Vitamin C prevents DNA mutation induced by oxidative stress

The precise role of vitamin C in the prevention of DNA mutations is controversial. Although ascorbic acid has strong antioxidant properties, it also has pro-oxidant effects in the presence of free transition metals. Vitamin C was recently reported to induce the decomposition of lipid hydroperoxides independent of metal interactions, suggesting that it may cause DNA damage. To directly address the role of vitamin C in maintaining genomic integrity we developed a genetic system for quantifying guanine base mutations induced in human cells under oxidative stress. The assay utilized a plasmid construct encoding the cDNA for chloramphenicol acetyl transferase modified to contain an amber stop codon, which was restored to wild type by G to T transversion induced by oxidative stress. The mutation frequency was determined from the number of plasmids containing the wild type chloramphenicol acetyl transferase gene rescued from oxidatively stressed cells. Cells were loaded with vitamin C by exposing them to dehydroascorbic acid, thereby avoiding transition metal-related pro-oxidant effects of ascorbic acid. We found that vitamin C loading resulted in substantially decreased mutations induced by H(2)O(2). Depletion of glutathione led to cytotoxicity and an increase in H(2)O(2)-induced mutation frequency; however, mutation frequency was prominently decreased in depleted cells preloaded with vitamin C. The mutation results correlated with a decrease in total 8-oxo-guanine measured in genomic DNA of cells loaded with vitamin C and oxidatively stressed. These findings directly support the concept that high intracellular concentrations of vitamin C can prevent oxidation-induced mutations in human cells.     

Genotoxicity is modulated by ascorbic acid: Studies using the wing spot test in Drosophila

The ability of ascorbic acid (Vitamin C) to modulate the genotoxic action of several mutagens was investigated in the wing spot test of Drosophila melanogaster. In this assay, 3-day-old transheterozygous larvae for the multiple wing hairs (mwh, 3-0.3) and flare (flr, 3-38.8) genes were treated with three reference mutagenic compounds, namely cobalt chloride (CoCl₂), 4-nitroquinoline 1-oxide (4-NQO) and potassium dichromate (K₂Cr₂O₇). The results obtained show that the three reference mutagens tested were clearly genotoxic in the Drosophila wing somatic mutation and recombination test (SMART). None of the three concentrations tested of ascorbic acid (25, 75 and 250 mM) induced significant increases in the frequency of the mutant clones recorded. When co-treatment experiments with ascorbic acid were carried out, different results were found. Thus, ascorbic acid was effective in reducing the genotoxicity of K₂Cr₂O₇ virtually to the control level; on the contrary, it did not show any antigenotoxic effect on the genotoxicity of 4-NQO. Finally, co-treatments with CoCl₂ and ascorbic acid show a significant increase in the frequency of mutant clones over the values obtained with CoCl₂ alone.     

Ascorbic Acid and Heterocyst Development in the Blue–Green Alga Amabaema ambigua

The nitrogen–fixing blue–green alga Anabaena ambigua was grown in a medium which contained either ammonium chloride as nitrogen source or molecular nitrogen. In the latter case the alga produced heterocysts. The material was analysed for ascorbic acid, dehydro-ascorbic acid and diketogulonic acid. The amount of a,scorbic acid was found always to he higher in the alga grown with molecular nitrogen. When the alga grown with combined nitrogen was transferred to the medium lacking it, there was an increase in the ascorbic acid content. Conversely, when material cultured on the nitrogen–free medium was suspended in the medium containing ammonium chloride, there was a decrease in the cellular ascorbic acid. Esogenously added ascorbic acid, up to 0.5 mg per ml, increased the heterocyst frequency to nearly three times that of the control. D–isoascorbic acid, an analogue of ascorbic acid, also showed an enhancement of heterocyst production. Algal extracts were fractionated by poiyacrylamide electro–phoresis, and the presence of ascorbic acid oxidase was detected on the gels. Two bands, with Rf values 0.34 and 1.0, were found to give positive test: for the enzyme. The total enzyme activity was 16.7 % higher in cells grown with molecular nitrogen than in those grown with combined nitrogen. The exact location of the enzyme in the alga ist not known although the heterocysts were earlier shown to contain ascorbic acid. Cytochemical tests, however, indicated strong per–oxidase activity in the heterocysts.     

