The formation of hydroxyproline in collagen by cells grown in the absence of serum

L-929 and 3T6 cells were conditioned to grow in a chemically defined medium lacking serum and ascorbate. Serum, when added, had a small stimulatory effect on the growth rate of the cells, but ascorbate had no effect either on the growth rate or on the rate of protein synthesis. These cells were also shown to lack gulonolactone oxidase activity and therefore could not synthesize their own ascorbate. Nevertheless, in the absence of serum and ascorbate both cell types were able to hydroxylate peptidyl proline to an appreciable extent. This suggest that reductant other than ascorbate can at least partially satisfy the requirement for a reductant in the prolyl hydroxylase reaction in vivo.     

Effect of ascorbate on oxygen uptake and growth of Escherichia coli B

The addition of ascorbate to aerobically growing cultures of Escherichia coli B caused only a short pause in growth and no subsequent change in the rate or extent of growth. The effect of ascorbate on oxygen uptake varied from inhibition in minimal medium to stimulation in rich medium. Cyanide-resistant growth and oxygen uptake were stimulated by ascorbate. Both the rate and extent of anaerobic growth were stimulated in proportion to the amount of ascorbate added when fumarate was the terminal electron acceptor. Ascorbate had no effect on any aspect of anaerobic growth in the absence of a terminal electron acceptor or in the presence of nitrate.     

Deleterious synergistic effects of ascorbate and copper on the development of Plasmodium falciparum: an in vitro study in normal and in G6PD-deficient erythrocytes

The effects of ascorbate and copper on the development of Plasmodium falciparum were studied in two modes: pretreatment of uninfected erythrocytes followed by infection by P. falciparum and treatment of parasitized erythrocytes. Pretreatment of G6PD(+) cells with ascorbate caused a slight enhancement in parasite development, while in G6PD(-) cells a suppressive effect on the plasmodia was demonstrated. Copper alone interfered with parasite growth in both cell types. The combination of copper and ascorbate arrested parasite maturation, an effect which was more pronounced in G6PD(-) cells. Synergism between copper and ascorbate was better demonstrated following the treatment of infected erythrocytes: while ascorbate alone supported parasite development and copper alone had only a marginal suppressive effect, the combination of copper and ascorbate yielded a marked inhibition of parasite growth. Ascorbate proved destructive to the parasites in the presence of adventitious copper, or on the second day of the parasite life cycle. In these cases it acted as a pro-oxidant, while in other systems, in particular in the presence of a chelator, ascorbate acted as an antioxidant and promoted parasite growth. The understanding of the role of transition metals and free radicals in parasite development and injury could shed light on novel approaches to fight malaria.     

Ascorbate free radical and its role in growth control

Summary Ascorbate and its free radical potentiates proliferation of HL-60 cells in serum-limiting media. Dehydroascorbate does not affect growth. This stimulation of growth is due to a general shortening of the cell cycle. The incubation of HL-60 cells with ascorbate free radical produces a significant change of the redox potential of cells. The presence of cells in culture media avoids the total oxidation of ascorbate, and also HL-60 cells induce the short-term stabilization of ascorbate. Ascorbate free radical potentiates also the onset of DNA synthesis in CCL39 cells induced by fetal calf serum, although itself does not affect quiescense to proliferation transition. This transition induced by fetal calf serum also potentiates the capacity of CCL39 cells to stabilize ascorbate. We discuss here the role of ascorbate free radical on growth control by its reduction by the plasma membrane redox system and its meaning for cell physioslogy.     

The effect of aging and increasing ascorbate concentrations on respiratory chain activity in cultured human fibroblasts

The specific activities of Complexes I‐III, II‐III, and IV of the respiratory chain, and citrate synthase, were determined in mitochondrial sonicates of six control passage 5 fibroblast cultures, cultivated in growth medium containing fetal calf serum as the only source of ascorbate. The enzymes were also assayed in serially subcultured fibroblasts which were characterized as aged at passage 20 and beyond. Results indicated a significant loss of all enzyme activities in aged cells at passage 20, 25, and 30. Further studies involved maintenance of serially subcultured cells in serum free media to which increasing ascorbate concentrations (100, 200, and 300 µmol 1^?1) were added. Results indicated that ascorbate at 100 µmol 1^?1 was not sufficient to restore any of the enzyme activities in aged cells. An ascorbate concentration of 200 µmol 1^?1 however, could totally restore Complex IV and citrate synthase activities, but had no effect on complexes I‐III and II‐III activities which required 300 µmol 1^?1 ascorbate to be partially or totally restored respectively. In conclusion, this study demonstrates an age related drop in mitochondrial respiratory chain activity in cultured human fibroblasts. Enzyme activities could be completely or partially restored in the presence of double or triple normal human plasma ascorbate concentrations. Copyright © 2010 John Wiley & Sons, Ltd.     

