Ascorbic acid reduces the frequency of iron induced micronuclei in bone marrow cells of mice

Iron is a potent oxidant that can lead to the formation of genotoxic lipid peroxides. Ascorbic acid, which enhances dietary iron absorption, has been suggested to enhance the oxidant effects of iron and to directly lead to the formation of lipid peroxides. The combined effects of dietary iron and ascorbic acid on genotoxicity were investigated by measuring the frequency of micronuclei in the bone marrow cells of C3H/He mice. In addition, liver iron concentration was measured in all treated groups. Three weeks old mice were fed diets for 3 weeks containing iron at 100 or 300 mg/kg diet in the form of FeSO(4) that were supplemented either with or without ascorbic acid (15 g/kg diet). The results of the bone marrow micronucleus test revealed that the high iron diet resulted in an increased frequency of micronucleated polychromatic erythrocytes (MnPCEs) as compared to low iron. Ascorbic acid supplementation in the low iron diet did not show any effect on incidence of MnPCEs and protected against the increased frequency of MnPCEs induced by the high iron diet. However, liver iron concentration was significantly increased only in the high iron treated and ascorbic acid supplemented group as compared to all other groups. These results demonstrate that ascorbic acid protects against the clastogenic effects of iron.     

Ascorbic acid promotes osteoclastogenesis from embryonic stem cells

Ascorbic acid (AA) is known to regulate cell differentiation; however, the effects of AA on osteoclastogenesis, especially on its early stages, remain unclear. To examine the effects of AA throughout the process of osteoclast development, we established a culture system in which tartrate-resistant acid phosphate (TRAP)-positive osteoclasts were induced from embryonic stem cells without stromal cell lines. In this culture system, the number of TRAP-positive cells was strongly increased by the addition of AA during the development of osteoclast precursors, and reducing agents, 2-mercaptoethanol, monothioglycerol, and dithiothreitol, failed to substitute for AA. The effect of AA was stronger when it was added during the initial 4 days during the development of mesodermal cells than when it was added during the last 4 days. On day 4 of the culture period, AA increased the total cell recovery and frequency of osteoclast precursors. Magnetic cell sorting using anti-Flk-1 antibody enriched osteoclast precursors on day 4, and the proportion of Flk-1-positive cells but not that of platelet-derived growth factor receptor alpha-positive cells was increased by the addition of AA. These results suggest that AA might promote osteoclastogenesis of ES cells through increasing Flk-1-positive cells, which then give rise to osteoclast precursors.     

Ascorbic acid inhibits the migration of walker 256 carcinosarcoma cells

The results of several experimental studies have shown that ascorbic acid inhibits tumor growth and metastasis. Ascorbic acid is an antioxidant that acts as a scavenger for a wide range of reactive oxygen species (ROS). Both tumour metastasis and cell migration have been correlated with the intracellular ROS level, so it was postulated that the inhibitory effect of ascorbic acid derivatives on cell motility may be caused by scavenging of ROS. Time-lapse analyses of Walker 256 carcinosarcoma cell migration showed that both the speed of movement and the cell displacement were inhibited by ascorbic acid applied in concentrations ranging from 10 to 250 μM. This effect correlated with a reduction in the intracellular ROS level in WC 256 cells, suggesting that ROS scavenging may be a mechanism responsible for the inhibition of WC 256 cell migration. However, another potent antioxidant, N-acetyl-L-cysteine, also efficiently decreased the intracellular ROS level in WC 256 cells, but did not affect the migration of the investigated cells. These results demonstrate that intact, unmodified ascorbic acid applied in physiologically relevant and nontoxicconcentrations exerts an inhibitory effect on the migration of WC 256 carcinosarcoma cells, and that this may be one of the factors responsible for the anti-metastatic activity of vitamin C. However, our data does not support the hypothesis that the scavenging of intracellular ROS is the main mechanism in the inhibition of cancer cell migration by ascorbic acid. Paper authored by participants of the international conference: XXXIV Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University, Zakopane, March 7–11, 2007, “The Cell and Its Environment”. Publication costs were covered by the organisers of this meeting.     