Rapid stimulation of free glucuronate formation by non-glucuronidable xenobiotics in isolated rat hepatocytes

Vitamin C synthesis in rat liver is enhanced by several xenobiotics, including aminopyrine and chloretone. The effect of these agents has been linked to induction of enzymes potentially involved in the formation of glucuronate, a precursor of vitamin C. Using isolated rat hepatocytes as a model, we show that a series of agents (aminopyrine, antipyrine, chloretone, clotrimazole, metyrapone, proadifen, and barbital) induced in a few minutes an up to 15-fold increase in the formation of glucuronate, which was best observed in the presence of sorbinil, an inhibitor of glucuronate reductase. They also caused an approximately 2-fold decrease in the concentration of UDP-glucuronate but little if any change in the concentration of UDP-glucose. Depletion of UDP-glucuronate with resorcinol or d-galactosamine markedly decreased the formation of glucuronate both in the presence and in the absence of aminopyrine, confirming the precursor-product relationship between UDP-glucuronate and free glucuronate. Most of the agents did not induce the formation of detectable amounts of glucuronides, indicating that the formation of glucuronate is not due to a glucuronidation-deglucuronidation cycle. With the exception of barbital (which inhibits glucuronate reductase), all of the above mentioned agents also caused an increase in the concentration of ascorbic acid. They had little effect on glutathione concentration, and their effect on glucuronate and vitamin C formation was not mimicked by glutathione-depleting agents such as diamide and buthionine sulfoximine. It is concluded that the stimulation of vitamin C synthesis exerted by some xenobiotics is mediated through a rapid increase in the conversion of UDP-glucuronate to glucuronate, which does not apparently involve a glucuronidation-deglucuronidation cycle.     

Aminopyrine uptake by guinea pig gastric mucosal cells. Mediation by cyclic AMP and interactions among secretagogues

The role of cyclic nucleotides in regulating acid secretion by dispersed mucosal cells from guinea-pig stomach was examined by measuring first the ability of histamine and carbachol to stimulate [dimethylamine-14C]aminopyrine uptake and cyclic nucleotide metabolism and secondly, the effect of exogenous cyclic nucleotides on basal and stimulated [14C]aminopyrine uptake. The [14C]aminopyrine was found in an acidic, osmotically sensitive compartment, probably associated with the initial steps in acid secretion by these cells. Although histamine increased [14C]aminopyrine uptake and cyclic AMP synthesis as expected, histamine was approx. 10-fold more potent in inducing [14C]aminopyrine uptake. This dissociation of [14C]aminopyrine uptake and cyclic AMP metabolism process was further manifested by the observation that prostaglandin E1 failed to increase [14C]aminopyrine uptake, although it did cause a rise in cellular cyclic AMP. Furthermore, prostaglandin E1 did not alter the [14C]-aminopyrine uptake caused by histamine. Carbachol was found to increase the [14C]aminopyrine uptake and also to potentiate the ability of histamine to increase [14C]aminopyrine uptake. Carbachol, however, affected neither the histamine-induced increase in cyclic AMP nor the binding of [3H]histamine to the cells. Cimetidine, a histamine H2 receptor antagonist, blocked the [14C]aminopyrine uptake induced either by histamine alone or by the potentiating combination of histamine plus carbachol. These results suggest that cyclic AMP is mediating the action of histamine on [14C]aminopyrine uptake but changes in cyclic AMP per se are not necessarily the cause for the potentiated increase in [14C]aminopyrine uptake. Furthermore, the potentiated response observed with histamine plus carbachol on [14C]aminopyrine uptake occurs at a biochemical step distal to and not obviously related to cyclic AMP generation.     