The effect of intracellular ascorbate on the susceptibility of HL60 and Jurkat cells to chemotherapy agents

Chemotherapy agents initiate tumour cell apoptosis and this is thought to involve oxidative stress. In this study we have investigated the effect of the important antioxidant Vitamin C (ascorbate) on the response of HL60 and Jurkat cells to three chemotherapy drugs, namely etoposide, melphalan and arsenic trioxide (As₂O₃). Cells grown in routine culture media are deficient in ascorbate and to determine its effect on chemotherapy drug-induced apoptosis we supplemented the cells prior to drug exposure. We found that ascorbate had a varied effect on apoptosis and cell cycle progression. Etoposide-induced apoptosis in HL60 cells was significantly increased in ascorbate-loaded cells as measured by caspase-3 activation and DNA degradation, and this appeared to reflect a decrease in the number of necrotic cells rather than increased cytotoxicity. In contrast, ascorbate had no effect on etoposide-induced apoptosis in Jurkat cells. In both cell types melphalan-induced apoptosis was unaffected by intracellular ascorbate, whereas both apoptosis and growth arrest with low concentrations of As₂O₃ were diminished. These results indicate that intracellular ascorbate can affect cell responses to chemotherapy drugs in a complex and somewhat unpredictable manner and that it may play an important role in the responsiveness of tumour cells to chemotherapy regimes. This study was supported by the Health Research Council of New Zealand.     

Copper catalyzed oxidation of ascorbate (vitamin C). Inhibitory effect of catalase, superoxide dismutase, serum proteins (ceruloplasmin, albumin, apotransferrin) and amino acids

The inhibitory effect of catalase and superoxide dismutase on copper catalyzed oxidation of ascorbate is probably due to a binding of copper ions. Scavengers of hydroxyl ions and singlet oxygen had no effect on the ascorbate oxidation rate. Copper binding serum proteins reduced the oxidation rate; the order of effectiveness being: Ceruloplasmin greater than human albumin = bovine albumin greater than apotransferrin. The excellent protection obtained with catalase and ceruloplasmin is possibly due to a strong affinity for cuprous ions generated during the reaction. Cupric ion binding amino acids (His, Thr, Glu, Gln, Tyr) had considerably weaker protective effect than the proteins studied. Apparently they do not compete favorably with ascorbate for cupric ions.     

Ascorbate enhances the toxicity of the photodynamic action of Verteporfin in HL-60 cells

As a reducing agent, ascorbate serves as an antioxidant. However, its reducing function can in some settings initiate an oxidation cascade, i.e., seem to be a "pro-oxidant." This dichotomy also seems to hold when ascorbate is present during photosensitization. Ascorbate can react with singlet oxygen, producing hydrogen peroxide. Thus, if ascorbate is present during photosensitization the formation of highly diffusible hydrogen peroxide could enhance the toxicity of the photodynamic action. On the other hand, ascorbate could decrease toxicity by converting highly reactive singlet oxygen to less reactive hydrogen peroxide, which can be removed via peroxide-removing systems such as glutathione and catalase. To test the influence of ascorbate on photodynamic treatment we incubated leukemia cells (HL-60 and U937) with ascorbate and a photosensitizer (Verteporfin; VP) and examined ascorbic acid monoanion uptake, levels of glutathione, changes in membrane permeability, cell growth, and toxicity. Accumulation of VP was similar in each cell line. Under our experimental conditions, HL-60 cells were found to accumulate less ascorbate and have lower levels of intracellular GSH compared to U937 cells. Without added ascorbate, HL-60 cells were more sensitive to VP and light treatment than U937 cells. When cells were exposed to VP and light, ascorbate acted as an antioxidant in U937 cells, whereas it was a pro-oxidant for HL-60 cells. One possible mechanism to explain these observations is that HL-60 cells express myeloperoxidase activity, whereas in U937 cells it is below the detection limit. Inhibition of myeloperoxidase activity with 4-aminobenzoic acid hydrazide (4-ABAH) had minimal influence on the phototoxicity of VP in HL-60 cells in the absence of ascorbate. However, 4-ABAH decreased the toxicity of ascorbate on HL-60 cells during VP photosensitization, but had no affect on ascorbate toxicity in U937 cells. These data demonstrate that ascorbate increases hydrogen peroxide production by VP and light. This hydrogen peroxide activates myeloperoxidase, producing toxic oxidants. These observations suggest that in some settings, ascorbate may enhance the toxicity of photodynamic action.     

