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Since the discovery of ferroptosis, it has been postulated that this type of cell death could be utilized in treatments for cancer. Unfortunately, several highly aggressive tumor models are resistant to the pharmacological induction of ferroptosis. However, with the use of combined therapies, it is possible to recover sensitivity to ferroptosis in certain cellular models. Here, we discovered that co-treatment with the metabolically stable ferroptosis inducer imidazole ketone erastin (IKE) and the oxidized form of vitamin C, dehydroascorbic acid (DHAA), is a powerful therapy that induces ferroptosis in tumor cells previously resistant to IKE-induced ferroptosis. We determined that DHAA and IKE + DHAA delocalize and deplete GPX4 in tumor cells, specifically inducing lipid droplet peroxidation, which leads to ferroptosis. Moreover, in vivo, IKE + DHAA has high efficacy with regard to the eradication of highly aggressive tumors such as glioblastomas. Thus, the use of IKE + DHAA could be an effective and safe therapy for the eradication of difficult-to-treat cancers., (© 2023. The Author(s).)

Black spot rot caused by Alternaria alternata is one of the major postharvest disease of apple fruit during logistic. This study evaluated in vitro inhibitory effect of 2-hydroxy-3-phenylpropanoic acid (PLA) at various concentrations on A. alternata and the possible mechanisms involved in its action. Results showed that different concentrations of PLA inhibited conidia germination and mycelial growth of A. alternata in vitro, and 1.0 g L -1 was the lowest effective concentration to suppress A. alternata growth. Moreover, PLA significantly reduced relative conductivity and increased malondialdehyde and soluble protein contents. PLA also increased H 2 O 2 and dehydroascorbic acid contents, but reduced ascorbic acid content. Additionally, PLA treatment inhibited catalase, ascorbate peroxidase, monodehydroascorbate acid reductase, dehydroascorbic acid reductase and glutathione reductase activities, whereas promoted superoxide dismutase activity. All these findings suggest that the possible mechanisms involved in the inhibitory effect of PLA on A. alternata included damaging the cell membrane integrity to cause electrolyte leakage and destroying reactive oxygen species balance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

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.

Efforts to develop effective drugs targeting PI3K and KRAS signaling pathways in PIK3CA/KRAS -mutant colorectal cancer stem cells (CRCSCs) remain challenging. Finding safe compounds that can easily enter CRCSCs with the ability to target metastasis-driver gene CXCR4 and pluripotency network genes as key upstream and downstream effectors of both PI3K and KRAS signaling pathways may provide promising results. PIK3CA/KRAS -mutant CRCSCs display high expression of glucose transporters (GLUTs) on their cell membrane and a glycolytic phenotype providing an opportunity to deliver antiglycolytic compounds into these cells via the GLUTs. CRC patients with low levels of vitamin C in their plasma show a shorter survival suggesting the ability of this vitamin at the physiologic levels for caspase-3 activation and apoptosis in CRCSCs. Vitamin C in an oxidized form (L-dehydroascorbic acid; L-DHA) with antiglycolytic activity can be taken up into CRC cells via the GLUTs. This may provide selective toxicity on CRCSCs and affect CXCR4 and stemness markers genes expression in these cells. To this end, we treated PIK3CA / KRAS -mutant LS174T cells with high glycolytic activity as an attractive model for CRCSCs with L-DHA equal to the pharmacological levels of vitamin C in human plasma, after which cell numbers, metabolic activity, proliferation-rate, CXCR4 and pluripotency network genes expression, caspase-3 activity with apoptosis were evaluated. 48 h post-treatment with 100- to 1000 µM L-DHA, cell numbers were decreased and measured to be 70-47% control. L-DHA with selective toxicity on LS174T cells diminished metabolic activity and cell proliferation-rate to 1.4-0.8 (Control OD = 1.5) and 92-54.5% respectively with no toxicity on PBMCs. L-DHA decreased CXCR4 , Bmi-1 , Sox -2 and Oct-4 expression to 45%, 85%, 45% and 48% control respectively followed by caspase-3 reactivation by 2.5 to 4.9-fold increases and induction of apoptosis ranging from 0.5% to 58.3% for 100- to 1000 µM L-DHA. According to our data, CRC stem-like cells were highly sensitive to L-DHA in in-vitro . L-DHA selectively targeted LS174T cells and successfully reactivated caspase-3 and apoptosis in these cells. CXCR4 , stemness marker genes and metabolic activity appear to be promising targets of L-DHA. Our results may provide a new therapeutic approach to target selectively GLUT-overexpressing PIK3CA/KRAS -mutant CRCSCs using L-DHA with no toxicity on normal cells.

Oxidative stress and inflammation are crucial factors that increase with age. In the progression of multiple age-related diseases, antioxidants and bioactive compounds have been recognized as useful antiaging agents. Oxidized or reduced vitamin C exerts different actions on tissues and has different metabolism and uptake. In this study, we analyzed the antiaging effect of vitamin C, both oxidized and reduced forms, in renal aging using laser microdissection, quantitative reverse-transcription polymerase chain reaction, and immunohistochemical analyses. In the kidneys of old SAM mice (10 months of age), a model of accelerated senescence, vitamin C, especially in the oxidized form (dehydroascorbic acid [DHA]) improves renal histology and function. Serum creatinine levels and microalbuminuria also decrease after treatment with a decline in azotemia. In addition, sodium-vitamin C cotransporter isoform 1 levels, which were increased during aging, are normalized. In contrast, the pattern of glucose transporter 1 expression is not affected by aging or vitamin C treatment. We conclude that oxidized and reduced vitamin C are potent antiaging therapies and that DHA reverses the kidney damage observed in senescence-accelerated prone mouse 8 to a greater degree., (© 2020 Wiley Periodicals LLC.)

Although tea catechins in green tea and green tea beverages must be stable to deliver good sensory quality and healthy benefits, they are always unstable during processing and storage. Ascorbic acid (AA) is often used to protect catechins in green tea beverages, and AA is easily oxidized to form dehydroascorbic acid (DHAA). However, the function of DHAA on the stability of catechins is not clear. The objective of this study was to determine the effects of DHAA on the stability of catechins and clarify the mechanism of effects by conducting a series of experiments that incubate DHAA with epigallocatechin gallate (EGCG) or catechins. Results showed that DHAA had a dual function on EGCG stability, protecting its stability by inhibiting hydrolysis and promoting EGCG consumption by forming ascorbyl adducts. DHAA also reacted with (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin (EGC) to form ascorbyl adducts, which destabilized them. After 9 h of reaction with DHAA, the depletion rates of EGCG, ECG, EC, and EGC were 30.08%, 22.78%, 21.45%, and 13.55%, respectively. The ability of DHAA to promote catechins depletion went from high to low: EGCG, ECG, EGC, and EC. The results are important for the processing and storage of tea and tea beverages, as well as the general exploration of synergistic functions of AA and catechins.

Opioids and their antagonists alter vitamin C metabolism. Morphine binds to glutathione (l-γ-glutamyl-l-cysteinyl-glycine), an intracellular ascorbic acid recycling molecule with a wide range of additional activities. The morphine metabolite morphinone reacts with glutathione to form a covalent adduct that is then excreted in urine. Morphine also binds to adrenergic and histaminergic receptors in their extracellular loop regions, enhancing aminergic agonist activity. The first and second extracellular loops of adrenergic and histaminergic receptors are, like glutathione, characterized by the presence of cysteines and/or methionines, and recycle ascorbic acid with similar efficiency. Conversely, adrenergic drugs bind to extracellular loops of opioid receptors, enhancing their activity. These observations suggest functional interactions among opioids and amines, their receptors, and glutathione. We therefore explored the relative binding affinities of ascorbic acid, dehydroascorbic acid, opioid and adrenergic compounds, as well as various control compounds, to glutathione and glutathione-like peptides derived from the extracellular loop regions of the human beta 2-adrenergic, dopamine D1, histamine H1, and mu opioid receptors, as well as controls. Some cysteine-containing peptides derived from these receptors do bind ascorbic acid and/or dehydroascorbic acid and the same peptides generally bind opioid compounds. Glutathione binds not only morphine but also naloxone, methadone, and methionine enkephalin. Some adrenergic drugs also bind to glutathione and glutathione-like receptor regions. These sets of interactions provide a novel basis for understanding some ways that adrenergic, opioid and antioxidant systems interact during anesthesia and drug abuse and may have utility for understanding drug interactions.

