Vitamin C inhibits hypoxia-induced damage and apoptotic signaling pathways in cardiomyocytes and ischemic hearts

Reactive oxygen species play a central role in myocardial ischemic injury and are a target for therapeutic intervention. Vitamin C is an essential antioxidant yet difficult to deliver in pharmacologic concentration to the myocardium. We found that adult rat cardiomyocytes accumulate vitamin C by transporting dehydroascorbic acid (DHA), the oxidized form of vitamin C, but do not transport ascorbic acid. Loading cells with vitamin C by DHA treatment resulted in resistance to hypoxia- and hypoxia/reoxygenation-induced cell death associated with the quenching of reactive oxygen species. When rats were injected with DHA before coronary occlusion, the ascorbic acid content in the heart was six to eight times higher than in untreated controls and myocardial infarction was reduced by 62%. DHA also provided significant protection when administered intravenously 2 h after coronary occlusion. In cardiomyocytes subjected to hypoxia/reoxygenation, DHA treatment resulted in decreased apoptosis associated with inhibition of Bax expression, caspase-3 activation, and cytochrome c translocation into the cytoplasm. DHA treatment also inhibited Jak2, STAT1, and STAT5 phosphorylation, and increased STAT3 phosphorylation, in hypoxic cardiomyocytes and ischemic myocardial tissue. Our findings suggest that DHA may be useful as a cardioprotectant in ischemic heart disease.     