Decreased extracellular matrix production in scurvy involves a humoral factor other than ascorbate

Our recent studies suggested that decreased collagen synthesis in bone and cartilage of scorbutic guinea pigs was not related to ascorbate-dependent proline hydroxylation. The decrease paralleled scurvy-induced weight loss and reduced proteoglycan synthesis. Those results led us to propose that the effects of ascorbate deficiency on extracellular matrix synthesis were caused by changes in humoral factors similar to those that occur in fasting. Here we present evidence for this proposal. Exposure of chick embryo chondrocytes to scorbutic guinea pig serum, in the presence of ascorbate, led to effects on extracellular matrix synthesis similar to those seen in scorbutic animals. The rates of collagen and proteoglycan synthesis were reduced to approximately 30-50% of the levels in cells cultured in normal guinea pig serum plus ascorbate, but proline hydroxylation and procollagen secretion were unaffected. Similar results were obtained with serum from fasted guinea pigs supplemented in vivo with ascorbate. The growth rate of the chondrocytes was not significantly affected by scorbutic guinea pig serum.     

Synergy between ascorbate and alpha-tocopherol on fibroblasts in culture

Ascorbate and tocopherol are important antioxidants that protect cells against oxidative stress. The interaction of ascorbate and alpha-tocopherol in cells is difficult to detect as both ascorbate and alpha-tocopherol are unstable in vitro in a biological medium. We examined the interactions between human dermal fibroblasts, ascorbate and alpha-tocopherol to determine the effects of the vitamins on growth and cell viability. The interaction of ascorbate and alpha-tocopherol was studied in a fibroblast culture medium during 48h. Ascorbate and alpha-tocopherol were detected by fluorimetry after high-performance liquid chromatography (HPLC). Cell growth and cell viability were studied by cell numeration after trypan blue staining. The ascorbate concentration fell in presence of alpha-tocopherol in cell culture medium under all experimental conditions, with or without cells. Ascorbate partly protected alpha-tocopherol but only in presence of cells. Cell viability was preserved by alpha-tocopherol whereas ascorbate enhanced fibroblast growth. The synergy between ascorbate and alpha-tocopherol corresponds to a consumption of ascorbate which spares alpha-tocopherol but only in presence of cells.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. Effects of the hexose monophosphate shunt as mediated by glutathione and ascorbate.

Lipid peroxidation and haemoglobin degradation were the two extremes of a spectrum of oxidative damage in red cells exposed to t-butyl hydroperoxide. The exact position in this spectrum depended on the availability of glucose and the ligand state of haemoglobin. In red cells containing oxy- or carbonmono-oxy-haemoglobin, hexose monophosphate-shunt activity was mainly responsible for metabolism of t-butyl hydroperoxide; haem groups were the main scavengers in red cells containing methaemoglobin. Glutathione, via glutathione peroxidase, accounted for nearly all of the hydroperoxide metabolizing activity of the hexose monophosphate shunt. Glucose protection against lipid peroxidation was almost entirely mediated by glutathione, whereas glucose protection of haemoglobin was only partly mediated by glutathione. Physiological concentrations of intracellular or extracellular ascorbate had no effect on consumption of t-butyl hydroperoxide or oxidation of haemoglobin. Ascorbate was mainly involved in scavenging chain-propagating species involved in lipid peroxidation. The protective effect of intracellular ascorbate against lipid peroxidation was about 100% glucose-dependent and about 50% glutathione-dependent. Extracellular ascorbate functioned largely without a requirement for glucose metabolism, although some synergistic effects between extracellular ascorbate and glutathione were observed. Lipid peroxidation was not dependent on the rate or completion of t-butyl hydroperoxide consumption but rather on the route of consumption. Lipid peroxidation appears to depend on the balance between the presence of initiators of lipid peroxidation (oxyhaemoglobin and low concentrations of methaemoglobin) and terminators of lipid peroxidation (glutathione, ascorbate, high concentrations of methaemoglobin).     