Ascorbic acid better known as vitamin C, is a reducing carbohydrate needed for a variety of functions in the human body. The most important characteristic of ascorbic acid is the ability to donate two electrons, predestining it as a major player in balancing the physiological redox state and as a necessary cofactor in multiple enzymatic hydroxylation processes. Ascorbic acid can be reversibly oxidized in two steps, leading to semidehydroascorbic acid and dehydroascorbic acid, respectively. Further degradation is irreversible and generates highly reactive carbonyl-intermediates. These intermediates are able to induce glycation of proteins, a non-enzymatic and unspecific reaction of carbonyls with amino groups involved to several age-related diseases. In this study, we investigated the effect of ascorbic acid- and dehydroascorbic acid-induced glycation on PC12 cells, which represent a model for neuronal plasticity. We found that both applications of ascorbic acid or dehydroascorbic acid leads to glycation of cellular proteins, but that ascorbic acid interferes more with viability and neurite outgrowth compared with dehydroascorbic acid., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Zucchini ( Cucurbita pepo subsp. pepo ) is a seasonal vegetable with high nutritional and medical values. Many useful properties of this fruit are attributed to bioactive compounds. Zucchini fruits ("Yellow" and "Light Green" varieties) and four distinctive components (lutein, β-carotene, zeaxanthin and dehydroascorbic acid) were selected. Firstly, the lutein, β-carotene, zeaxanthin and dehydroascorbic acid contents were determined in these fruits. Then, in order to evaluate the safety and suitability of their use, different assays were carried out: (i) genotoxicity and anti-genotoxicity tests to determine the safety and DNA-protection against hydrogen peroxide; (ii) cytotoxicity; and (iii) DNA fragmentation and Annexin V/PI (Propidium Iodide) assays to evaluate the pro-apoptotic effect. Results showed that: (i) all the substances were non-genotoxic; (ii) all the substances were anti-genotoxic except the highest concentration of lutein; (iii) "Yellow" zucchini epicarp and mesocarp exhibited the highest cytotoxic activity (IC 50 > 0.1 mg/mL and 0.2 mg/mL, respectively); and (iv) "Light Green" zucchini skin induced internucleosomal DNA fragmentation, β-carotene being the possible molecule responsible for its pro-apoptotic activity. To sum up, zucchini fruit could play a positive role in human health and nutrition due to this fruit and its components were safe, able to inhibit significantly the H₂O₂-induced damage and exhibit anti-proliferative and pro-apoptotic activities toward HL60 (human promyelocytic leukemia cells) tumor cells. The information generated from this research should be considered when selecting potential accessions for breeding program purposes., Competing Interests: The authors declare no conflict of interest.

The high level of dehydroascorbic acid (DHA) in the lenticular tissue is an important risk factor for the development of age-related cataracts. In this study, the effects of DHA on structure and function of lens crystallins were studied in the presence of carnosine using gel mobility shift assay, different spectroscopic techniques, and lens culture analysis. The DHA-induced unfolding and aggregation of lens proteins were largely prevented by this endogenous dipeptide. The ability of carnosine to preserve native protein structure upon exposure to DHA suggests the essential role of this dipeptide in prevention of the senile cataract development. Although the DHA-modified α-crystallin was characterized by altered chaperone activity, functionality of this protein was significantly restored in the presence of carnosine. The increased proteolytic instability of DHA-modified lens proteins was also attenuated in the presence of carnosine. Furthermore, the assessment of lens culture suggested that DHA can induce significant lens opacity which can be prevented by carnosine. These observations can be explained by the pleiotropic functions of this endogenous and pharmaceutical compound, notably by its anti-glycation and anti-aggregation properties. In summary, our study suggests that carnosine may have therapeutic potential in preventing senile cataracts linked with the increased lenticular DHA generation, particularly under pathological conditions associated with the oxidative stress.

Introduction: Accumulation of hypoxia inducible factor-1 alpha (HIF-1α) in malignant tissue is known to contribute to oncogenic progression and is inversely associated with patient survival. Ascorbic acid (AA) depletion in malignant tissue may contribute to aberrant normoxic activity of HIF-1α. While AA supplementation has been shown to attenuate HIF-1α function in malignant melanoma, the use of dehydroascorbic acid (DHA) as a therapeutic means to increase intracellular AA and modulate HIF-1α function is yet to be evaluated. Here we compared the ability of AA and DHA to increase intracellular vitamin C content and decrease the malignant potential of human melanoma by reducing the activity of HIF-1α., Methods: HIF-1α protein accumulation was evaluated by western blot and transcriptional activity was evaluated by reporter gene assay using a HIF-1 HRE-luciferase plasmid. Protein expressions and subcellular localizations of vitamin C transporters were evaluated by western blot and confocal imaging. Intracellular vitamin C content following AA, ascorbate 2-phosphate (A2P), or DHA supplementation was determined using a vitamin C assay. Malignant potential was accessed using a 3D spheroid Matrigel invasion assay. Data was analyzed by One or Two-way ANOVA with Tukey's multiple comparisons test as appropriate with p<0.05 considered significant., Results: Melanoma cells expressed both sodium dependent vitamin C (SVCT) and glucose (GLUT) transporters for AA and DHA transport respectively, however advanced melanomas responded favorably to AA, but not DHA. Physiological glucose conditions significantly impaired intracellular vitamin C accumulation following DHA treatment. Consequently, A2P and AA, but not DHA treated cells demonstrated lower HIF-1α protein expression and activity, and reduced malignant potential. The ability of AA to regulate HIF-1α was dependent on SVCT2 function and SVCT2 was not significantly inhibited at pH representative of the tumor microenvironment., Conclusions: The use of ascorbic acid as an adjuvant cancer therapy remains under investigated. While AA and A2P were capable of modulating HIF-1α protein accumulation/activity, DHA supplementation resulted in minimal intracellular vitamin C activity with decreased ability to inhibit HIF-1α activity and malignant potential in advanced melanoma. Restoring AA dependent regulation of HIF-1α in malignant cells may prove beneficial in reducing chemotherapy resistance and improving treatment outcomes., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Ascorbic acid (AA), the reduced form of vitamin C, is incorporated into neurons via the sodium ascorbate co-transporter SVCT2. However, this transporter is not expressed in astrocytes, which take up the oxidized form of vitamin C, dehydroascorbic acid (DHA), via the facilitative hexose transporter GLUT1. Therefore, neuron and astrocyte interactions are thought to mediate vitamin C recycling in the nervous system. Although astrocytes are essential for the antioxidant defense of neurons under oxidative stress, a condition in which a large amount of ROS is generated that may favor the extracellular oxidation of AA and the subsequent neuronal uptake of DHA via GLUT3, potentially increasing oxidative stress in neurons. This study analyzed the effects of oxidative stress and DHA uptake on neuronal cell death in vitro. Different analyses revealed the presence of the DHA transporters GLUT1 and GLUT3 in Neuro2a and HN33.11 cells and in cortical neurons. Kinetic analyses confirmed that all cells analyzed in this study possess functional GLUTs that take up 2-deoxyglucose and DHA. Thus, DHA promotes the death of stressed neuronal cells, which is reversed by incubating the cells with cytochalasin B, an inhibitor of DHA uptake by GLUT1 and GLUT3. Additionally, the presence of glial cells (U87 and astrocytes), which promote DHA recycling, reverses the observed cell death of stressed neurons. Taken together, these results indicate that DHA promotes the death of stressed neurons and that astrocytes are essential for the antioxidative defense of neurons. Thus, the astrocyte-neuron interaction may function as an essential mechanism for vitamin C recycling, participating in the antioxidative defense of the brain.