Effect of copperglycinate on the radiation induced micronuclei formation in mice bone marrow

Summary The frequency of micronuclei were determined in the bone marrow of Swiss albino mice treated with or without copperglycinate 15 min before exposure to 4.5 Gy gamma radiation. The frequency of micronuclei increased from day 1/4 to day 1 post-irradiation in both the irradiated groups and declined thereafter, with the frequency of micronuclei remaining significantly lower in the copperglycinate treated animals.     

Comparison of micronuclei frequency in bone marrow cells of three rat lines

The aim of this paper is to compare the spontaneous and induced with cyclophosphamide micronucleus indexes in bone marrow cells of the Sprague Dawley, Lewis and Wistar rat lines. Five experimental groups were formed (10 animals of each sex and of each line, in every group). The first group was used as the negative control (intact animals), the second one was exposed to oral administration of drugs; other conditions were the same as for the other groups. The third group was treated with 2% Tween 65 and the fourth group was treated with 0.9% NaCl. Both substances were administered by oral way to 2 mL/kg during 14 days. The fifth group was treated intraperitoneally with strong mutagen cyclophosphamide in the dose of 50 mg/kg (10 mL/kg in solution), on 48th and 24th hours before euthanasia. The Sprague Dawley line (both sexes) was significantly different from the other lines. Rats of this line had lower index of spontaneous formation of micronuclei, higher index of cyclophosphamide-induced micronucle formation, percent of micronucleated erythrocytes in bone marrow and the index of cytotoxicity. The results obtained make it possible to identify the most appropriate line of rats as model animals for studies of genotoxicity. It will allow also to obtain more accurate estimates of genotoxicity of various substances.     

Ascorbic acid reverses the prolonged anesthetic action of pentobarbital in Akr1a-knockout mice

Aims

Aldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, is highly expressed in the liver and is involved in both the detoxification of carbonyl compounds and ascorbic acid biosynthesis. By comparison with wild-type mice, Akr1a-knockout (Akr1a^−/−) mice and human Akrla-transgenic (Akr1a^tg/+) mice experience different anesthetic actions from pentobarbital—prolonged in Akr1a-knockout (Akr1a^−/−) mice and shortened in human Akrla-transgenic (Akr1a^tg/+) mice.

Main methods

We investigated this alteration in the anesthetic efficacy of pentobarbital in Akr1a genetically modified mice.

Key findings

Neither the cytosolic protein of wild-type mouse liver nor purified rat AKR1A directly reduced pentobarbital. Ascorbic acid administration neutralized the prolonged duration of the loss of the righting reflex (LORR) in Akr1a^−/− mice, but preincubation of pentobarbital with ascorbic acid prior to administration did not change the anesthetic effect. Those results indicated that ascorbic acid does not directly reduce pentobarbital. Enzymatic activities and levels of the proteins of some cytochrome P450s that make up a potent detoxification system for pentobarbital showed no changes in the genetically modified mice examined. Thus, ascorbic acid also had no effect on the detoxification system in the liver. The prolonged duration of LORR in the Akr1a^−/− mice caused by pentobarbital and the neutralization of the anesthetic effect by ascorbic acid together with other results imply that ascorbic acid alters the responses of the neuronal system to anesthetics.

Significance

Pentobarbital action is increased under conditions of ascorbic acid deficiency, and this may have to be taken into account when anesthetizing malnourished patients.     

Ascorbic acid decreases oxidant stress in endothelial cells caused by the nitroxide tempol