Effect of certain inhibitors of nucleic acid and protein synthesis on the growth and development of amine oxidase and ascorbate oxidase in germinating peas

5-Fluorodeoxyuridine(FUDR), actinomycin D(Act-D), puromycinaminonucleoside( puromycin) and 5-fluorouracil(5-FU) were usedto analyze the development of amine oxidase and ascorbate oxidasein, and the growth of pea seedlings. Both ascorbate oxidasedevelopment and growth were inhibited by administration of FUDR,and this effect was alleviated by the addition of thymidine.The development of amine oxidase was less sensitive to FUDR.Act-D and puromycin significantly inhibited both developmentof the enzyme and growth of the seedlings. On the other hand,5-FU inhibited growth of the seedlings and ascorbate oxiadsedevelopment, but had no remarkable effect on the synthesis ofamine oxidase. (Received September 30, 1974; )     

Partial reversal by sodium ascorbate of hyperoxia-induced damage to HEp-2 cell cultures

Summary Hyperoxia induced cellular damage was used as an experimental model system for examining the ameliorative role of antioxidants. Multiplication of HEp-2 cells in monolayer culture was inhibited after exposure to 100% O₂ either hyperbarically at 3 atm absolute (atma) or normobarically at 1 atma for periods from 15 s to 4 h. The inhibition was characterized by a slower rate of replication for a period from 1 to 3 d after exposure than in unexposed cultures, and then massive cellular death. Less killing followed exposure to normobaric O₂ than to hyperbaric O₂, and the shorter the period of exposure to hyperoxia the less killing. Addition of 100 μg/ml of sodiuml-ascorbate to unexposed cultures enhanced growth (cell number at 6 d) almost twofold. When added ascorbate was present only during hyperoxic exposure (but not afterward), subsequent growth in air was enhanced 1.6-fold. However, when cells were exposed without added ascorbate, there was from 2 to 12-fold greater growth in air in the presence of the added ascorbate (as compared to exposed controls). This greater growth was always only a partial reversal of the lethal effect resulting from hyperoxia. Addition of 25 μg/ml catalase did not affect control or exposed cultures. Addition of ascorbate plus catalase was not as effective as ascorbate alone in promoting growth; the catalase moiety antagonized some of the growth enhancing influence of ascorbate. This suggests that extracellular H₂O₂ was not a factor in the lethal effect resulting from hyperoxia.     

Kinetic studies of copper-induced oxidation of urate, ascorbate and their mixtures

Urate and ascorbate are the major water-soluble low molecular weight antioxidants in serum. Much attention has been devoted to the effect of these antioxidants on lipoprotein peroxidation in vivo and on their effect on copper-induced peroxidation ex vivo. These studies revealed that urate inhibits ascorbate oxidation in vitro, whereas the effect of ascorbate on urate oxidation has not been systematically studied thus far. The present study addresses mechanistic aspects of the kinetics of copper-induced oxidation of both these antioxidants and their mutual effects in aqueous solutions. We found that: (i) ascorbate becomes oxidized much faster than urate. (ii) Urate inhibits the oxidation of ascorbate but, even in the presence of excess urate, ascorbate becomes oxidized much faster than urate. (iii) Ascorbate, as well as the products of its oxidation (and/or hydrolysis) inhibit the copper-induced oxidation of urate. All these results are consistent with the hypothesis that the rate of ascorbate oxidation is determined by the rate of reoxidation of reduced copper (Cu(I)) to Cu(II) by molecular oxygen, whereas the rate of urate oxidation is governed by the rate of oxidation of urate within a 2:1 urate/copper complex. We think that the mutual effects of urate and ascorbate on each other's oxidation are likely to enhance their inhibitory effect on lipid peroxidation in biologically relevant systems including membranes and lipoproteins.     

The in situ kinetics of dopamine beta-hydroxylase in bovine adrenomedullary chromaffin cells. Intravesicular compartmentation reduces apparent affinity for the cofactor ascorbate

The Km of dopamine beta-hydroxylase for its cofactor, ascorbic acid, was determined in situ in primary cultures of bovine adrenomedullary chromaffin cells and in isolated chromaffin vesicles. A range of intravesicular ascorbate concentrations in chromaffin cell cultures (1.1-31.2 mM) was achieved by varying the number and concentration of ascorbate additions to the culture media. The rate of octopamine synthesis from tyramine displayed a Michaelis-Menten relationship with respect to ascorbate concentration and an apparent Km of dopamine beta-hydroxylase for ascorbate of 15.0 +/- 2.0 mM was determined. In isolated chromaffin vesicles, with an initial intravesicular ascorbate concentration of approximately 10 mM, ascorbate consumption during beta-hydroxylation occurred as a first order process. This indicated that dopamine beta-hydroxylase was not saturated at this initial ascorbate concentration. When isolated chromaffin vesicles were prepared with different intravesicular ascorbate concentrations, the rate of octopamine synthesis displayed a Michaelis-Menten relationship with respect to ascorbate with an apparent Km of 17.0 +/- 5.0 mM. Ascorbate consumption also occurred as a first order process in ascorbate-loaded chromaffin-vesicle ghosts which had initial ascorbate concentrations of approximately 30 mM but which were depleted of other small molecules such as catecholamines. These results indicate that the in situ Km of dopamine beta-hydroxylase for ascorbate (approximately 15 mM) is 25-fold higher than it is for the purified or partially purified enzyme assayed under optimal conditions in vitro (0.6 mM). The factor(s) which decreases the enzyme affinity for ascorbate, relative to in vitro, resides in the chromaffin vesicle interior and is also retained in chromaffin-vesicle ghosts. The mechanism of this effect remains to be determined. The Km value determined in these experiments is close to the estimated intravesicular ascorbate concentration of bovine chromaffin granules in vivo (4), suggesting that the availability of ascorbate could become a factor in regulating the rate of dopamine beta-hydroxylation.     