Due to its anti-obesity effects, an adipocyte-derived hormone, leptin, has become important for the treatment of obesity. However, most obese subjects are in a state of leptin resistance, and endoplasmic reticulum (ER) stress is suggested to be involved in the pathophysiology of leptin resistance. Dehydroascorbic acid (DHAA), an oxidized form of vitamin C, was found to be increased in diabetes. In the present study, we investigated the possible effects of DHAA on the activation of ER stress and leptin resistance. A human neuroblastoma cell line, stably transfected with the Ob-Rb leptin receptor (SH-SY5Y-ObRb), was treated with DHAA. We found that DHAA upregulated ER stress-related genes such as GRP78, CHOP, and spliced XBP1. Moreover, leptin-induced STAT3 phosphorylation was hindered by DHAA. These results suggested that increases in the levels of DHAA might be harmful to neurons, contributing to defective leptin-responsive signaling., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Exposure of U937 cells to low concentrations of L-ascorbic acid (AA) is associated with a prompt cellular uptake and a further mitochondrial accumulation of the vitamin. Under the same conditions, dehydroascorbic acid (DHA) uptake was followed by rapid reduction and accumulation of identical intracellular levels of AA, however, in the absence of significant mitochondrial uptake. This event was instead observed after exposure to remarkably greater concentrations of DHA. Furthermore, experiments performed in isolated mitochondria revealed that DHA transport through hexose transporters and Na(+) -dependent transport of AA were very similar. These results suggest that the different subcellular compartmentalization of the vitamin is mediated by events promoting inhibition of mitochondrial AA transport, possibly triggered by low levels of DHA. We obtained results in line with this notion in intact cells, and more direct evidence in isolated mitochondria. This inhibitory effect was promptly reversible after DHA removal and comparable with that mediated by established inhibitors, as quercetin. The results presented collectively indicate that low intracellular concentrations of DHA, because of its rapid reduction back to AA, are a poor substrate for direct mitochondrial uptake. DHA concentrations, however, appear sufficiently high to mediate inhibition of mitochondrial transport of AA/DHA-derived AA., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Arsenite directly triggers cytochrome c and Smac/DIABLO release in mitochondria isolated from U937 cells. These effects were not observed in mitochondria pre-exposed for 15 min to 10 µM L-ascorbic acid (AA). In other experiments, intact cells treated for 24-72 h with arsenite were found to die by apoptosis through a mechanism involving mitochondrial permeability transition. Pre-exposure (15 min) to low micromolar concentrations of AA and dehydroascorbic acid (DHA), resulting in identical cytosolic levels of the vitamin, had a diverse impact on cell survival, as cytoprotection was only observed after treatment with AA. Also the mitochondrial accumulation of the vitamin was restricted to AA exposure. An additional indication linking cytoprotection to the mitochondrial fraction of the vitamin was obtained in experiments measuring susceptibility to arsenite in parallel with loss of mitochondrial and cytosolic AA at different times after vitamin exposure. Finally, we took advantage of our recent findings that DHA potently inhibits AA transport to demonstrate that DHA abolishes all the protective effects of AA, under the same conditions in which the mitochondrial accumulation of the vitamin is prevented without affecting the overall cellular accumulation of the vitamin., (© 2015 International Union of Biochemistry and Molecular Biology.)

Enhanced glucose requirement of cancer cells is associated with an increased glucose transport across plasma membrane that is mediated by a family of facilitated glucose transporter proteins, named GLUTs. GLUT1 is the main transporter in thyroid cancer cells. Glucose is the principal physiological substrate of GLUT1; however, it is also capable of transporting of oxidized form of vitamin C [i.e., dehydroascorbic acid (DHAA) which inside the cells is reduced to ascorbic acid (AA)]. The objective of this study was to determine the effect of normo-, hypo-, and hyperglycemia conditions on GLUT1-dependent intracellular ascorbate accumulation and viability of thyroid cancer cells. GLUT1 seems to be the main DHAA transporter in thyroid cancer cells because its knockdown by RNAi reduced DHAA accumulation by more than 80%. The results showed that in thyroid cancer cells high glucose inhibits both transport of AA and DHAA. Inhibition of vitamin C transport by glucose had a cytotoxic effect on the cells. However, stabilization of vitamin C in one of 2 forms (i.e., AA or DHAA) abolished this effect. These results suggest that cytotoxic effect is rather associated with extracellular accumulation of vitamin C and changes of its oxidation state than with intracellular level of ascorbate.

Water dispersible and biologically important molecule dehydroascorbic acid (DHA, capable to cross the blood brain barrier) coated Fe3O4 superparamagnetic nanoparticles having an average size of ∼6 nm were synthesized through one pot aqueous coprecipitation method under atmospheric air. An antioxidant ascorbic acid (AA) used in the synthesis oxidized itself to dehydroascorbic acid (DHA) to consume dissolved or available oxygen in reaction mixture which died away the oxidative impact of atmospheric air and formed DHA encapsulated the Fe3O4 nanoparticles which stabilized the Fe3O4 nanoparticles and significantly enhanced their colloidal solubility in water. Fe3O4 phase, superparamagnetic property, DHA coating and stable colloidal solubility in water were confirmed by means of XPS, VSM, IR and zeta potential analysis respectively. T1, T2 and T2(∗) weighted magnetic resonance imaging (MRI) and corresponding relaxivity (r1=0.416, r2=50.28 and r2(∗)=123.65 mM(-1) and r2/r1=120.86, r2(∗)r1=297.23) of colloidally dispersed DHA-coated nanoparticle water phantom revealed a strong contrast enhancement in T2 and T2(∗) weighted images. The compatibility of DHA-coated Fe3O4 nanoparticles toward human blood cells was examined by means of cell counting and cell morphological analysis with the use of optical microscope and scanning electron microscope imaging., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Resuscitation from cardiac arrest is partly limited by progressive reduction in left ventricular distensibility, leading to decreased hemodynamic efficacy of cardiopulmonary resuscitation (CPR). Reduction in left ventricular distensibility has been linked to loss of mitochondrial bioenergetic function that can result from oxidative injury. Attenuation of oxidative injury by administration of vitamin C during CPR may help maintain left ventricular distensibility and favor resuscitability and survival. Ventricular fibrillation was electrically induced in 2 series of 16 rats each and left untreated for 10 minutes. Resuscitation was attempted by 8 minutes of CPR and delivery of electrical shocks. Dehydroascorbate (DHA)-an oxidized form of vitamin C that enters the cell via glucose transporters-was used in series 1 and ascorbic acid (AA)-the reduced form of vitamin C that enters the cell via specialized AA transporters-in series 2. In each series, rats were randomized 1:1 to receive a 250 mg/kg right atrial bolus of DHA or AA or vehicle immediately before chest compression. Left ventricular distensibility-measured as the ratio between coronary perfusion pressure and compression depth-was numerically lower (not significant) in rats that received DHA (1.6 ± 0.2 vs. 1.9 ± 0.7 mm Hg/mm) and AA (1.8 ± 0.6 vs. 1.9 ± 0.3 mm Hg/mm). In addition, resuscitability was compromised by DHA (2/8 vs. 7/8; P = 0.041) and by AA (0/8 vs. 5/8; P = 0.026). AA levels in mitochondria were no different than control. Vitamin C failed to preserve left ventricular distensibility during CPR and had detrimental effects on resuscitability, suggesting possible disruption of protective signaling mechanisms during oxidative stress by vitamin C.