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

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

Anti-cancer effects of vitamin C revisited

Vitamin C was first suggested to have cancer-fighting properties in the 1930s and has been the subject of controversy ever since. Despite repeated reports of selective cancer cell toxicity induced by high-dose vitamin C treatment in vitro and in mouse models, the mechanism of action has remained elusive.Yun et al.¹ have recently shed light on what was until now the elusive mechanism by which vitamin C (aka ascorbate) induces toxicity in selected oncogene-driven cancers. They reported that in cells with mutations of KRAS or BRAF, death is not caused by vitamin C itself, but rather by its oxidized form dehydroascorbate (DHA). Whereas vitamin C enters cells through sodium cotransporters, DHA competes with glucose for uptake through glucose transporters (particularly GLUT1 and GLUT4) and then is reduced back to vitamin C in cells² (Figure 1). It was previously observed that while melanoma cell lines take up DHA at much higher rates than vitamin C, normal melanocytes do not, demonstrating that transformation-driven upregulation of GLUTs leads to increased uptake of DHA³. More recently, using Magnetic Resonance Spectroscopy Imaging, it was demonstrated that hyperpolarized ¹³C-labeled DHA is rapidly taken up by cancer cells and converted to vitamin C, illustrating the tumors'' reducing state⁴. Yun et al.¹ now show that the reduction of DHA back to vitamin C is at the crux of the vitamin C-induced cell death observed in these cancer cells.Open in a separate windowFigure 1Mechanistic overview of proposed vitamin C toxicity in CRCs driven by KRAS and BRAF mutations. KRAS and BRAF mutations induce metabolic reprogramming by upregulating GLUT1, glucose uptake, and glycolytic flux. Upon vitamin C treatment and its extracellular oxidation, DHA (the oxidized form of vitamin C) is taken up through GLUT1 and is reduced back to vitamin C in the cells, depleting GSH and NADPH. Consequently, an increase in ROS leads to GAPDH oxidation, and with it, to a decrease in glycolytic flux. In parallel, ROS-mediated oxidative DNA damage induces PARP activation and subsequently, NAD⁺ levels fall and cause additional inhibition of GAPDH and glycolysis, resulting in energy crisis and cell death.Mutations in KRAS or BRAF are found in approximately half of the cases of colorectal cancer (CRC) and their expression correlates with an increase in GLUT1 expression, glucose uptake and reliance on glycolysis. Yun et al.¹ observed that vitamin C is oxidized to DHA in tissue culture media and that KRAS or BRAF mutated CRC cell lines take up more DHA compared to their wild-type counterparts. More importantly, they found that DHA induces death in the mutant lines, but not in wild-type counterparts overexpressing GLUT1, suggesting that additional oncogenic reprogramming is necessary for DHA-induced toxicity. The authors then profiled metabolic changes after treatment with vitamin C. In cells with KRAS or BRAF mutations, they found an accumulation of the glycolytic intermediates upstream of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) whereas those downstream of GAPDH were depleted. This indicated a decrease in GAPDH activity and a concomitant diversion of glucose into the oxidative phase of the pentose phosphate pathway (PPP), a metabolic shift that, upon oxidative stress, helps restore NADPH levels and cellular reducing potential (Figure 1). Indeed, Yun and co-workers found that the intracellular reduction of DHA back to vitamin C depleted the cellular stores of reduced glutathione (GSH, the major antioxidant in cells), leading to an increase in reactive oxygen species (ROS). Furthermore, they found that, upon exposure to DHA, GAPDH itself was oxidized (on Cys¹⁵²), and consequently inhibited. Inhibition of GAPDH caused energy stress in the highly glycolytic mutant cell lines, leading to cell death. In mice harboring tumor xenografts with either KRAS or BRAF mutations, treatment with high doses of vitamin C reduced tumor size. Treatment also reduced the number and size of polyps in an Apc-driven transgenic mouse model of intestinal cancer, but again, only in tumors expressing mutant Kras. Moreover, in addition to showing the direct inhibition of GAPDH by oxidation, the authors demonstrated that its activity is further hindered by DHA-induced NAD⁺ depletion, since GAPDH activity relies on the availability of NAD⁺ as a co-substrate. The major NAD⁺ consumer, the DNA repairing enzyme poly ADP-ribose polymerase (PARP) was then investigated. It was found that the increase in ROS after high-dose vitamin C treatment also induced DNA damage and PARP activation in KRAS or BRAF transformed cells. Providing the cells with a PARP inhibitor or an NAD⁺ precursor partially rescued their viability. Thus, ROS cause the inhibition of GAPDH activity in cells on two fronts: first, via inducing its direct oxidation and second, by causing NAD⁺ depletion (Figure 1). Yun et al.¹ thus demonstrated an intricate mechanism by which oncogenic reprogramming, which causes glycolysis addiction, induces a metabolic vulnerability which can be exploited with high doses of DHA that elevate intracellular ROS as it is converted back to vitamin C.Despite numerous clinical studies, the anti-cancer property of vitamin C has remained controversial. Potential translation of the mechanism presented by Yun et al.¹ to therapeutic application raises concerns regarding toxicities of high-dose vitamin C treatment. Though the authors do not report side effects in their mice (treated daily with 4-8 g/kg body weight IP), the upper dose equates to over half a kilogram for the average person. High-dose oral supplementation of vitamin C is associated with increased kidney stone incidence, and clinical studies demonstrated significant renal, cardiac, and metabolic toxicity upon vitamin C administration. Still, overall reports of toxicity are variable, poorly graded, and therefore inconclusive^5,6. Affinity studies of DHA for GLUT1 may help establish a lower effective dose, though unwanted side effects in tissues that highly express GLUT1 need to be considered. The brain obtains vitamin C by uptake of DHA through GLUT1 at the blood-brain barrier and its subsequent reduction⁷. Erythrocytes express high levels of GLUT1 and are crucial for ascorbate recycling, keeping DHA levels low⁸. Importantly, erythrocytes rely solely on glycolysis for energy production. Thus, high DHA levels may induce brain toxicity and haemolysis via mechanisms similar to those described by Yun et al.¹.Though vitamin C oxidizes rapidly in tissue culture media, it acts mainly as an antioxidant in vivo. It remains unclear how and where circulating vitamin C is oxidized in vivo, an issue not addressed by Yun et al.¹. Oxidation of vitamin C to DHA by tumor stroma has been suggested⁹, complicating the ability to predict tumor responsiveness to the treatment. As such, without being able to control the extent of vitamin C oxidation to DHA, effectiveness and toxicity of vitamin C treatment cannot be predicted.Finally, the authors report that, following DHA uptake, NAD⁺ depletion by PARP activation contributes to the inhibition of glycolysis (and potentially to the stimulation of oxidative PPP flux). The observation that PARP inhibition partly rescues vitamin C-treated cells may suggest that the toxic effect of DHA uptake is not caused by ROS alone, since restoring NAD⁺ levels and glycolysis with the PARP inhibitor may actually decrease PPP flux and NADPH production, and aggravate the redox stress. It remains to be demonstrated that PARP inhibition indeed restores NAD⁺ levels in vitamin C-treated cells, and how this affects the balance between energy and redox metabolism. This also raises the question whether PARP activity in BRCA1/2-deficient tumors produces a similar metabolic phenotype via NAD⁺ depletion and whether the use of a PARP inhibitor (e.g., olaparib) to treat these tumors¹⁰ might restore NAD⁺ levels and counterbalance GAPDH inhibition by other oxidative agents.In summary, Yun et al.¹ show that, in glycolysis-addicted KRAS and BRAF driven cancer cells, high-dose vitamin C treatment induces cell death via the uptake and reduction of its oxidized form DHA back to vitamin C. DHA reduction, through scavenging GSH, induces oxidative stress, leading to GAPDH inactivation, inhibition of glycolysis and the subsequent energy crisis and cell death. This study further elucidates the mechanism by which ROS can induce cell death, and neatly shows how vitamin C, an antioxidant, can work as a double edged sword. However, further work is necessary to determine whether there is a therapeutic potential for vitamin C in cancer patients.     