Ascorbate interacts with reduced glutathione to scavenge phenoxyl radicals in HL60 cells

We have compared the abilities of ascorbate and reduced glutathione (GSH) to act as intracellular free radical scavengers and protect cells against radical-mediated lipid peroxidation. Phenoxyl radicals were generated in HL60 cells, through the action of their myeloperoxidase, by adding H2O2 and phenol. Normally cultured cells, which contain no ascorbate; cells that had been preloaded with ascorbate; and those that had been depleted of GSH with buthionine sulfoximine were investigated. Generation of phenoxyl radicals resulted in the oxidation of ascorbate and GSH. Ascorbate loss was much greater in the absence of GSH, and adding glucose gave GSH-dependent protection against ascorbate loss. Ascorbate, or glucose metabolism, had little effect on the GSH loss. Glutathionyl radical formation was detected by spin trapping with DMPO in cells lacking ascorbate, and the signal was suppressed by ascorbate loading. Addition of phenol plus H2O2 to the cells caused lipid peroxidation, as measured with C11-BODIPY. Peroxidation was greatest in cells that lacked both ascorbate and GSH. Either scavenger alone gave substantial inhibition but optimal protection was seen with both present. These results indicate that GSH and ascorbate can each act as an intracellular radical scavenger and protect against lipid peroxidation. With both present, ascorbate is preferred and acts as the ultimate radical sink for phenoxyl or glutathionyl radicals. However, GSH is still consumed by metabolically recycling dehydroascorbate. Thus, recycling scavenging by ascorbate does not spare GSH, but it does enable the two antioxidants to provide more protection against lipid peroxidation than either alone.     

High serum levels interfere with the normal differentiated state of avian tendon cells by altering translational regulation

In low serum (0.2%) medium, ascorbate stimulates primary avian tendon cells to increase procollagen synthesis from 12 to 50% of total protein synthesis. This is reversibly blocked by an increase of serum levels from 0.2 to 3%. Ascorbate in low serum medium has been shown previously to stimulate the procollagen pathway by sequentially increasing by sixfold the secretion rate constant, then translation rates, and finally mRNA levels. We now show that addition of ascorbate to cultures containing 3% serum induces a sixfold increase in the secretion rate constant but translation rates and mRNA levels remain unchanged. In fully induced cells, an increase in serum levels causes a down-regulation of procollagen synthesis. In this case, the translational products of the induced cell are rapidly altered (less than 1 h), with noncollagen protein synthesis being stimulated preferentially over procollagen synthesis. This change is not reflected in procollagen mRNA levels since they remain constant for at least 6 h following addition of high serum. After 48 h in high serum, the induction of procollagen synthesis by ascorbate is reversed and the level of procollagen mRNA drops to that of uninduced cells. The data are consistent with the model that serum acts primarily at the translational level. High serum levels break the coupling in the ascorbate induction process that ties the stimulation of procollagen secretion rates to the increase in procollagen translation rates, and this prevents the maintenance of the induced state.     

The ascorbate-driven reduction of extracellular ascorbate free radical by the erythrocyte is an electrogenic process

Erythrocytes can reduce extracellular ascorbate free radicals by a plasma membrane redox system using intracellular ascorbate as an electron donor. In order to test whether the redox system has electrogenic properties, we studied the effect of ascorbate free radical reduction on the membrane potential of the cells using the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide. It was found that the erythrocyte membrane depolarized when ascorbate free radicals were reduced. Also, the activity of the redox system proved to be susceptible to changes in the membrane potential. Hyperpolarized cells could reduce ascorbate free radical at a higher rate than depolarized cells. These results show that the ascorbate-driven reduction of extracellular ascorbate free radicals is an electrogenic process, indicating that vectorial electron transport is involved in the reduction of extracellular ascorbate free radical.