Effect of ascorbic acid on stimulatory status of activated mouse peritoneal phagocytes

Mouse peritoneal macrophages (MPM) when elicited by the antioxidant ascorbic acid have been found to be significantly stimulatory, exhibiting marked alteration at the cellular and enzyme levels. Alterations recorded were as follows--cellular yield per mouse, their protein content, lysosomal acid hydrolase levels and capability to phagocyte, all were significantly enhanced. The new stimulant was observed to produce no synergistic action on MPM when thioglycollate, BCG or endotoxin along with the same stimulated the latter. Levels of antioxidants like ascorbic acid and glutathione were found to be enhanced in elicited macrophages whereas superoxide dismutase levels varied when the three above stimulators were administered. However, the ascorbic acid elicited cells showed an increase in glutathione levels and a decrease in SOD levels but no change in total intracellular ascorbic acid levels. Further, though ascorbic acid interaction enhanced the phagocytic capability of MPM as compared to resident cells, no significant boosting of phagocytic process could be observed when each of three stimulators coupled with ascorbic acid was used for macrophage elicitation.     

Effect of exogenous ascorbic acid intake on biosynthesis of ascorbic acid in mice

The effect of exogenous ascorbic acid intake on biosynthesis of ascorbic acid in mice has been studied. After the mice were on diets containing added ascorbic acid for two months, the activities of ascorbic acid synthesizing enzymes in the mouse liver homogenates were measured using L-gulono-gamma-lactone as a substrate. Exogenous ascorbic acid intake (0.5, 1 or 5% in the diet) was able to increase the concentration of ascorbic acid in the blood and to decrease the activities of ascorbic acid synthesizing enzymes in mouse liver. The results suggest that ascorbic acid synthesis was controlled by local regulatory mechanism or by the concentration of ascorbic acid in the hepatic portal blood. Ingestion of dietary erythorbic acid, a stereoisomer of ascorbic acid, had no effect on the activities of ascorbic acid synthesizing enzymes.     

Developmental response of <Emphasis Type="Italic">Euplectrus comstockii</Emphasis> to ascorbic acid in the diet of the larval host, <Emphasis Type="Italic">Heliothis virescens</Emphasis>

Recent developments in genetic engineering have paved the way for researchers to produce crops of high nutritional and yield value, in addition to being resistant to diseases and pests. Ascorbic acid content is one of the parameters researchers are trying to enhance in plants. This study investigated the effect of different levels of dietary ascorbic acid of a beneficial wasp, Euplectrus comstockii Howard (Hymenoptera: Eulophidae), by measuring life history parameters of the wasp when reared on lepidopteran larvae fed a basal diet containing low and high levels of ascorbic acid. Odds and odds ratio analyses showed that the probability of egg hatch and adult emergence for the wasp increased with the amount of ascorbic acid in the diet of the host, and that the rate of development and probability of female or male progeny was similar for most levels of ascorbic acid tested. This would indicate that as the ascorbic acid concentration increases in the pest insect the effectiveness of the wasp is likely to increase and when, by comparison with other published findings, the effectiveness of microbial pathogens is likely to decrease.     

Attachment of Entamoeba histolytica to glass in a defined maintenance medium: specific requirement for cysteine and ascorbic acid

Cysteine and ascorbic acid were previously shown to be required by Entamoeba histolytica trophozoites for attachment to glass, elongation, and ameboid movement as well as for short-term (12-24 h) survival in a balanced salt solution containing bovine serum albumin and a vitamin solution (Maintenance Medium 1). If the only function of cysteine and ascorbate was to decrease the redox potential, other reducing agents should be effective. However, the requirement for cysteine in the presence of ascorbic acid was highly specific. Equally effective were D- and L-cysteine; however, of many other compounds tested, only thioglycolic acid, ascorbic acid, or L-cystine (in decreasing order) were somewhat active. Under N2 atmosphere, cysteine and ascorbic acid were still required, although their concentrations could be halved. The ability to attach in the maintenance medium was irreversibly lost after only 5 min of cysteine-ascorbic acid deprivation; however, there was no decrease in viability when the amebae were transferred to growth medium within 30 min. Cysteine thiol groups in the medium were oxidized rapidly regardless of the concentration of ascorbic acid or the presence of amebae; however, ascorbic acid prolonged attachment of amebae.     

