR. The overexpression of cytosolic DHAR significantly increased DHAR activities and AsA contents in potato leaves and tubers, whereas chloroplastic DHAR overexpression only increased DHAR activities and AsA contents in leaves, and did not change them in tubers. These results indicated that AsA content of potato can be elevated by enhancing recycling ascorbate via DHAR overexpression, moreover, cytosolic DHAR might play main important roles in improving the AsA contents of potato tubers.

Exposure to excess dissolved iron in vivo affects oxidative status in the bivalve Mya arenaria.

The effect of in vivo Fe exposure on the oxidative metabolism of the bivalve Myaarenaria was studied. Fe was supplemented in natural seawater and resulted in a significant increase in the total Fe content in the bivalve digestive gland (DG) between 9 to 17days of exposure. Mortality of treated animals increased drastically after day 18. Oxidative stress conditions were characterized in DG through assessment of the generation of reactive oxygen species (ROS) and ascorbyl radical (A) content. Both parameters were affected following a biphasic profile showing significant increases by days 2 and 9 of Fe exposure. The content of 2-thiobarbituric acid reactive substances (TBARS) was significantly increased over control values by days 2, 9 and 17 of treatment. The labile Fe pool (LIP) in isolated DG was elevated over control values by day 7, and maintained this increase until day 17 of Fe exposure. The content of NO, assessed by EPR spin trapping, was 60% lower in DG of animals exposed for 2days to Fe than in control values, with no further changes. The biphasic profile of oxidative stress response to Fe exposure in DG suggests that at early stages of Fe supplementation the cellular control mechanisms, such as CAT activity, were operative to limit oxidative damage, but further Fe exposure overwhelmed these abilities. Moreover, the second phase could be understood as the consequence of the exhaustion of cellular protective systems that could also involve NO.

[Comparative research of different Bupleurum chinense composition to influence of hepatotoxicity of rats and oxidative damage mechanism].

OBJECTIVE: To compare the influence of different Bupleurun chinense composition to the degree of hepatotoxicity damage to rats and oxidative damage mechanism. METHOD: To successively lavage alcohol extracted and water extracted B. chinense composition to rats for 30 days, the general conditions were observed and the related index of liver function, the content of total-SH in serum, the content of MDA, the activity of SOD and the content and activity of GSH and GSH-Px in serum and liver tissue were detected. RESULT: Alcohol and water extracted B. chinense composition all could induce the increases of the activity of ALT and AST in serum, liver weight and the ratio of liver to body, and the content of MDA and induce the decreses of the content of total -SH in serum, the content of GSH, and the activity of SOD and GSH-Px in serum and liver tissue. The above-mentioned changes gradually aggravated with dose increasing, and there was significant difference compared with control group with distilled water. CONCLUSION: The different B. chinense composition all can induce hepatotoxicity damage, and the channel of hepatic damage is related with the peroxidative damage mechanism. The degree of hepatotoxicity damage caused by the alcohol extracted composition is more serious than that by the water extracted composition.

Molecular characterization of a dehydroascorbate reductase from Pinus bungeana.

Abstract Dehydroascorbate reductase (DHAR) plays a critical role in the ascorbate-glutathione recycling reaction for most higher plants. To date, studies on DHAR in higher plants have focused largely on Arabidopsis and agricultural plants, and there is virtually no information on the molecular characteristics of DHAR in gymnosperms. The present study reports the cloning and characteristics of a DHAR (PbDHAR) from a pine, Pinus bungeana Zucc. ex Endl. The PbDHAR gene encodes a protein of 215 amino acid residues with a calculated molecular mass of 24.26 kDa. The predicted 3-D structure of PbDHAR showed a typical glutathione S-transferase fold. Reverse transcription-polymerase chain reaction revealed that the PbDHAR was a constitutive expression gene in P. bungeana. The expression level of PbDHAR mRNA in P. bungeana seedlings did not show significant change under high temperature stress. The recombinant PbDHAR was overexpressed in Escherichia coli following purification with affinity chromatography. The recombinant PbDHAR exhibited enzymatic activity (19.84 micromol/min per mg) and high affinity (a K(m) of 0.08 mM) towards the substrates dehydroascorbate (DHA). Moreover, the recombinant PbDHAR was a thermostable enzyme, and retained 77% of its initial activity at 55 degrees C. The present study is the first to provide a detailed molecular characterization of the DHAR in P. bungeana.

