Recycling of vitamin C from its oxidized forms by human endothelial cells

Endothelial cells encounter oxidant stress due to their location in the vascular wall, and because they generate reactive nitrogen species. Because ascorbic acid is likely involved in the antioxidant defenses of these cells, we studied the mechanisms by which cultures of EA.hy926 endothelial cells recycle the vitamin from its oxidized forms. Cell lysates reduced the ascorbate free radical (AFR) by both NADH- and NADPH-dependent mechanisms. Most NADH-dependent AFR reduction occurred in the particulate fraction of the cells. NADPH-dependent reduction resembled that due to NADH in having a high affinity for the AFR, but was mediated largely by thioredoxin reductase. Reduction of dehydroascorbic acid (DHA) required GSH and was both direct and enzyme dependent. The latter was saturable, half-maximal at 100 microM DHA, and comparable to rates of AFR reduction. Loading cells to ascorbate concentrations of 0.3-1.6 mM generated intracellular DHA concentrations of 20-30 microM, indicative of oxidant stress in culture. Whereas high-affinity AFR reduction is the initial and likely the preferred mechanism of ascorbate recycling, any DHA that accumulates during oxidant stress will be reduced by GSH-dependent mechanisms.     

Induction of radioresistance by a nitric oxide-mediated bystander effect

To elucidate whether nitric oxide secreted from irradiated cells affects cellular radiosensitivity, we examined the accumulation of inducible nitric oxide synthase, TP53 and HSP72, the concentration of nitrite in the medium of cells after X irradiation, and cellular radiosensitivity using two human glioblastoma cell lines, A-172, which has a wild-type TP53 gene, and a transfectant of A-172 cells, A-172/mp53, bearing a mutated TP53 gene. Accumulation of inducible nitric oxide synthase was caused by X irradiation of the mutant TP53 cells but not of the wild-type TP53 cells. Accumulation of TP53 and HSP72 in the wild-type TP53 cells was observed by cocultivation with irradiated mutant TP53 cells, and the accumulation was abolished by the addition of an inhibitor for inducible nitric oxide synthase, aminoguanidine, to the medium. Likewise, accumulation of these proteins was observed in the wild-type TP53 cells after exposure to conditioned medium from irradiated mutant TP53 cells, and the accumulation was abolished by the addition of a specific nitric oxide scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, to the medium. The radiosensitivity of wild-type TP53 cells was reduced when the cells were cultured in conditioned medium from irradiated mutant TP53 cells compared to conventional fresh growth medium. Collectively, these findings indicate the potential importance of an intercellular signal transduction pathway initiated by nitric oxide in the cellular response to ionizing radiation.     

Radiation-induced bystander effect

Data concerning induction mechanisms, the objects and methods of investigation of a non-target radiobiological phenomenon bystander effect, its role in radiation-induced genomic instability and oncogenesis are summarized.     

Inhibition of benzo(a)pyrene-induced carcinogenesis by vitamin C alone and by vitamin C/vitamin E and selenium/glutathione

The reduction on peroxidation caused by benzopyrenes by some naturally occurring antineoplastic agents was studied in this experimental work. Inhibition/reduction of experimental carcinogenesis induced by benzo(a)pyrene by vitamin C alone and by vitamin C/vitamin E and selenium/ glutathione was attempted in 224 female Wistar rats divided in four groups. Injected with 10.08 mL benzo(a)pyrene, the animals were treated with some naturally occurring substances like vitamin C alone and a combination of anticarcinogens. By calculating the carcinogenic potency of benzo(a)pyrene and the anticarcinogenic potency of substances used as well as histological examination of developed tumors and survival time of treated animals, it was found that vitamin C exerts a significant anticarcinogenic effect of 8.3 units and that the combination of the two anticarcinogens used produced a significant prolongation of the animals survival time with anticarcinogenic potency of 22.1 and 22.2 units, respectively. This is considered a potent anticarcinogenic effect. The question of an additional supportive administration of such agents complementary to the conventional cancer chemotherapy in humans is raised. Of course, further studies are needed.     