Early effects of hypoxia/reoxygenation on VEGF,Ang-1, Ang-2 and their receptors in the rat myocardium: Implications for myocardial angiogenesis

The oxidized form of vitamin C (dehydroascorbic acid, DHA) completely and irreversibly inactivates recombinant human hexokinase type I, in a pseudo-first order fashion. The inactivation reaction occurs without saturation, indicating that DHA does not form a reversible complex with hexokinase. Further characterization of this response revealed that the inactivation does not require oxygen and that dithiothreitol, while able to prevent the DHA-mediated loss of enzyme activity, failed to restore the activity of the DHA-inhibited enzyme. Inactivation was not associated with cleavage of the peptide chain or cross-linking. The decay in enzymatic activity was however both dependent on deprotonation of a residue with an alkaline pK_a and associated with covalent binding of DHA to the protein. In addition, inactivation of hexokinase decreased or increased, respectively, in the presence of the substrates glucose or MgATP. Finally, amino acid analysis of the DHA-modified hexokinase revealed a decrease of cysteine residues.Taken together, the above results are consistent with the possibility that covalent binding of the reagent with a thiol group of cysteine is a critical event for the DHA-mediated loss of hexokinase activity.     

Vitamin C is a kinase inhibitor: dehydroascorbic acid inhibits IkappaBalpha kinase beta

Reactive oxygen species (ROS) are key intermediates in cellular signal transduction pathways whose function may be counterbalanced by antioxidants. Acting as an antioxidant, ascorbic acid (AA) donates two electrons and becomes oxidized to dehydroascorbic acid (DHA). We discovered that DHA directly inhibits IkappaBalpha kinase beta (IKKbeta) and IKKalpha enzymatic activity in vitro, whereas AA did not have this effect. When cells were loaded with AA and induced to generate DHA by oxidative stress in cells expressing a constitutive active IKKbeta, NF-kappaB activation was inhibited. Our results identify a dual molecular action of vitamin C in signal transduction and provide a direct linkage between the redox state of vitamin C and NF-kappaB signaling events. AA quenches ROS intermediates involved in the activation of NF-kappaB and is oxidized to DHA, which directly inhibits IKKbeta and IKKalpha enzymatic activity. These findings define a function for vitamin C in signal transduction other than as an antioxidant and mechanistically illuminate how vitamin C down-modulates NF-kappaB signaling.     