A U937 cell clone, in which low micromolar concentrations of ascorbic acid (AA) and dehydroascorbic acid (DHA) are taken up at identical rates, was used to investigate possible interactions between transport systems mediating cellular uptake of the two forms of the vitamin. Results obtained with different experimental approaches showed that DHA potently and reversibly inhibits AA uptake through Na(+)-AA cotransporters. Hence, a progressive increase in extracellular DHA concentrations in the presence of a fixed amount of AA caused an initial decrease in the net amount of vitamin C accumulated, and eventually, at higher levels, it caused an accumulation of the vitamin solely based on DHA uptake through hexose transporters. DHA-dependent inhibition of AA uptake was also detected in various other cell types. Taken together, our results provide evidence of a novel biological effect mediated by concentrations of DHA compatible with those produced at inflammatory sites., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Vitamin C is an essential factor for neuronal function and survival, existing in two redox states, ascorbic acid (AA), and its oxidized form, dehydroascorbic acid (DHA). Here, we show uptake of both AA and DHA by primary cultures of rat brain cortical neurons. Moreover, we show that most intracellular AA was rapidly oxidized to DHA. Intracellular DHA induced a rapid and dramatic decrease in reduced glutathione that was immediately followed by a spontaneous recovery. This transient decrease in glutathione oxidation was preceded by an increase in the rate of glucose oxidation through the pentose phosphate pathway (PPP), and a concomitant decrease in glucose oxidation through glycolysis. DHA stimulated the activity of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the PPP. Furthermore, we found that DHA stimulated the rate of lactate uptake by neurons in a time- and dose-dependent manner. Thus, DHA is a novel modulator of neuronal energy metabolism by facilitating the utilization of glucose through the PPP for antioxidant purposes., (© 2014 International Society for Neurochemistry.)

Chlamydomonas reinhardtii tolerates relatively high H2 O2 levels that induce an array of antioxidant activities. However, rather than rendering the cells more resistant to oxidative stress, the cells become far more sensitive to an additional H2 O2 dose. If H2 O2 is provided 1.5-9 h after an initial dose, it induces programmed cell death (PCD) in the wild-type, but not in the dum1 mutant impaired in the mitochondrial respiratory complex III. This mutant does not exhibit a secondary oxidative burst 4-5 h after the inducing H2 O2 , nor does it activate metacaspase-1 after the second H2 O2 treatment. The intracellular dehydroascorbate level, a product of ascorbate peroxidase, increases under conditions leading to PCD. The addition of dehydroascorbate induces PCD in the wild-type and dum1 cultures, but higher levels are required in dum1 cells, where it is metabolized faster. The application of dehydroascorbate induces the expression of metacaspase-2, which is much stronger than the expression of metacaspase-1. The presence or absence of oxidative stress, in addition to the rise in internal dehydroascorbate, may determine which metacaspase is activated during Chlamydomonas PCD. Cell death is strongly affected by the timing of H2 O2 or dehydroascorbate admission to synchronously grown cultures, suggesting that the cell cycle phase may distinguish cells that perish from those that do not., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Otherwise nontoxic levels of peroxynitrite promote toxicity in U937 cells pre-exposed to low micromolar concentrations of l-ascorbic acid (AA). This event was associated with the mitochondrial accumulation of the vitamin and with the early formation of secondary reactive oxygen species and DNA single-strand breaks. The same concentrations of peroxynitrite, however, failed to elicit detectable effects in cells pre-exposed to dehydroascorbic acid (DHA), in which mitochondrial accumulation of vitamin C did not occur despite the identical cytosolic levels. Coherently, oxidation of extracellular AA failed to affect the intracellular concentration of the vitamin, but nevertheless prevented its mitochondrial localization as well as the enhanced response to peroxynitrite. Furthermore, in cells postincubated in vitamin C-free medium, time-dependent loss of mitochondrial AA was paralleled by a progressive decline of susceptibility to peroxynitrite, under the same conditions in which cells retained about half of the initial AA. Using different experimental approaches, we finally showed that the enhancing effects of AA are mediated by events associated with peroxynitrite-dependent superoxide/H2 O2 formation in the mitochondrial respiratory chain. Collectively, these results indicate that mitochondria actively take up vitamin C as AA and respond to otherwise inactive concentrations of peroxynitrite with the mitochondrial formation of secondary species responsible for DNA damage and toxicity. DHA preloading, while leading to the accumulation of identical levels of vitamin C, fails to produce these effects because of the poor mitochondrial accumulation of the vitamin., (© 2013 International Union of Biochemistry and Molecular Biology, Inc.)

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., (© 2013 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: Duodenal cytochrome b (Dcytb) is a mammalian plasma ferric reductase enzyme that catalyses the reduction of ferric to ferrous ion in the process of iron absorption. The current study investigates the relationship between Dcytb, iron, dehydroascorbate (DHA) and Hif-2α in cultured cell lines., Methods: Dcytb and Hif-2α protein expression was analysed by Western blot technique while gene regulation was determined by quantitative PCR. Functional analyses were carried out by ferric reductase and (59)Fe uptake assays., Results: Iron and dehydroascorbic acid treatment of cells inhibited Dcytb mRNA and protein expression. Desferrioxamine also enhanced Dcytb mRNA level after cells were treated overnight. Dcytb knockdown in HuTu cells resulted in reduced mRNA expression and lowered reductase activity. Preloading cells with DHA (to enhance intracellular ascorbate levels) did not stimulate reductase activity fully in Dcytb-silenced cells, implying a Dcytb-dependence of ascorbate-mediated ferrireduction. Moreover, Hif-2α knockdown in HuTu cells led to a reduction in reductase activity and iron uptake., Conclusions: Taken together, this study shows the functional regulation of Dcytb reductase activity by DHA and Hif-2α., General Significance: Dcytb is a plasma membrane protein that accepts electrons intracellularly from DHA/ascorbic acid for ferrireduction at the apical surface of cultured cells and enterocytes., (© 2013.)

Recently, many clinical reports have suggested that the ascorbyl free radical (Asc(∙)) can be treated as a noninvasive, reliable, real-time marker of oxidative stress, but its generation mechanisms in human blood have rarely been discussed. In this study, we used upstream substances, enzyme inhibitors, and free radical scavengers to delineate the mechanisms of Asc(∙) formation in human platelet-rich plasma (PRP). Our results show that the doublet signal was detected in PRP samples by using electron spin resonance, and the hyperfine splitting of the doublet signal was a(H) = 1.88 gauss and g-factor = 2.00627, which was determined to be the Asc(∙). We observed that the inhibitors of NADPH oxidase (NOX), cyclooxygenase (COX), lipoxygenase (LOX), cytochrome P450 (CYP450), mitochondria complex III, and nitric oxide synthase (NOS), but not xanthine oxidase, diminished the intensity of the Asc(∙) signal dose dependently. All enzyme inhibitors showed no obvious antioxidant activity during a Fenton reaction assay. In summary, the obtained data suggest that Asc(∙) formation is associated with NOX, COX, LOX, CYP450, eNOS, and mitochondria in human PRP.