Evaluation of Insulin and Ascorbic Acid Effects on Expression of Bcl-2 Family Proteins and Caspase-3 Activity in Hippocampus of STZ-Induced Diabetic Rats

Aims Effects of insulin and ascorbic acid on expression of Bcl-2 family proteins and caspase-3 activity in hippocampus of diabetic rats were evaluated in this study. Methods Diabetes was induced in Wistar male rats by streptozotocin (STZ). Six weeks after verification of diabetes, the animals were treated for 2 weeks with insulin or/and ascorbic acid in separate groups. Hippocampi of rats were removed and evaluation of Bcl-2, Bcl-x_L, and Bax proteins expression in frozen hippocampi tissues were done by SDS-PAGE electrophoresis and blotting. The Bcl-2, Bcl-x_L, and Bax proteins bands were visualized after incubation with specific antibodies using enhanced chemiluminescences method. Caspase-3 activity was determined using the caspase-3/CPP32 Fluorometric Assay Kit. Results Diabetic rats showed increase in Bax protein expression and decrease in Bcl-2 and Bcl-x_L proteins expression. The Bax/Bcl-2 and Bax/Bcl-x_L ratios were found higher compared with non-diabetic control group. Treatments with insulin and/or ascorbic acid were resulted in decrease in Bax protein expression and increase in Bcl-2 and Bcl-x_L proteins expression. The Bcl-2/Bax and Bcl-x_L/Bax ratios were found higher in treated groups than untreated diabetic group. Caspase-3 activity level was found higher in diabetic group compared with non-diabetic group. Treatment with insulin and ascorbic acid did downregulated caspase-3 activity. Conclusions Our data provide supportive evidence to demonstrate the antiapoptotic effects of insulin and ascorbic acid on hippocampus of STZ-induced diabetic rats.     

Phytoremediation of Cr(VI) by Spirodela polyrrhiza (L.) Schleiden employing reducing and chelating agents

Phytoremediation of Cr(VI) by Spirodela polyrrhiza in binary combinations with low molecular weight organic compounds (LMWOCs) with a reducing or chelating potential, viz., ascorbic acid, citric acid, tartaric acid, oxalic acid, lactic acid, and glycerol was studied in Cr(VI) containing hydroponic media. Significant increase in the relative dry weight of plants with respect to Cr(VI) treated controls was observed with ascorbic acid and glycerol. The uptake of chromium by S. polyrrhiza followed Michaelis-Menten kinetics of active ion uptake. Interaction between Cr and ascorbic acid, oxalic acid, and lactic acid decreased Cr uptake, whereas citric acid, glycerol, and tartaric acid increased it. Supplementation of LMWOCs to Cr(VI) containing media decreased the MDA content of the plants. Multiple regression models revealed that LMWOCs decrease lipid peroxidation independently, as well as that induced by Cr(VI). It was found that superoxide dismutase (SOD), guaiacol peroxidase (GPX), and catalase (CAT) activities were increased significantly in plants growing in media containing Cr(VI). The study established that lactic acid, citric acid, ascorbic acid, and glycerol were most effective in increasing the Cr(VI) phytoremediating potential of S. polyrrhiza and LMWOCs with reducing or chelating properties decrease Cr(VI) stress in S. polyrrhiza.     

Attachment of Entamoeba histolytica to Glass in a Defined Maintenance Medium: Specific Requirement for Cysteine and Ascorbic Acid*

SYNOPSIS. Cysteine and ascorbic acid were previously shown to be required by Entamoeba histolytica trophozoites for attachment to glass, elongation, and ameboid movement as well as for short-term (12–24 h) survival in a balanced salt solution containing bovine serum albumin and a vitamin solution (Maintenance Medium 1). If the only function of cysteine and ascorbate was to decrease the redox potential, other reducing agents should be effective. However, the requirement for cysteine in the presence of ascorbic acid was highly specific. Equally effective were D- and L-cysteine; however, of many other compounds tested, only thioglycolic acid, ascorbic acid, or L-cystine (in decreasing order) were somewhat active. Under N₂ atmosphere, cysteine and ascorbic acid were still required, although their concentrations could be halved. The ability to attach in the maintenance medium was irreversibly lost after only 5 min of cysteine-ascorbic acid deprivation; however, there was no decrease in viability when the amebae were transferred to growth medium within 30 min. Cysteine thiol groups in the medium were oxidized rapidly regardless of the concentration of ascorbic acid or the presence of amebae; however, ascorbic acid prolonged attachment of amebae.     

Isolated Medicago truncatula mutants with increased calcium oxalate crystal accumulation have decreased ascorbic acid levels.