D-4F reduces EO6 immunoreactivity, SREBP-1c mRNA levels, and renal inflammation in LDL receptor-null mice fed a Western diet

LDL receptor-null (LDLR(-/-)) mice on a Western diet (WD) develop endothelial dysfunction and atherosclerosis, which are improved by the apolipoprotein A-I (apoA-I) mimetic peptide D-4F. Focusing on the kidney, LDLR(-/-)mice were fed a WD with D-4F or the inactive control peptide scrambled D-4F (ScD-4F) added to their drinking water. The control mice (ScD-4F) developed glomerular changes, increased immunostaining for MCP-1/CCL2 chemokine, increased macrophage CD68 and F4/80 antigens, and increased oxidized phospholipids recognized by the EO6 monoclonal antibody in both glomerular and tublo-interstitial areas. All of these parameters were significantly reduced by D-4F treatment, approaching levels found in wild-type C57BL/6J or LDLR(-/-) mice fed a chow diet. Sterol-regulatory element binding protein-1c (SREBP-1c) mRNA levels and triglyceride levels were elevated in the kidneys of the control mice (ScD-4F) fed the WD compared with C57BL/6J and LDLR(-/-) mice on chow (P < 0.001 and P < 0.001, respectively) and compared with D-4F-treated mice on the WD (P < 0.01). There was no significant difference in plasma lipids, lipoproteins, glucose, blood pressure, or renal apoB levels between D-4F- and ScD-4F-treated mice. We conclude that D-4F reduced renal oxidized phospholipids, resulting in lower expression of SREBP-1c, which, in turn, resulted in lower triglyceride content and reduced renal inflammation.     

Human choroid plexus papilloma cells efficiently transport glucose and vitamin C

In vitro and in vivo studies suggest that the basolateral membrane of choroid plexus cells, which is in contact with blood vessels, is involved in the uptake of the reduced form of vitamin C, ascorbic acid (AA), through the sodium‐vitamin C cotransporter, (SVCT2). Moreover, very low levels of vitamin C were observed in the brains of SVCT2‐null mice. The oxidized form of vitamin C, dehydroascorbic acid (DHA), is incorporated through the facilitative glucose transporters (GLUTs). In this study, the contribution of SVCT2 and GLUT1 to vitamin C uptake in human choroid plexus papilloma (HCPP) cells in culture was examined. Both the functional activity and the kinetic parameters of GLUT1 and SVCT2 in cells isolated from HCPP were observed. Finally, DHA uptake by GLUT1 in choroid plexus cells was assessed in the presence of phorbol‐12‐myristate‐13‐acetate (PMA)‐activated human neutrophils. A marked increase in vitamin C uptake by choroid plexus cells was observed that was associated with superoxide generation and vitamin C oxidation (bystander effect). Thus, vitamin C can be incorporated by epithelial choroid plexus papilloma cells using the basolateral polarization of SVCT2 and GLUT1. This mechanism may be amplified with neutrophil infiltration (inflammation) of choroid plexus tumors.

     

Ascorbic acid depletion enhances expression of the sodium-dependent vitamin C transporters, SVCT1 and SVCT2, and uptake of ascorbic acid in livers of SMP30/GNL knockout mice

In this study, we examined whether ascorbic acid (AA) and dehydroascorbic acid (DHA), the oxidized form of AA, levels in tissues regulate the AA transporters, sodium-dependent vitamin C transporters (SVCT) 1 and SVCT2 and DHA transporters, glucose transporter (GLUT) 1, GLUT3, GLUT4 mRNA by using senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice. These mice are incapable of synthesizing AA in vivo. AA depletion enhanced SVCT1 and SVCT2 mRNA expression in the liver and SVCT1 and GLUT4 mRNA expression in the small intestine, but not in the cerebrum or kidney. Next, we examined the actual impact of AA uptake by using primary cultured hepatocytes from SMP30/GNL KO mice. In the AA-depleted hepatocytes from SMP30/GNL KO mice, AA uptake was significantly greater than in matched cultures from wild-type mice. These results strongly affirm that intracellular AA is an important regulator of SVCT1 and SVCT2 expression in the liver.     