The effects of L-ascorbic acid and its stable analogue L-ascorbic acid 2-glucoside on the restoration of liver mass and recovery of liver function after 70% partial hepatectomy (PH), were compared with other natural vitamin C analogues in rats in vivo. L-Ascorbic acid (100 mg/kg/d, intraperitoneally (i.p.))- and L-ascorbic acid 2-glucoside (50 mg/kg/d, i.p.)-treated rats showed an approximately 1.3-fold increase in the ratio of liver weight (LW) to body weight (BW), when compared to saline (as control)-, L-dehydroascorbic acid (150 mg/kg/d, i.p.)- and D-isoascorbic acid (150 mg/kg/d, i.p.)-administrated rats on day 3 after PH. Accordingly, 5-bromo-2-deoxyuridine-labeling index in the regenerating liver was significantly higher in L-ascorbic acid- and L-ascorbic acid 2-glucoside-treated rats compared with saline-, L-dehydroascorbic acid and D-isoascorbic acid-treated rats on day 1. In control rats, liver-related serum alanine aminotransferase (ALT) activity was rapidly elevated on day 1, and then decreased to near pre-operative levels on day 5 following PH. L-Ascorbic acid and L-ascorbic acid 2-glucoside significantly lowered the serum ALT on day 1 after PH compared with saline-, L-dehydroascorbic acid- and D-isoascorbic acid-administered rats. These results demonstrate that L-ascorbic acid and L-ascorbic acid 2-glucoside significantly promote the regeneration of liver mass and function with full recovery after liver injury.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimer transcription factor whose elevated activity in many cancers helps them to survive under hypoxic conditions and enhances their capacity to grow invasively, establish metastases, and survive chemo- or radiotherapy. Optimal intracellular levels of ascorbate suppress the level and transcriptional activity of HIF-1under normoxic or mildly hypoxic conditions by supporting the activity of proly and asparagyl hydroxylases that target HIF-1alpha. High intracellular ascorbate can also work in various ways to down-regulate activation of NF-kappaB which, like HIF-1 is constitutively active in many cancers and promotes aggressive behavior - in part by promoting transcription of HIF-1alpha. Yet recent evidence suggests that, even in the context of adequate ascorbate nutrition, the intracellular ascorbate content of many aggressive cancers may be supoptimal for effective HIF-1 control. This likely reflects low expression or activity of the SVCT2 ascorbate transporter. The expression of SVCT2 in cancers has so far received little study; but the extracellular acidity characteristic of many tumors would be expected to reduce the activity of this transporter, which has a mildly alkaline pH optimum. Unfortunately, since SVCT2 has a high affinity for ascorbate, and its activity is nearly saturated at normal healthy serum levels of this vitamin, increased oral administration of ascorbate would be unlikely to have much impact on the intracellular ascorbate content of tumors. However, cancers in which HIF-1 is active express high levels of glucose transporters such as GLUT-1, and these transporters can promote influx of dehydroascorbic acid (DHA) via facilitated diffusion; once inside the cell, DHA is rapidly reduced to ascorbate, which effectively is "trapped" within the cell. Hence, episodic intravenous infusions of modest doses of DHA may have potential for optimizing the intracellular ascorbate content of cancers, potentially rendering them less aggressive. Indeed, several published studies have concluded that parenteral DHA--sometimes in quite modest doses--can retard the growth of transplanted tumors in rodents. As an alternative or adjunctive strategy, oral administration of sodium bicarbonate, by normalizing the extracellular pH of tumors, has the potential to boost the activity of SCTV2 in tumor cells, thereby promoting increased ascorbate uptake. Indeed, the utility of oral sodium bicarbonate for suppressing metastasis formation in nude mice xenografted with a human breast cancer has been reported. Hence, oral sodium bicarbonate and intravenous DHA may have the potential to blunt the aggressiveness of certain cancers in which suboptimal intracellular ascorbate levels contribute to elevated HIF-1 activity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The Myelodysplastic Syndromes are stem cell heterogeneous disorders characterized by peripheral cytopenias and hypercellular bone marrow, which can evolute to acute leukaemia. Vitamin C can act as an antioxidant, ascorbic acid (AA) donates two electrons and becomes oxidized to dehydroascorbic acid (DHA). Under physiological conditions, vitamin C predominantly exists in its reduced (AA) form but also exists in trace quantities in the oxidized form (DHA). This study evaluates the therapeutic potential of vitamin C in Myelodysplastic Syndromes (MDSs). F36P cells (MDS cell line) were treated with ascorbate and dehydroascorbate alone and in combination with cytarabine. Cell proliferation and viability were assessed by trypan blue assay and cell death was evaluated by optical microscopy and flow cytometry. The role of reactive oxygen species, mitochondrial membrane potential, BAX, BCL-2 and cytochrome C were also assessed. Vitamin C decreases cell proliferation and viability in a concentration, time and administration dependent-manner inducing cell death by apoptosis, which was shown to be associated to an increased in superoxide production, mitochondrial membrane depolarization. These compounds modulate BCL-2, BAX and cytochrome C release. These results suggest that vitamin C induces cell death trough apoptosis in F36P cells and may be a new therapeutic approach in Myelodysplasia., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Ascorbic acid enhances synthesis of norepinephrine from dopamine in adrenal chromaffin cells by serving as a co-factor for chromaffin granule dopamine β-hydroxylase (DβH). However, there is controversy regarding in situ kinetics of the ascorbate effect in chromaffin cells, as well as whether they apply to neuronal cells. In this study we evaluated the stimulation of norepinephrine synthesis from dopamine in cultured SH-SY5Y neuroblastoma cells. These cells contained neither ascorbate nor norepinephrine in culture, but when provided with dopamine, they generated intracellular norepinephrine at rates that were stimulated several-fold by intracellular ascorbate. Ascorbate-induced increases in norepinephrine synthesis in dopamine-treated cells were linear over 60 min, despite saturation of intracellular ascorbate. Norepinephrine accumulation after 60 min of incubation with 100 μM dopamine was half-maximal at intracellular ascorbate concentrations of 0.2-0.5 mM, which fits well with the literature K(m) for ascorbate of DβH using dopamine as a substrate. Moreover, these ascorbate concentrations were generated by initial extracellular ascorbate concentrations of less than 25 μM due to concentrative accumulation by the ascorbate transporter. Treatment with 100 μM dopamine acutely increased cellular superoxide generation, which was prevented by ascorbate loading, but associated with a decrease in intracellular ascorbate when the latter was present at concentrations under 1 mM. These results show that ascorbate promptly enhances norepinephrine synthesis from dopamine by neuronal cells that it does so at physiologic intracellular concentrations in accord with the kinetics of DβH, and that it both protects cells from superoxide and by providing electrons to DβH., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Thawed ram spermatozoa were incubated at 37°C in the presence of dehydroascorbic acid (DHA), TEMPOL (TPL), N-acetyl-cysteine (NAC) and rutin (RUT), at 0.1 and 1 mm, in order to test their effects on sperm physiology. Cryopreserved spermatozoa from four rams were thawed, pooled, washed and incubated in TALP-Hepes with 1 mm or 0.1 mm of each antioxidant, performing a replicate with induced oxidative stress (Fe(2+) /ascorbate). Motility (CASA), viability and mitochondrial membrane potential (flow cytometry) were analysed at 2 and 4 h. Lipoperoxidation (MDA production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were analysed at 4 h. Antioxidants, except DHA 0.1 mm, decreased motility and kinematic parameters, but had little effect on viability or mitochondrial activity. Except 1 mm DHA, the antioxidants reduced ROS at 4 h. Moreover, NAC 1 mm, rutin and TEMPOL reduced ROS and DNA damage in the presence of oxidative stress. N-acetyl-cysteine, rutin 1 mm and TEMPOL reduced lipoperoxidation in the presence of oxidative stress. However, DHA did not affect lipoperoxidation. At 1 mm, DHA increased DNA damage in the absence of oxidative stress. Dehydroascorbic acid effects could arise from spermatozoa having a low capacity for reducing it to ascorbic acid, and it may be tested in the presence of other antioxidants or reducing power. Future research should focus in testing whether the inhibition of motility observed for NAC, rutin and TEMPOL is reversible. These antioxidants might be useful at lower temperatures (refrigerated storage or cryopreservation) when their protective effects could be advantageous., (© 2012 Blackwell Verlag GmbH.)

Stoma is surrounded by two guard cells, and regulates the contents of water and CO(2) in plant, its opening and closing was affected by various factors. Recently, dehydroascorbate was found to induce stomata closure and H(2)O(2) generation. However, the mechanism of H(2)O(2) production is not clear. DPI and imidazole inhibit the flavoprotein and the b(-type) cytochrome components of the NADPH oxidase complex. Application of DPI or imidazole with DHA together impaired stomatal closure and elevation of H(2)DCF-DA fluorescent intensity induced by DHA in guard cells. CoCl(2) and PD98059, as the blocker of calcium channel and the inhibitor of MAPKKK, both impaired stomatal closure induced by DHA. The results suggested that DHA-induced H(2)O(2) generation via activation of NADPH oxidase, and thus resulting in stomatal closure. Moreover, Ca(2+) channel and MAPK cascades were involved in stomatal closure induced by DHA., (Crown Copyright © 2011. Published by Elsevier Masson SAS. All rights reserved.)

Vitamin C, available in its reduced form (ascorbic acid; AA) and in its oxidized form (dehydroascorbic acid; DHA), may act in physiological conditions as an antioxidant or pro-oxidant. The aim of this study is to evaluate the cytotoxic effects of pharmacological doses of AA in a human colorectal adenocarcinoma cell line (WiDr) in vitro, through spectrophotometry, clonogenic assays and flow cytometry, and in vivo with xenotransplanted Balb/c nu/nu mice. The results show that the reduced form of vitamin C induces an anti-proliferative and cytotoxic effect in adenocarcinoma colorectal cells under study. The results obtained in vivo after treatment with AA showed a large reduction in the rate of tumor growth. Such understanding can guide decisions about which colorectal cancer patients might potentially benefit from vitamin C pharmacologic therapy.