Inhibition of benzo(a)pyrene-induced carcinogenesis by vitamin C alone and by vitamin C/vitamin E and selenium/glutathione

The reduction on peroxidation caused by benzopyrenes by some naturally occurring antineoplastic agents was studied in this experimental work. Inhibition/reduction of experimental carcinogenesis induced by benzo(a)pyrene by vitamin C alone and by vitamin C/vitamin E and selenium/glutathione was attempted in 224 female Wistar rats divided in four groups. Injected with 10.08 mL benzo(a)pyrene, the animals were treated with some naturally occurring substances like vitamin C alone and a combination of anticarcinogens. By calculating the carcinogenic potency of benzo(a)pyrene and the anticarcinogenic potency of substances used as well as histological examination of developed tumors and survival time of treated animals, it was found that vitamin C exerts a significant anticarcinogenic effect of 8.3 units and that the combination of the two anticarcinogens used produced a significant prolongation of the animals survival time with anticarcinogenic potency of 22.1 and 22.2 units, respectively. This is considered a potent anticarcinogenic effect. The question of an additional supportive administration of such agents complementary to the conventional cancer chemotherapy in humans is raised. Of course, further studies are needed.     

Synergistic effect of vitamin C on DNA damage induced by cadmium.

Salts of divalent cadmium are well-known human mutagens and carcinogens. In the present work, the ability of vitamin C to modulate genotoxic effects of cadmium chloride on human lymphocytes was assessed using single cell gel electrophoresis (comet assay). Vitamin C at 20 and 100 micromol/l and cadmium at 5, 30 and 150 micromol/l significantly increased the tail moment of lymphocytes. Vitamin C also increased the tail moment of cells exposed to cadmium. This effect was concentration-dependent: the higher the vitamin C concentration the greater the tail moment. The combined effects of cadmium and vitamin C were more pronounced at all concentrations tested than the sum of the effects of the compounds applied separately (p < 0.05), so cadmium and vitamin C can be considered to have synergistic effects. The results obtained can be partly explained by the participation of cadmium in the Fenton reaction and reduction of its oxidized form by vitamin C.     

Radiation-induced “bystander” effect

Research data on the developmental mechanisms and subjects and methods of studying a nontarget radiation phenomenon, called the “bystander effect,” are generalized and analyzed, and its role in radiation-induced genomic instability and oncogenesis is studied. A natural model system is developed for identifying radiation-induced cytogenetic bystander effect in human somatic cells. These cells should be collected from a population of peripheral blood lymphocytes that has been irradiated in vitro or in vivo (as the source of the damaging signal) along with a population of nonirradiated lymphocytes of individuals of the opposite sex, which are used as “bystanders.”     

A stochastic model of cancer initiation including a bystander effect

A stochastic model of cancer initiation is considered. The model is used to evaluate whether a bystander effect may be important in the pre-malignant and malignant stages of carcinogenesis, and furthermore, on the basis of epidemiological data, to estimate the mutation rates of genes involved in the development of oral leukoplakias. The bystander effect is defined here as the capability of oncogenic mutations to increase the mutation probability of neighbouring (bystander) cells, thus leading potentially to a cascade of neighbouring mutated and neoplastic cells as a pre-stage in the development to leukoplakias and cancer. We find that incidence data for oral cancer are indeed in accordance with a significant bystander effect, operating either alone or in combination with genomic instability in the early stages of carcinogenesis, i.e. the development of neoplasia. Simulations performed gave a picture of how mutations and neoplasia may spread in a tissue, to form characteristic leukoplakias with a core of neoplastic cells. The model also showed that the probability of finding at least one neoplastic cell in the tissue after a given number of years is more sensitive to changes in genomic instability within the cell itself than to changes in a bystander effect. Based on epidemiological data we also calculate the maximum number of oncogenic genes that may be involved in the bystander effect and development of genomic instability. Even if capable of explaining the initial development of oncogenic mutations towards neoplastic cells, the bystander model could not reproduce the observed incidence rates of leukoplakia without assuming a carcinogen mutation probability per cell per year of neoplastic cells practically equal to one. This means that the bystander effect, to be of substantial importance in the final development of neoplastic cells towards leukoplakias, requires a very significant increase in mutation probabilities for bystanders to neoplastic cells. Alternatively, additional mechanisms such as abnormal cell differentiation and uncontrolled proliferation and apoptotis in the neoplastic stage may be of major importance during the development to cancerization.     