The mechanisms controlling oxalate biosynthesis and calcium oxalate formation in plants remain largely unknown. As an initial step toward gaining insight into these regulatory mechanisms we initiated a mutant screen to identify plants that over-accumulate crystals of calcium oxalate. Four new mutants were identified, from an ethyl methanesulfonate (EMS)-mutagenized Medicago truncatula (cv. Jemalong genotype A17) population, that over-accumulated calcium oxalate crystals. The increased calcium oxalate content of these new mutants, as with the previously isolated mutant cod4, resulted from an increase in druse crystals accumulated within the mesophyll cells of leaves. Complementation and segregation analysis revealed that each mutant was affected at a different locus. This was confirmed through the genetic mapping of each mutation to different linkage groups. Together, these findings emphasize the complexity of factors that can contribute to oxalate biosynthesis and crystal formation in these plants. In addition, each mutant showed a common decrease in ascorbic acid content providing genetic support for ascorbic acid as a precursor in the oxalate biosynthetic pathway for druse crystal formation. Further support was obtained by the ability of an exogenous supply of ascorbate to induce druse crystal formation while other tested organic acids did not induce crystal production.     

Unequivocal evidence in support of the nonenzymatic redox coupling between glutathione/glutathione disulfide and ascorbic acid/dehydroascorbic acid.

Experiments were performed to evaluate the nonenzymatic reaction between glutathione (GSH) and dehydroascorbic acid (DHA). Though both ascorbic acid and glutathione disulfide (GSSG) are formed from this reaction, previous work has focused almost exclusively on measurements of ascorbic acid. In contrast, there is very little information about the formation of GSSG under the same conditions as those used to produce ascorbic acid. The emphasis on ascorbic acid stems from the fact that a spectrophotometric technique is available for its measurement, whereas 1H-NMR or an amino acid analyzer has been used to measure GSSG. The present experiments use a simple, rapid method for accurately and precisely measuring the concentrations of GSSG in a solution. The spectrophotometric (340 nm) procedure uses NADPH and glutathione reductase; analysis time is very short, many replicate samples can be tested and as little as 0.05-0.1 mM GSSG can be detected. Using this method, it is shown that there is an equimolar production of GSSG and ascorbic acid from GSH and DHA and that the decrease in GSH is stoichiometrically related to the increase in the concentration of GSSG. The present findings provide additional insight into the interaction between the GSH/GSSG redox couple and the ascorbic acid/DHA redox couple.     

Short-term Effects of Ethanol Intoxication on Ascorbic Acid and Lipid Metabolism and on Drug-metabolizing Enzymes in Liver of Rats

The effects of one-time ethanol intoxication on ascorbic acid and lipid metabolism and on drug-metabolizing enzymes in liver of rats were investigated. Male Donryu rats that had been fed semi-purified feed were given 5 g/kg ethanol solution (25%, w/v) via a stomach tube and killed 16 h after intubation. The amount of ascorbic acid excreted in the urine after ethanol administration increased, but renal and adrenal concentrations of ascorbic acid decreased. The serum levels of total cholesterol, high-density-lipoprotein cholesterol, triglycerides, phospholipids, and non-esterified fatty acids were elevated in rats given ethanol, but hepatic level of total lipids, cholesterol, triglycerides, phospholipids were not. The hepatic concentrations of cytochrome P-450 and cytochrome b₅ did not increase, but this large dose of ethanol increased the activities of aminopyrine N-demethylase and cytochrome c reductase.

These results indicated that the single dose of ethanol affected the ascorbic acid and lipid metabolism of rats, and induced drug-metabolizing enzymes in their liver.     

The effects of haloperidol and amphetamine on ascorbic acid and uric acid in caudate and nucleus accumbens of rats as measured by voltammetry in vivo

The ability of haloperidol (0.1 mg/kg) to reduce the amphetamine-induced (2 and 5 mg/kg) increase in ascorbic and uric acid in anterior caudate and in nucleus accumbens was tested using voltammetry in vivo. In both areas, haloperidol reduced the amphetamine-induced increase in uric acid. In both areas, haloperidol only marginally affected the amphetamine-induced increase in ascorbic acid. Amphetamine-induced increases in uric acid were more nearly dose-related than changes in ascorbic acid. Of the two compounds, uric acid seems more likely to be associated with dopamine.     