Diabetes is associated with an increased risk of heart failure, in part explained by endoplasmic reticulum stress and apoptosis. Protein disulfide isomerase (PDI) prevents stressed cardiomyocytes apoptosis. We hypothesized that diabetes impairs PDI function by an alteration in its oxido-reductive state. Myocardial biopsies harvested from the anterolateral left ventricular wall from diabetic (n = 7) and nondiabetic (n = 8) patients were used to assess PDI expression and cardiomyocyte death. A mouse model of diabetes (streptozotocin injection, 130 mg/mL) was used to study PDI expression and its redox state after ischemia/reperfusion injury induced by 30-min occlusion of the left anterior coronary artery followed by reperfusion. Transthoracic echocardiography was performed to assess cardiac remodeling after 1 wk. Western blot analysis was used to analyze PDI expression, and methoxy-polyethyleneglycol-maleimide was used to assess its redox state. Dehydroascorbate (DHA) administration was used to restore the PDI redox state. Diabetic patients had a greater number of transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells than nondiabetic patients despite a greater myocardial PDI expression suggesting altered PDI function. Diabetic mice had a worse postinfarction remodeling associated with an altered PDI redox state. DHA treatment restored functional PDI redox state and ameliorated post-myocardial infarction remodeling. An increase in PDI levels with a paradoxical decrease of its active form occurs in the diabetic heart after ischemia and may explain the lack of protective effects of PDI in diabetes. Restoration of PDI redox state prevents adverse remodeling. The potential significance of these findings deserves to be validated in a clinical setting.

Hydrogen sulfide is enzymatically produced in mammalian tissues and functions as a gaseous transmitter. However, H(2)S is also highly toxic as it inhibits mitochondrial respiration at the level of cytochrome c oxidase, which additionally is involved in sulfide oxidation. The accumulation of toxic sulfide levels contributes to the pathology of some diseases. This paper demonstrates that sulfide toxicity can be modified, and dehydroascorbic acid functions as an effector in this process. It significantly reduces the inhibitory effect of sulfide on cytochrome c oxidase, resulting in higher rates of respiration and sulfide oxidation in rat mitochondria. After the addition of dehydroascorbic acid mitochondria maintained more than 50% of the oxygen consumption and ATP production rates with different substrates in the presence of high concentrations of sulfide that would normally lead to complete inhibition. Dehydroascorbic acid significantly increased the sulfide concentration necessary to cause half maximal inhibition of mitochondrial respiration and thus completely prevented inhibition at low, physiological sulfide concentrations. In addition, sulfide oxidation was stimulated and led to ATP production even at high concentrations. The decrease in sulfide toxicity was more pronounced when analyzing supermolecular functional units of the respiratory chain than in isolated cytochrome c oxidase activity. Furthermore, the protective effect of dehydroascorbic acid at high sulfide concentrations was completely abolished by quantitative solubilization of mitochondrial membrane proteins with dodeclymaltoside. These results suggest that binding of cytochrome c oxidase to other proteins probably within respiratory chain supercomplexes is involved in the modulation of sulfide oxidation and toxicity by dehydroascorbic acid., (2011 Elsevier B.V. All rights reserved.)

The effects of ascorbic acid (AA) and dehydroascorbic acid (DHA), one of products of the disproportionation of monodehydroascorbate (MDHA) by AA oxidase (AAO, EC 1.10.3.3), on the gravitropic curvature of Arabidopsis roots were characterized by biochemical and genetic approaches. Exogenously applied AA and DHA both stimulated root gravitropic responses in a concentration-dependent fashion. AA also changed the Indole-3-acetic acid (IAA) distribution in the roots after gravistimulation. In an effort to determine the relationship between AA and DHA in the gravitropic response, changes in the amount of reduced AA were evaluated in Arabidopsis under a variety of conditions. The expression level of an AAO gene (AAO1) was increased upon gravistimulation. Brassinolide (BL), indole-3-acetic acid (IAA), and AA also increased the transcript levels of this gene. Root elongation and the gravitropic response were both suppressed in the AA biosynthesis mutant, vtc1, which has a greatly reduced level of total AA. Furthermore, the line of AAO double mutants (aao1-1 X aao3-1, 41-21) showed a reduced gravitropic response and reduced root elongation. Taken together, the results of this study imply that both AA and DHA help to determine the redox environment for the root gravitropic response, but DHA, rather than AA, is a major player in the regulation of the gravitropic response mediated by AA in the roots of Arabidopsis thaliana., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

In this work, we studied the behaviours of urease in the presence of l-ascorbic acid (AA) and dehydroascorbic acid (DHA) in different conditions. The inactivations of urease were carried out in an unbuffered and buffered system. We show that in the unbuffered system AA inactivated urease in a biphasic manner by denaturation brought about by AA-lowered pH. Further, we show that in the buffered system neither AA nor DHA themselves are inhibitors of urease. The inhibitory action of AA and DHA was revealed in the presence of Fe(3+) ions and most importantly, unlike reported in the literature, it was found to be primarily mediated by H(2)O(2). The resulting inhibition by DHA-Fe(3+) consisted of enzyme thiol oxidation and its effectiveness grew with increasing pH. The results may shed light on the roles of AA in therapies applied in ureolytic bacteria infections, notably those with Helicobacter pylori.

Purpose: While the benefits of ascorbic acid (vitamin C, ascorbate) as an essential nutrient are well established, its effects on tumor cells and in tumor treatment are controversial. In particular, conflicting data exist whether ascorbate may increase the cytotoxic effects of antineoplastic drugs or may rather exert adverse effects on drug sensitivity during cancer treatment. Findings are further obscured regarding the distinction between ascorbate and dehydroascorbate (DHA). Thus, the purpose of this study was to evaluate and directly compare the cytotoxic efficacy of ascorbate compared to DHA, and to analyse if ascorbate at pharmacological concentrations affects the efficacy of antineoplastic agents in prostate carcinoma cells., Methods: We directly compare the effects of ascorbate (supplied as 'Pascorbin solution for injection') and DHA on tumor cell viability, and determine IC(50) values for various cell lines. At concentrations well below the IC(50), ascorbate effects on cell proliferation and cell cycle are analysed. We furthermore determine changes in cellular sensitivity towards various cytostatic drugs upon pre-treatment of cells with ascorbate., Results: We demonstrate higher therapeutic efficacy of ascorbate over DHA in various cell lines, independent of cell line-specific differences in ascorbate sensitivity, and identify the extracellular generation of H(2)O(2) as critical mechanism of ascorbate action. We furthermore show that, in addition to pro-apoptotic effects described previously, ascorbate treatment already at concentrations well below the IC(50) exerts anti-proliferative effects on tumor cells. Those are based on interference with the cell cycle, namely by inducing a G(0)/G(1) arrest. Pre-treatment of tumor cells with ascorbate leads to increased cellular sensitivity towards Docetaxel, Epirubicin, Irinotecan and 5-FU, but not towards Oxaliplatin and Vinorelbin. For Docetaxel and 5-FU, a linear correlation between this sensitizing effect and the ascorbate dosage is observed., Conclusions: The redox-active form of vitamin C, ascorbate, shows therapeutic efficacy in tumor cells. These antitumor effects of ascorbate are mainly based on its extracellular action and, in addition to the induction of apoptosis, also include an anti-proliferative effect by inducing cell cycle arrest. Furthermore, ascorbate treatment specifically enhances the cytostatic potency of certain chemotherapeutics, which implicates therapeutic benefit during tumor treatment.