Some vertebrates go with the GLO

Most vertebrates synthesize vitamin C (ascorbate) de novo from glucose, but humans and certain other mammals cannot. In this issue, Montel-Hagen et al. (2008) demonstrate that erythrocytes from these ascorbate auxotrophs switch the preference of their glucose transporter Glut1 from glucose to dehydroascorbate (DHA), the oxidized form of vitamin C. This substrate preference switch is mediated by the membrane protein stomatin and is an evolutionary adaptation to vitamin C deficiency.     

Effects of organosulfurs, isothiocyanates and vitamin C towards hydrogen peroxide-induced oxidative DNA damage (strand breaks and oxidized purines/pyrimidines) in human hepatoma cells

The aim of this study was to evaluate the effects of organosulfurs, isothiocyanates and vitamin C towards hydrogen peroxide-induced DNA damage (DNA strand breaks and oxidized purines/pyrimidines) in human hepatoma cells (HepG2), using the Comet assay. Treatment with hydrogen peroxide (H(2)O(2)) increased the levels of DNA strand breaks and oxidized purine and pyrimidine bases, in a concentration and time dependent manner. Organosulfur compounds (OSCs) reduced DNA strand breaks induced by H(2)O(2). In addition, OSCs also decreased the levels of oxidized pyrimidines. However, none of the OSCs tested reduced the levels of oxidized purines. Isothiocyanates compounds (ITCs) and vitamin C showed protective effects towards H(2)O(2)-induced DNA strand breaks and oxidized purine and pyrimidine bases. The results indicate that removal of oxidized purine and pyrimidine bases by ITCs was more efficient than by OSCs and vitamin C. Our findings suggest that OSCs, ITCs and vitamin C could exert their protective effects towards H(2)O(2)-induced DNA strand breaks and oxidative DNA damage by the free radical-scavenging efficiency of these compounds.     

Effects of vitamin C intake on gingival oxidative stress in rat periodontitis

Increased levels of oxidative stress due to excessive production of reactive oxygen species are involved in the pathogenesis of periodontitis. Studies suggest a negative association between plasma vitamin C level and the severity of periodontitis. We hypothesized that increases in plasma vitamin C levels after vitamin C intake might clinically reduce gingival oxidative stress in a rat periodontitis model. A ligature was placed around rat mandibular molars for 4 weeks to induce periodontitis, and the rats were then given drinking water with or without 1 g/L vitamin C for 2 weeks after the ligature was removed. The periodontitis-induced rats showed a 149% increase in 8-hydroxydeoxyguanosine level and a 40% decrease in reduced:oxidized glutathione ratio in gingival tissue. Vitamin C intake induced a 175% increase in plasma vitamin C level, resulting in an improvement in the gingival 8-hydroxydeoxyguanosine level (decreased) and in the reduced:oxidized glutathione ratio (increased). Furthermore, in ligature-induced periodontitis lesions, gene expression encoding inflammation, including interleukin-1 alpha and interleukin-1 beta, was more than twofold down-regulated by vitamin C intake. The results suggest that systemic administration of vitamin C could be clinically beneficial in improving periodontitis-induced oxidative stress by down-regulating inflammatory gene expression.     