Role of glucose metabolism in acid formation by isolated gastric glands

The role played by glucose in providing energy for acid formation was studied in isolated gastric glands from rabbit. The widely-used inhibitors of glycolysis, iodoacetic acid and iodoacetamide were found to inhibit glucose oxidation as well as the indicators of acid formation, respiration and accumulation of aminopyrine. However, the potent inhibition of acid formation was found to involve a nonspecific mechanism other than the simple inhibition of glycolysis. An alternative approach involved use of the glucose transport inhibitor, phloretin. Phloretin blocked glucose oxidation and also inhibited functional responses. Acid formation was restored easily by the addition of pyruvate or various other oxidizable substrates. Measurement of lactate formation in the absence of exogenous glucose showed that the gastric glands contain very little glycogen. Addition of external glucose resulted in a 10-fold increase in lactate formation and this rate was stimulated further by histamine and rotenone. Rotenone also inhibited both respiration and aminopyrine accumulation; however, the inhibition was not complete. Phloretin treatment resulted in total inhibition of the residual aminopyrine accumulation after rotenone treatment. The results are interpreted to indicate that gastric glands are dependent almost totally on external substrate supply to support acid formation; and, that while anaerobic glucose metabolism can sustain a very low level of acid formation, the major role of glucose is to yield pyruvate equivalents for subsequent oxidation.     

Effects of common inhibitors of gastric acid secretion on secretagogue-induced respiration and aminopyrine accumulation in isolated gastric glands.

1. The effects of three inhibitors of gastric acid secretion, atropine, burimamide and thiocyanate, have been studied in isolated glands from the rabbit gastric mucosa. The glands were either resting or stimulated by carbachol, histamine or dibutyryl cyclic AMP. The effects were determined from changes in oxygen consumption and accumulation of the weak base aminopyrine. The latter gives an indirect measurement of the acid production in the glands. 2. Atropine (10 (-6) M) almost totally inhibited the transient response induced by carbachol (10 (-4) M) in both measured parameters. The histamine-induced increase in respiration was inhibited when the atropine concentration was raised to 10 (-4) M. To a lesser extent also, histamine-induced aminopyrine accumulation was reduced. The dibutyryl cyclic AMP stimulated oxygen consumption was not affected by atropine. 3. Burimamide competitively inhibited the histamine responses but was without effect on those of carbachol and dibutyryl cyclic AMP. 4. Thiocyanate (10 (-2) M) inhibited the increase in oxygen consumption induced by all three secretagogues but not down the prestimulatory level, in spite of total abolishment of the aminopyrine accumulation. 5. In unstimulated glands, burimamide (10 (-3) M) or atropine (10 (-4) M) did not alter the normal aminopyrine ratio (aminopyrine in intraglandular water/ aminopyrine in extraglandular water) of approximately 50. This may indicate the existence of preformed acid in resting parietal cells. Thiocyanate, on the other hand, lowered the aminopyrine ratio in unstimulated glands from 46 to 2. Possible mechanisms for the thiocyanate effect are discussed in terms of an inability to separate acid and base in the secreting membrane.     

镍对水稻离体叶片脂质过氧化作用的影响

杂交水稻离体叶片用１０－３ｍｏｌ／ＬＮｉＳＯ４处理后,镍阻抑了叶片在衰老过程中ＳＯＤ、ＣＡＴ酶活性的下降和ＡｓＡ氧化酶活性的上升,ＡｓＡ和叶绿素含量的下降得到延缓,减少了膜脂过氧化程度。     

Ascorbic Acid Turnover in Developing Chick Embryonic Tissues and its Metabolic Significance

A histochemical investigation on the content of ascorbic acid in chick embryonic tissues was undertaken using an improved technique. The concentration of ascorbic acid was found to be higher in the tissues which are involved in active biosynthetic mechanisms. Although the overall localization of ascorbic acid in intestine and proventriculus was less yet their secretory areas were rich in this vitamin. A significant successive increase in intensity of staining for ascorbic acid in the tissues from 15th to 21st day was evident from the data. The maximum ascorbic acid deposition was found in almost all the tissues of 21st day embryo as compared to the other age groups. This increase is correlated with the hatching process.