Folic acid or combination of folic acid and vitamin B(12) prevents short-term arsenic trioxide-induced systemic and mitochondrial dysfunction and DNA damage.

The effect of folic acid and folic acid + vitamin B(12) supplementation upon short-term arsenic-induced systemic and pancreatic islet cell mitochondria oxidative stress was investigated in male rats. Arsenic trioxide was administered orally at a dose of 3 mg kg body weight(-1) day(-1) for 30 days, and folic acid and vitamin B(12) were administered at a dose of 36 and 0.63 microg kg body weight(-1) day(-1), respectively, for 30 days. Compared to control, arsenic-treated group showed a significant increase in the levels of systemic oxidative markers, malondialdehyde (MDA), nitric oxide (NO), and hydroxyl radical (OH(-)) formation, which were found decreased significantly after supplementation either with folic acid or a combination of folic acid + vitamin B(12). Similar supplementations were found effective against arsenic-induced oxidative marker changes (MDA, NO, and OH(-)) in pancreatic islet cell mitochondria. Also, low activities of antioxidant defense enzymes such as superoxide dismutase and catalase, and level of antioxidant glutathione, all could regain significantly on supplementations both against systemic and islet cell mitochondria oxidative stress. Results of agarose-gel electrophoresis of DNA from lymphocytes and islet cells of arsenic-exposed rats showed DNA smearing, which could be reduced with simultaneous administration either with folic acid or a combination of folic acid + vitamin B(12). Significantly, similar supplementations were found effective in increasing the urinary clearance of arsenic. Together, these results indicate that folic acid and vitamin B(12) may be effective to reduce the arsenic-induced damage at molecular target level.

Copper-induced changes in the growth, oxidative metabolism, and saponin production in suspension culture roots of Panax ginseng in bioreactors.

Roots of Panax ginseng exposed to various concentrations of Cu (0.0, 5, 10.0, 25.0, and 50.0 microM) accumulated high amounts of Cu in a concentration-dependent and duration-dependent manner. Roots treated with 50 microM Cu resulted in 52% and 89% growth inhibition after 20 and 40 days, respectively. Saponin synthesis was stimulated at a Cu concentration between 5 and 25 muM but decreased at 50 microM Cu. Malondialdehyde content (MDA), lipoxygenase activity (LOX), superoxide ion (O2*-) accumulation, and H2O2 content at 5 and 10 microM Cu-treated roots were not increased but strongly increased at 50 microM Cu resulting in the oxidation of ascorbate (ASC) and glutathione (GSH) to dehydroascorbate (DHA) and glutathione disulfide (GSSG), respectively indicating a clear oxidative stress. Seven well-resolved bands of superoxide dismutase (SOD) were detected in the gel and an increase in SOD activity seemed to be mainly due to the induction of Fe-SOD 3. Five to 10 microM Cu slightly induced activity of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR), guaiacol peroxidase (G-POD) but inhibited monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) enzyme activities. No changes in catalase (CAT) activity and in activity gel were found up to 25 microM Cu, but both G-POD and CAT activities were inhibited at 50 microM Cu. Glutathione metabolism enzymes such as gamma-glutamylcysteine synthetase (gamma-GCS), glutathione-S-transferase (GST), and glutathione peroxidase activities (GPx) were activated at 5 and 10 microM Cu but were strongly inhibited at 50 microM Cu due to the Cu accumulation in root tissues. The strong depletion of GSH at 50 microM Cu was associated to the strong induction of gamma-glutamyltranspeptidase (gamma-GGT) activity. These results indicate that plant could grow under Cu stress (5-25 microM) by modulating the antioxidant defense mechanism for combating Cu induced oxidative stress.

Modulation of cadmium-induced oxidative stress in Ceratophyllum demersum by zinc involves ascorbate-glutathione cycle and glutathione metabolism.