Radiation induces senescence and a bystander effect through metabolic alterations

Cellular senescence is a state of irreversible growth arrest; however, the metabolic processes of senescent cells remain active. Our previous studies have shown that radiation induces senescence of human breast cancer cells that display low expression of securin, a protein involved in control of the metaphase–anaphase transition and anaphase onset. In this study, the protein expression profile of senescent cells was resolved by two-dimensional gel electrophoresis to investigate associated metabolic alterations. We found that radiation induced the expression and activation of glyceraldehyde-3-phosphate dehydrogenase that has an important role in glycolysis. The activity of lactate dehydrogenase A, which is involved in the conversion of pyruvate to lactate, the release of lactate and the acidification of the extracellular environment, was also induced. Inhibition of glycolysis by dichloroacetate attenuated radiation-induced senescence. In addition, radiation also induced activation of the 5′-adenosine monophosphate-activated protein kinase (AMPK) and nuclear factor kappa B (NF-κB) pathways to promote senescence. We also found that radiation increased the expression of monocarboxylate transporter 1 (MCT1) that facilitates the export of lactate into the extracellular environment. Inhibition of glycolysis or the AMPK/NF-κB signalling pathways reduced MCT1 expression and rescued the acidification of the extracellular environment. Interestingly, these metabolic-altering signalling pathways were also involved in radiation-induced invasion of the surrounding, non-irradiated breast cancer and normal endothelial cells. Taken together, radiation can induce the senescence of human breast cancer cells through metabolic alterations.     

Stimulatory effect of vitamin C on autophagy in glial cells

Intracellular accumulation of damaged or abnormal proteins is a common event associated with numerous neurodegenerative diseases and other age-related pathologies. Increasing the activity of the intracellular proteolytic systems normally responsible for the removal of these abnormal proteins might be beneficial in lessening the severity or development of those pathologies. In this study we have used human astrocyte glial cells to investigate the effect of vitamin C (ascorbate) on the intracellular turnover of proteins. Supplementation of the culture medium with physiological concentrations of vitamin C did not affect protein synthesis, but did increase the rate of protein degradation by lysosomes. Vitamin C accelerated the degradation of intra- and extracellular proteins targeted to the lysosomal lumen by autophagic and heterophagic pathways. At the doses analyzed, vitamin C lowered and stabilized the acidic intralysosomal pH at values that result in maximum activation of the lysosomal hydrolases.     

Recycling of pyridoxine (vitamin B6) by PUP1 in Arabidopsis

Vitamin B6 is a cofactor for more than 140 essential enzymatic reactions and was recently proposed as a potent antioxidant, playing a role in the photoprotection of plants. De novo biosynthesis of the vitamin has been described relatively recently and is derived from simple sugar precursors as well as glutamine. In addition, the vitamin can be taken up from exogenous sources in a broad range of organisms, including plants. However, specific transporters have been identified only in yeast. Here we assess the ability of the family of Arabidopsis purine permeases (PUPs) to transport vitamin B6. Several members of the family complement the growth phenotype of a Saccharomyces cerevisiae mutant strain impaired in both de novo biosynthesis of vitamin B6 as well as its uptake. The strongest activity was observed with PUP1 and was confirmed by direct measurement of uptake in yeast as well as in planta, defining PUP1 as a high affinity transporter for pyridoxine. At the tissue level the protein is localised to hydathodes and here we use confocal microscopy to illustrate that at the cellular level it is targeted to the plasma membrane. Interestingly, we observe alterations in pyridoxine recycling from the guttation sap upon overexpression of PUP1 and in a pup1 mutant, consistent with the role of the protein in retrieval of pyridoxine. Furthermore, combining the pup1 mutant with a vitamin B6 de novo biosynthesis mutant (pdx1.3) corroborates that PUP1 is involved in the uptake of the vitamin.     

The bystander effect in C3H 10T cells and radon-induced lung cancer.