Glutathione (GSH) and ascorbate (ascorbic acid, vitamin C, ASC) are two critical water-soluble antioxidants in aerobic organisms. The reaction of ascorbate with reactive oxygen species leads to dehydroascorbate. It is generally accepted that GSH reduces dehydroascorbate (DHA) to give oxidized glutathione (GSSG) and ASC (2GSH+DHA-->GSSG+ASC) as a chemical pathway of ascorbate regeneration. Here, we report the formation of a novel conjugation product between GSH and the decomposition of DHA denoted as GS-DHA*. On the basis of MS and NMR analyses, the structure of GS-DHA* consists of an intact GSH moiety attached via the thiol group to a five-carbon fragment of DHA. The conjugation product appears as a mixture of four diastereomers with very similar proton and carbon chemical shifts. The formation of GS-DHA* adducts is demonstrated in Jurkat cells upon incubation with DHA in culture followed by analysis of the whole-cell extracts by HPLC coupled to tandem mass spectrometry. This novel conjugation product may be a useful biomarker of DHA stress and model system of protein modification., (Copyright 2010 Elsevier Inc. All rights reserved.)

Depending on the circumstances, autophagy can be either a protective or damaging cellular process. The role of autophagy in photodynamic therapy (PDT), a photo-chemotherapy that utilizes light to activate a photosensitizer drug to achieve localized cellular damage, has been explored in recent years. It has been reported that autophagy in PDT is significantly influenced by the treatment protocol. In this work, the role of Photofrin, a well-established clinical photosensitizer, in regulating cellular autophagy was investigated. The effects of Photofrin on cellular autophagy induced by conventional starvation or rapamycin techniques were studied. By fluorescence imaging, Western blotting and cell viability assays, it was found that Photofrin can effectively inhibit cellular autophagy induced by starvation or rapamycin. This autophagy blocking is independent of the photosensitizing property of the drug. With Baf-A1, a well-established agent that inhibits autophagosome from fusing with lysosome, we also found that, the observed phenomenon is not due to accelerated degradation of existing autophagosomes, thus proving that the drug Photofrin alone, without light excitation, can truly block autophagy.

Ascorbate (vitamin C) is the major electron donor to a tPMET (transplasma membrane electron transport) system that was originally identified in human erythrocytes. This plasma membrane redox system appears to transfer electrons from intracellular ascorbate to extracellular oxidants (e.g. non-transferrin-bound iron). Although this phenomenon has been observed in nucleated cells, its mechanism and regulation are not well understood. In the present study we have examined both facets of this phenomenon in K562 cells and primary astrocyte cultures. Using ferricyanide as the analytical oxidant we demonstrate that tPMET is enhanced by dehydroascorbate uptake via facilitative glucose transporters, and subsequent accumulation of intracellular ascorbate. Additionally, we demonstrate that this stimulation is not due to ascorbate that is released from the cells, but is dependent only on a restricted intracellular pool of the vitamin. Substrate-saturation kinetics suggest an enzyme-catalysed reaction across the plasma membrane by an as-yet-unidentified reductase that relies on extensive recycling of intracellular ascorbate. Inhibition of ascorbate-stimulated tPMET by the NHE (Na(+)/H(+)-exchanger) inhibitors amiloride and 5-(N-ethyl-N-isopropyl)amiloride, which is diminished by bicarbonate, suggests that tPMET activity may be regulated by intracellular pH. In support of this hypothesis, tPMET in astrocytes was significantly inhibited by ammonium chloride-pulse-induced intracellular acidification, whereas it was significantly stimulated by bicarbonate-induced intracellular alkalinization. These results suggest that ascorbate-dependent tPMET is enzyme-catalysed and is modulated by NHE activity and intracellular pH.

Oxidative stress-induced mitochondrial dysfunction is a common consequence of severe sepsis. However, oxidative stress also activates signalling cascades which enable protection of cells against subsequent oxidative damage. This study hypothesized that cellular uptake of vitamin C as dehydroascorbic acid rather than ascorbic acid would up-regulate antioxidant enzyme systems and impart a protective effect to mitochondria in cells subsequently exposed to lipopolysaccharide (LPS) in an iron free environment. Treatment of monocytes with dehydroascorbic acid, but not ascorbic acid, caused oxidative stress (p< 0.001). Dehydroascorbic acid exposure also resulted in increased manganese superoxide dismutase (p= 0.018) and catalase (p= 0.003) expression. Pre-treatment of monocytes with dehydroascorbic acid followed by LPS resulted in higher mitochondrial membrane potentials than cells without pre-treatment (p< 0.0001). Lower cytochrome c in cytosol (p< 0.05) and higher mitochondrial expression of the anti-apoptotic Bcl-2 protein (p= 0.029) was also found in monocytes pre-treated before subsequent LPS exposure, compared to cells without pre-treatment. In conclusion, acute exposure of monocytes to dehydroascorbic acid in an iron free environment induces cytoprotective antioxidant enzymes and protected mitochondria from the harmful effects of oxidative stress prior to a septic insult, which was abrogated when cells were pre-incubated with the DHA uptake inhibitor cytocholasin B.

The antioxidant activities of the extract and its relevant fraction as well as isolated compounds from the dead bark of Araucaria angustifolia are presented. This tree represents the Mixed Ombrophile Forest, which is endangered due to extensive logging. The dead bark of Araucaria is naturally discarded by the tree, and its hydroalcoholic crude extract has exhibited protective qualities against stress induced by H(2)O(2) in cell culture. Using several in vitro models, here we describe the antioxidant potential of the crude extract and its component ethyl acetate fraction and also of some compounds isolated from the ethyl acetate fraction. We provide the first description of the isolation of two natural product afzelechin derivatives. The extract and isolated compounds, particularly epiafzelechin protocatechuate (5), displayed very high antioxidant activity, as did compound 4, quercetin, which is well known for its antioxidant properties. In a DPPH assay, compound 5 exhibited an IC(50) of 0.7 μM; in a lipid peroxidation assay; IC(50) values of 21 μM and 35 μM were obtained when the oxidation was induced by UV and ascorbate free radicals, respectively.

Oxidative stress has been proposed as a mechanism of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The aim of this research was to evaluate the protective effects of increased intracellular ascorbate levels against TCDD acute toxicity in the insulin-secreting beta-cell line INS-1E. Ascorbate is considered a potent antioxidant, but its therapeutic efficacy is greatly limited by its slow achievement of high intracellular levels. This might be circumvented by administration of dehydroascorbate (DHA), which is transported at a much higher rate and undergoes rapid intracellular reduction to ascorbate. Indeed, 30 min incubation of INS-1E cells with various concentrations of DHA caused a remarkable, dose-related increase of the intracellular ascorbate levels. INS-1E cells preincubated with 0.5 and 1.0mM DHA showed a greater viability than control cells after 1h exposition to cytotoxic TCDD concentrations. In our experimental conditions, TCDD surprisingly failed to increase ROS production in INS-1E cells, but induced a dose-related mitochondrial depolarisation which was significantly improved by DHA preincubation. Furthermore, DHA preincubation completely prevented the low dose TCDD-induced inhibition of glucose-stimulated insulin secretion. Thus, our results suggest that DHA preincubation protects INS-1E cells against TCDD acute toxicity by partially preserving mitochondrial function.

The incidence and prevalence of kidney stones are notable and are projected to increase over the next decade. Risk factors for kidney stones abound, but a prominent risk factor is hyperoxaluria, which has numerous etiologies, including vitamin C (ascorbic acid) dietary supplement intake. This randomized, double-blind, crossover study examined the effects of two different vitamin C formulations and found that vitamin C with metabolites (Ester-C) significantly reduced urine oxalate levels compared to ascorbic acid. This is a potential novel finding that requires further clinical evaluation.