To understand the interaction between Zn, an essential micronutrient and Cd, a non-essential element, Cd-10 microM and Zn supplemented (10, 50, 100, and 200 microM) Cd 10 microM treated Ceratophyllum demersum L. (Coontail), a free floating freshwater macrophyte was chosen for the study. Cadmium at 10 microM concentration decreased thiol content, enhanced oxidation of ascorbate (AsA) and glutathione (GSH) to dehydroascorbate (DHA) and glutathione disulfide (GSSG), respectively, a clear indication of oxidative stress. Zinc supplementation to Cd (10 microM) treated plants effectively restored thiols, inhibited oxidation of AsA and GSH maintaining the redox molecules in reduced form. Cd-10 microM slightly induced ascorbate peroxidase (APX, E.C. 1.11.1.11) but inhibited monodehydroascorbate reductase (MDHAR, E.C. 1.6.5.4), dehydroascorbate reductase (DHAR, E.C. 1.8.5.1) and glutathione reductase (GR, E.C. 1.6.4.2), enzymes of ascorbate-glutathione cycle (AGC). Zn supplementation restored and enhanced the functional activity of all the AGC enzymes (APX, MDHAR, DHAR and GR). Gamma-glutamylcysteine synthetase (gamma-GCS, E.C. 6.3.2.2) was not affected by Cd as well as Zn, but Zn supplements increased glutathione-S-transferase (GST, E.C. 2.5.1.18) activity to a greater extent than Cd and simultaneously restored glutathione peroxidase (GSH-PX, E.C. 1.11.1.9) activity impaired by Cd toxicity. Zn-alone treatments did not change above investigated parameters. These results clearly indicate the protective role of Zn in modulating the redox status of the plant system through the antioxidant pathway AGC and GSH metabolic enzymes for combating Cd induced oxidative stress.

Changes in intracellular and apoplastic peroxidase activity, ascorbate redox status, and root elongation induced by enhanced ascorbate content in Allium cepa L.

Onions (Allium cepa L.) treated with external ascorbic acid or with the immediate precursor of its synthesis L-galactono-gamma-lactone show a stimulated elongation rate of the roots and an increase in the number of new radicles appearing at the bulb base. Treatment with both molecules resulted in an enhanced accumulation of ascorbate and dehydroascorbate along the root axis, but the distribution of these redox forms was not uniform along the root, as detected in intracellular (symplastic) and extracellular (apoplastic) compartments. Thus, those radicular zones metabolically more active, such as the meristem and the elongation zone, accumulated the highest amount of both redox forms of ascorbate. On the other hand, ascorbate and L-galactono-gamma-lactone also stimulated cytosolic glucose-6-phosphate dehydrogenase activity and inhibited peroxidase activity as deduced from in vivo and in vitro experiments. Differences were also found when comparing apoplastic and symplastic activities. These results are compatible with the idea of an ascorbate-mediated stimulation of root growth by inhibiting cell wall stiffening and increasing root metabolism.

Dehydroascorbate uptake activity correlates with cell growth and cell division of tobacco bright yellow-2 cell cultures.

Recently, ascorbate (ASC) concentration and the activity of a number of enzymes from the ASC metabolism have been proven to correlate with differences in growth or cell cycle progression. Here, a possible correlation between growth and the activity of a plasma membrane dehydroascorbate (DHA) transporter was investigated. Protoplasts were isolated from a tobacco (Nicotiana tabacum) Bright Yellow-2 cell culture at different intervals after inoculation and the activity of DHA transport was tested with (14)C-labeled ASC. Ferricyanide (1 mM) or dithiothreitol (1 mM) was included in the test to keep the external (14)C-ASC in its oxidized respectively reduced form. Differential uptake activity was observed, correlating with growth phases of the cell culture. Uptake of DHA in cells showed a peak in exponential growth phase, whereas uptake in the presence of dithiothreitol did not. The enhanced DHA uptake was not due to higher endogenous ASC levels that are normally present in exponential phase because preloading of protoplasts of different ages did not affect DHA uptake. Preloading was achieved by incubating cells before protoplastation for 4 h in a medium supplemented with 1 mM DHA. In addition to testing cells at different growth phases, uptake of DHA into the cells was also followed during the cell cycle. An increase in uptake activity was observed during M phase and the M/G1 transition. These experiments are the first to show that DHA transport activity into plant cells differs with cell growth. The relevance of the data to the action of DHA and ASC in cell growth will be discussed.