Little, M. P. and Wakeford, R. The Bystander Effect in C3H 10T(1/2) Cells and Radon-Induced Lung Cancer. Radiat. Res. 156, 695-699 (2001).Bystander effects, whereby cells that are not directly exposed to radiation exhibit adverse biological effects, have been observed in a number of experimental systems, including C3H 10T(1/2) cells exposed to alpha-particle radiation. The bystander effect implies that risks from exposure to low doses of radiation obtained by linear extrapolation from data for high-dose exposures might be substantial underestimates. The best estimate of the ratio of the lung cancer risk among persons exposed to low (residential) doses of radon daughters to that among persons (underground miners) exposed to high doses of radon daughters is in the range of 2.4-4.0, with an upper 95% confidence limit of about 14. Assuming that the bystander effect observed in the in vitro C3H 10T(1/2) cell system applies to human lung cells in vivo, these epidemiological data imply that the central estimate of the number of neighboring cells that can contribute to the bystander effect is between 0 and 1, with an upper 95% confidence limit of about 7. As a consequence, the bystander effect observed in the experimental C3H 10T(1/2) cell system probably does not play a large part in the process of radon-induced lung carcinogenesis in humans.     

The effect of vitamin C on oxygen radical-induced sister-chromatid exchanges

We studied the effects of vitamin C (sodium ascorbate) on the genotoxicity of oxygen radicals to tissue culture cells. Chinese hamster ovary cells (CHO cells), when exposed to an enzymatic oxygen radical generating system (xanthine oxidase plus hypoxanthine), develop increased numbers of sister-chromatid exchanges (SCEs). Inclusion of ascorbate in these incubations resulted in significant, but variable effects. In some cases, ascorbate (less than 0.1 mM) was protective and fewer SCEs were produced. In others, significant augmentation of oxygen radical-induced SCEs occurred. These experiments illustrate the complexity of the interactions of ascorbate in biologic systems and the difficulty of predicting a desirable or harmful effect.     

The radiation-induced bystander effect for clonogenic survival

It has long been accepted that the radiation-induced heritable effects in mammalian cells are the result of direct DNA damage. Recent evidence, however, suggests that when a cell population is exposed to a low dose of alpha particles, biological effects occur in a larger proportion of cells than are estimated to have been traversed by alpha particles. Experiments involving the Columbia University microbeam, which allows a known fraction of cells to be traversed by a defined number of alpha particles, have demonstrated a bystander effect for clonogenic survival and oncogenic transformation in C3H 10T(1/2) cells. When 1 to 16 alpha particles were passed through the nuclei of 10% of a C3H 10T(1/2) cell population, more cells were unable to form colonies than were actually traversed by alpha particles. Both hit and non-hit cells contributed to the outcome of the experiments. The present work was undertaken to assess the bystander effect of radiation in only non-hit cells. For this purpose, Chinese hamster V79 cells transfected with hygromycin- or neomycin-resistance genes were used. V79 cells stably transfected with a hygromycin resistance gene and stained with a nuclear dye were irradiated with the charged-particle microbeam in the presence of neomycin-resistant cells. The biological effect was studied in the neomycin-resistant V79 cells after selective removal of the hit cells with geneticin treatment.     

Reductive activation of mitomycins A and C by vitamin C

The anticancer drug mitomycin C produces cytotoxic effects after being converted to a highly reactive bis-electrophile by a reductive activation, a reaction that a number of 1-electron or 2-electron oxidoreductase enzymes can perform in cells. Several reports in the literature indicate that ascorbic acid can modulate the cytotoxic effects of mitomycin C, either potentiating or inhibiting its effects. As ascorbic acid is a reducing agent that is known to be able to reduce quinones, it could be possible that the observed modulatory effects are a consequence of a direct redox reduction between mitomycin C and ascorbate. To determine if this is the case, the reaction between mitomycin C and ascorbate was studied using UV/Vis spectroscopy and LC/MS. We also studied the reaction of ascorbate with mitomycin A, a highly toxic member of the mitomycin family with a higher redox potential than mitomycin C. We found that ascorbate is capable to reduce mitomycin A efficiently, but it reduces mitomycin C rather inefficiently. The mechanisms of activation have been elucidated based on the kinetics of the reduction and on the analysis of the mitosene derivatives formed after the reaction. We found that the activation occurs by the interplay of three different mechanisms that contribute differently, depending on the pH of the reaction. As the reduction of mitomycin C by ascorbate is rather inefficiently at physiologically relevant pH values we conclude that the modulatory effect of ascorbate on the cytotoxicity of mitomycin C is not the result of a direct redox reaction and therefore this modulation must be the consequence of other biochemical mechanisms.