Humans use two sodium-ascorbate cotransporters (hSVCT1 and hSVCT2) for transporting the dietary essential micronutrient ascorbic acid, the reduced and active form of vitamin C. Although the human liver plays a pivotal role in regulating and maintaining vitamin C homeostasis, vitamin C transport physiology and regulation of the hSVCT systems in this organ have not been well defined. Thus, this research used a human hepatic cell line (HepG2), confirming certain results with primary human hepatocytes and determined the initial rate of ascorbic acid uptake to be Na(+) gradient, pH dependent, and saturable as a function of concentration over low and high micromolar ranges. Additionally, hSVCT2 protein and mRNA are expressed at higher levels in HepG2 cells and native human liver, and the cloned hSVCT2 promoter has more activity in HepG2 cells. Results using short interfering RNA suggest that in HepG2 cells, decreasing hSVCT2 message levels reduces the overall ascorbic acid uptake process more than decreasing hSVCT1 message levels. Activation of PKC intracellular regulatory pathways caused a downregulation in ascorbic acid uptake not mediated by a single predicted PKC-specific amino acid phosphorylation site in hSVCT1 or hSVCT2. However, PKC activation causes internalization of hSVCT1 but not hSVCT2. Examination of other intracellular regulatory pathways on ascorbic acid uptake determined that regulation also potentially occurs by PKA, PTK, and Ca(2+)/calmodulin, but not by nitric oxide-dependent pathways. These studies are the first to determine the overall ascorbic acid uptake process and relative expression, regulation, and contribution of the hSVCT systems in human liver epithelial cells.

In the present study, ascorbic acid weakly inhibited the multiplication of viruses of three different families: herpes simplex virus type 1 (HSV-1), influenza virus type A and poliovirus type 1. Dehydroascorbic acid, an oxidized form of ascorbic acid and hence without reducing ability, showed much stronger antiviral activity than ascorbic acid, indicating that the antiviral activity of ascorbic acid is due to factors other than an antioxidant mechanism. Moreover, addition of 1 mM Fe3+, which oxidizes ascorbic acid to dehydroascorbic acid and also enhances the formation of hydroxyl radicals by ascorbic acid in the culture media, strongly enhanced the antiviral activity of ascorbic acid to a level significantly stronger than that of dehydroascorbic acid. Although both ascorbic acid and dehydroascorbic acid showed some cytotoxicity, the degree of cytotoxicity of the former was 10-fold higher than the latter, suggesting that the observed antiviral activity of ascorbic acid with and without ferric ion is, at least in part, a secondary result of the cytotoxic effect of the reagent, most likely due to the free radicals. However, the possibility that oxidation of ascorbic acid also contributed to the antiviral effects of ascorbic acid exists, in particular in the presence of ferric ion, since dehydroascorbic acid exhibited a very strong antiviral activity. Characterization of the mode of antiviral action of dehydroascorbic acid revealed that the addition of the reagent even at 11 h post infection almost completely inhibited the formation of progeny infectious virus in the infected cells, indicating that the reagent inhibits HSV-1 multiplication probably at the assembly process of progeny virus particles after the completion of viral DNA replication.

Sulfide oxidation in the lugworm, Arenicola marina (L.), is most likely localized in the mitochondria, which can either produce ATP with sulfide as a substrate or detoxify it via an alternative oxidase. The present study identified selective activators of the energy-conserving and the detoxifying sulfide oxidation pathways respectively. In the presence of the ROS scavengers glutathione (GSH) and ascorbate, isolated lugworm mitochondria rapidly oxidized up to 100 micromoll(-1) sulfide with maximal oxygen consumption rates but did not produce any ATP in the process. Under these conditions, salicylhydroxamic acid (SHAM), which is an inhibitor of the alternative oxidase of plant mitochondria, completely blocked oxygen consumption whereas inhibitors of complex III and IV had hardly any effect. By contrast, dehydroascorbate (DHA) enabled the mitochondria to gain ATP from sulfide oxidation even if the sulfide concentration far exceeded the threshold for inhibition of cytochrome oxidase. In the presence of dehydroascorbate, respiratory rates were independent of sulfide concentrations, with a respiratory control ratio of 2.1+/-0.2, and both oxygen consumption and ATP production were completely inhibited by myxothiazol and sodium azide but only marginally by SHAM. The present data indicate that a redox mechanism may contribute to the regulation of sulfide oxidation in lugworm mitochondria in vivo. Thus, mitochondria are presumably much more sulfide resistant in a cellular context than previously thought.

Nitrogen fixation in legumes is dramatically inhibited by abiotic stresses, and this reduction is often associated with oxidative damage. Although ascorbate (ASC) has been firmly associated with antioxidant defence, recent studies have suggested that the functions of ASC are related primarily to developmental processes. This study examines the hypothesis that ASC is involved in alleviating the oxidative damage to nodules caused by an increase in reactive oxygen species (ROS) under water stress. The hypothesis was tested by supplying 5mM ASC to pea plants (Pisum sativum L.) experiencing moderate water stress (ca. -1 MPa) and monitoring plant responses in relation to those experiencing the same water stress without ASC. A supply of exogenous ASC increased the nodule ASC+dehydroascorbate (DHA) pool compared to water-stressed nodules without ASC, and significantly modulated the response to water stress of the unspecific guaiacol peroxidase (EC 1.11.1.7) in leaves and nodules. However, ASC supply did not produce recovery from water stress in other nodule antioxidant enzymes, nodule carbon and nitrogen enzymes, or nitrogen fixation. The supply of the immediate ASC precursor, galactono-1,4-lactone (GL), increased the nodule ASC+DHA pool, but also failed to prevent the decline of nitrogen fixation and the reduction of carbon flux in nodules. These results suggest that ASC has a limited role in preventing the negative effects of water stress on nodule metabolism and nitrogen fixation.

Objective: Oxidative damage has been implicated in the pathogenesis of cerebral ischemia. We previously demonstrated that exogenously supplied dehydroascorbic acid (DHA), an oxidized, blood-brain barrier transportable form of the antioxidant ascorbic acid (AA), improves outcome after experimental stroke., Methods: To investigate the neuroprotective effect of DHA therapy, we measured cerebral AA levels using a novel assay, quantified markers of lipid peroxidation, and evaluated infarct volume after reperfused stroke in a murine model. All experiments were performed using a new citrate/sorbitol-stabilized DHA formulation to improve the stability of the compound., Results: Intraparenchymal AA levels declined after cerebral ischemia/reperfusion and were repleted in a dose-dependent fashion by postischemic administration of intravenous DHA (P < 0.01). Repletion of these levels was associated with reductions in cerebral malondialdehyde levels (P < 0.05), which were also elevated after reperfused stroke. DHA repletion of interstitial AA levels and reduction in cerebral lipid peroxidation was associated with dose-dependent reductions in infarct volume (P < 0.05)., Conclusion: Together, these results indicate that an intravenous cerebroprotective dose of citrate/sorbitol-stabilized DHA is correlated with increased brain ascorbate levels and a suppression of excessive oxidative metabolism.

A capillary apparatus for in vitro microdialysis was used to investigate melatonin and ascorbic acid effects on dopamine (DA) autoxidation or nitric oxide (NO)-mediated oxidation in suspended PC12 cells. Following high K+ (KCl 75 mm) infusion, secreted DA underwent a partial autoxidation or peroxynitrite-mediated oxidation when the potential peroxynitrite generator 3-morpholinosydnonimine (SIN-1, 1.0 mm) was co-infused with KCl. Ascorbic acid was supplied to the medium by means of intracellular reduction of infused dehydroascorbic acid (DHAA) (5.0 mm). Melatonin (50 microm) and DHAA showed a synergistic effect in inhibiting DA autoxidation and peroxynitrite-mediated DA oxidation. Moreover, melatonin increased dialysate recovery of ascorbic acid released from PC12 cells. Endogenous melatonin was depleted in rats maintained on a 24-hr light cycle for 1 wk. In melatonin-depleted rats, baseline levels of dialysate ascorbic acid were lower than controls, while those of DA were unaffected. In these rats, intrastriatal infusion of 5.0 mm SIN-1 induced DA increases significantly lower than in controls; in addition, dialysate ascorbic acid concentrations exhibited significant decreases. Melatonin co-infusion restored SIN-1 effects on dialysate DA and antagonized SIN-1-induced ascorbic acid decreases. Melatonin-depleted rats were allowed to recover. In these rats, striatal baseline ascorbic acid, as well as SIN-1-induced increases in dialysate DA did not differ from controls. Taken together, these findings suggest that endogenous melatonin is an active component of the striatal extracellular antioxidant pool, as it maintains endogenous ascorbic acid in its reduced status and co-operates with ascorbic acid in protecting extracellular DA from exogenous NO-mediated oxidation.