Role of dietary amino acid balance in diet restriction‐mediated lifespan extension,renoprotection, and muscle weakness in aged mice

Extending healthy lifespan is an emerging issue in an aging society. This study was designed to identify a dietary method of extending lifespan, promoting renoprotection, and preventing muscle weakness in aged mice, with a focus on the importance of the balance between dietary essential (EAAs) and nonessential amino acids (NEAAs) on the dietary restriction (DR)‐induced antiaging effect. Groups of aged mice were fed ad libitum, a simple DR, or a DR with recovering NEAAs or EAAs. Simple DR significantly extended lifespan and ameliorated age‐related kidney injury; however, the beneficial effects of DR were canceled by recovering dietary EAA but not NEAA. Simple DR prevented the age‐dependent decrease in slow‐twitch muscle fiber function but reduced absolute fast‐twitch muscle fiber function. DR‐induced fast‐twitch muscle fiber dysfunction was improved by recovering either dietary NEAAs or EAAs. In the ad libitum‐fed and the DR plus EAA groups, the renal content of methionine, an EAA, was significantly higher, accompanied by lower renal production of hydrogen sulfide (H₂S), an endogenous antioxidant. Finally, removal of methionine from the dietary EAA supplement diminished the adverse effects of dietary EAA on lifespan and kidney injury in the diet‐restricted aged mice, which were accompanied by a recovery in H₂S production capacity and lower oxidative stress. These data imply that a dietary approach could combat kidney aging and prolong lifespan, while preventing muscle weakness, and suggest that renal methionine metabolism and the trans‐sulfuration pathway could be therapeutic targets for preventing kidney aging and subsequently promoting healthy aging.     

Preferential uptake and accumulation of oxidized vitamin C by THP-1 monocytic cells.

THP-1 cells preferentially accumulate vitamin C in its oxidized form. The uptake displays first-order kinetics and leads to a build-up of an outward concentration gradient which is stable in the absence of extracellular vitamin. The transport is faster than reduction by extracellular glutathione or by added cytosolic extract, and glutathione-depleted cells show the same uptake rates as control cells. In addition, energy depletion or oxidation of intracellular sulfhydryls does not inhibit accumulation of ascorbate. The accumulation, however, always occurs in the reduced form. The affinity for dehydroascorbate is lower (Km 450 microM vs 60 microM) than for reduced ascorbate, but the maximal rate is more than 30 times higher (581 compared to 19 pmol.min-1 per 106 cells), and it is independent of sodium, whereas the uptake of ascorbate is not. The sodium gradient also allows accumulation of reduced ascorbate. Inhibitors of glucose transport by the GLUT-1 transporter also inhibit uptake of dehydroascorbate (DHA), but there are some inconsistencies, because the Ki-values are higher than reported for the isolated transporter and one inhibitor (deoxyglucose) is noncompetitive. The preferential uptake of the dehydro-form of the vitamin may be useful for situations where this short-lived metabolite is formed by oxidation in the environment.     

Effects of diet and age on oxidative damage products in healthy subjects

Damage of molecules as a consequence of oxidative stress has been implicated in the pathogenesis of chronic diseases related to aging. Diet is a key environmental factor affecting the incidence of many chronic diseases. Antioxidant substances in diet enhance the DNA, lipid and protein protection by increasing the scavenging of free radicals. Products of oxidative damage of DNA (DNA strand breaks with oxidized purines or oxidized pyrimidines), lipids (conjugated dienes of fatty acids) and proteins (carbonyls) in relation to nutrition (vegetarian diet vs. non-vegetarian, traditional mixed diet) were measured in young women aged 20-30 years (46 vegetarians, 48 non-vegetarians) vs. older women aged 60-70 years (33 vegetarians, 34 non-vegetarians). In young subjects, no differences in values of oxidative damage as well as plasma values of antioxidative vitamins (C,beta-carotene) were observed between vegetarian and non-vegetarian groups. In older vegetarian group significantly reduced values of DNA breaks with oxidized purines, DNA breaks with oxidized pyrimidines and lipid peroxidation and on the other hand, significantly increased plasma values of vitamin C and beta-carotene were found compared to the respective non-vegetarian group. Significant age dependences of measured parameters (increase in all oxidative damage products and decrease in plasma vitamin concentrations in older women) were noted only in non-vegetarians. Vegetarian values of older women vs. young women were similar or non-significantly changed. The results suggest that increase of oxidative damage in aging may be prevented by vegetarian nutrition.     

Cellular pathways for transport and efflux of ascorbate and dehydroascorbate

The mechanisms allowing the cellular transport of ascorbic acid represent a primary aspect for the understanding of the roles played by this vitamin in pathophysiology. Considerable research effort has been spent in the field, on several animal models and different cell types. Several mechanisms have been described to date, mediating the movements of different redox forms of ascorbic acid across cell membranes. Vitamin C can enter cells both in its reduced and oxidized form, ascorbic acid (AA) and dehydroascorbate (DHA), utilizing respectively sodium-dependent transporters (SVCT) or glucose transporters (GLUT). Modulation of SVCT expression and function has been described by cytokines, steroids and post-translational protein modification. Cellular uptake of DHA is followed by its intracellular reduction to AA by several enzymatic and non-enzymatic systems. Efflux of vitamin C has been also described in a number of cell types and different pathophysiological functions were proposed for this phenomenon, in dependence of the cell model studied. Cellular efflux of AA is mediated through volume-sensitive (VSOAC) and Ca²⁺-dependent anion channels, gap-junction hemichannels, exocytosis of secretory vesicles and possibly through homo- and hetero-exchange systems at the plasma membrane level. Altogether, available data suggest that cellular efflux of ascorbic acid - besides its uptake - should be taken into account when evaluating the cellular homeostasis and functions of this important vitamin.     

Crosstissue coexpression network of aging

Aging is characterized by the interlocking decay of biological functions over time. Microarrays have been successful in elucidating some of the genome-wide changes that occur with age. Using the AGEMAP dataset that catalogs changes in gene expression as a function of age in 16 tissues in mice, we identified tissue-specific aging genes. Coordinated aging processes across different tissues then were clarified in crosstissue coexpression networks on both the gene and pathway levels. Our findings provide more concrete information about coordinated aging across different tissues. By bridging gene-level and tissue-level research, this study could help identify targets for attenuation of critical aging-related genes, pathways, or networks for antiaging intervention.     

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.     

Vitamin C and vitamin E restore the resistance of GSH-depleted lens cells to H2O2

A decline in reduced glutathione (GSH) levels is associated with aging and many age-related diseases. The objective of this study was to determine whether other antioxidants can compensate for GSH depletion in protection against oxidative insults. Rabbit lens epithelial cells were depleted of > 75% of intracellular GSH by 25-200 microM buthionine sulfoximine (BSO). Depletion of GSH by BSO alone had little direct effect on cell viability, but resulted in an approximately 30-fold increase in susceptibility to H(2)O(2)-induced cell death. Experimentally enhanced levels of nonprotein sulfhydryls other than GSH (i.e., N-acetylcysteine) did not protect GSH-depleted cells from H(2)O(2)-induced cell death. In contrast, pretreatment of cells with vitamin C (25-50 microM) or vitamin E (5-40 microM), restored the resistance of GSH-depleted cells to H(2)O(2). However, concentrations of vitamin C > 400 microM and vitamin E > 80 microM enhanced the toxic effect of H(2)O(2). Although levels of GSH actually decreased by 10-20% in cells supplemented with vitamin C or vitamin E, the protective effects of vitamin C and vitamin E on BSO-treated cells were associated with significant ( approximately 70%) decreases in oxidized glutathione (GSSG) and concomitant restoration of the cellular redox status (as indicated by GSH:GSSG ratio) to levels detected in cells not treated with BSO. These results demonstrate a role for vitamin C and vitamin E in maintaining glutathione in its reduced form. The ability of vitamin C and vitamin E in compensations for GSH depletion to protect against H(2)O(2)-induced cell death suggests that GSH, vitamin C, and vitamin E have common targets in their actions against oxidative damage, and supports the preventive or therapeutic use of vitamin C and E to combat age- and pathology-associated declines in GSH. Moreover, levels of these nutrients must be optimized to achieve the maximal benefit.