Stable nitroxide radicals have been considered as therapeutic antioxidants because they can scavenge more toxic radicals in biologic systems. However, as radicals they also have the potential to increase oxidant stress in cells and tissues. We studied the extent to which this occurs in cultured EA.hy926 endothelial cells exposed to the nitroxide Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl). Tempol was rapidly reduced by the cells, as manifest by an increase in the ability of the cells to reduce extracellular ferricyanide and by disappearance of the Tempol EPR signal. Cells loaded with ascorbic acid, which directly reacts with Tempol, showed increased rates of Tempol-dependent ferricyanide reduction, and a more rapid loss of the Tempol EPR signal than cells not containing ascorbate. In this process, intracellular ascorbate was oxidized, and was depleted at lower Tempol concentrations than was GSH, another important intracellular low molecular weight antioxidant. Further evidence that Tempol concentrations of 100-1000 μM induced an oxidant stress was that it caused an increase in the oxidation of dihydrofluorescein in cells and inhibited ascorbate transport at concentrations as low as 50-100 μM. The presence of intracellular ascorbate both prevented dihydrofluorescein oxidation and spared GSH from oxidation by Tempol. Such sparing was not observed when GSH was depleted by other mechanisms, indicating that it was likely due to protection against oxidant stress. These results show that whereas Tempol may scavenge other more toxic radicals, care must be taken to ensure that it does not itself induce an oxidant stress, especially with regard to depletion of ascorbic acid.     

Lack of micronuclei formation in bone marrow of rats after oral exposure to thiocyclam insecticide

In this study, a nereistoxin analogue insecticide, thiocyclam, was administered to adult male albino rats by gavage dose of 135, 270 and 540 mg/kg b.w. repeated for 5 days at 24 h intervals. Control animals received only water. Thiocyclam was tested for its potential to cause genotoxic effects in rat bone marrow cells using an in vivo micronucleus assay. After 24 h of the last treatment, rats from all dose levels were sacrificed. Bone marrow cells were collected and assayed for the presence of micronuclei. Thiocyclam did not cause any increase in the incidence of micronucleated polychromatic erythrocytes in rats bone marrow at any of the dose levels. The polychromatic erythrocytes/normochromatic erythrocytes (PCE:NCE) ratio was found to be in the range from 0.50 ± 0.11 to 0.55 ± 0.02. The results of this study demonstrate that the effect of thiocyclam is not significant in the rat in vivo micronucleus assay.     

Ascorbic acid spares alpha-tocopherol and decreases lipid peroxidation in neuronal cells

Ascorbic acid is considered an antioxidant in the central nervous system, but direct evidence that ascorbate protects neuronal cells from oxidant stress is lacking. Differentiated SH-SY5Y cells in culture took up ascorbic acid on the sodium-dependent vitamin C transporter Type 2 and retained it much more effectively than dehydroascorbic acid. Intracellular ascorbate spared alpha-tocopherol, both in cells loaded with alpha-tocopherol in culture and in cells under oxidant stress due to extracellular ferricyanide. Sparing of alpha-tocopherol in response to ferricyanide was associated with protection against lipid peroxidation in cell membranes. These results show that neuronal cells concentrate ascorbate, and that intracellular ascorbate, either directly or through sparing of alpha-tocopherol, protects them against oxidant stress.     

Ascorbic acid effect on the onset of cell proliferation in pea root

The ability of ascorbic acid to induce cell proliferation of non-cycling cells was investigated in quiescent embryo root of Pisum sativum L. cv. Lincoln, as well as in the active plantlet root meristem, where a minor portion of the cells is non-proliferating. Quiescent embryo cells speeded up the G₀–G₁ transition during germination in the presence of ascorbic acid. In addition, proliferating cells present in the root tip of 3-day-old plantlets, arrested at the G₁/S boundary by hydroxyurea, resumed the cycle earlier than the control, when treated with ascorbic acid. In contrast, ascorbic acid was unable to induce the proliferation of non-cycling cells present in the active meristem. Therefore, these data suggest that the ability of ascorbic acid lo induce cell proliferation depends on the physiological status of the cell. In particular the data indicate that ascorbic acid is involved in cell proliferation as a factor necessary to enable already competent cells to progress through the cell cycle phases, but not as a factor able to induce non-competent cells to overcome proliferation arrest.     

Two structurally distinct inhibitors of glycogen synthase kinase 3 induced centromere positive micronuclei in human lymphoblastoid TK6 cells

Glycogen synthase kinase 3 (GSK3) is an attractive novel pharmacological target. Inhibition of GSK3 is recently regarded as one of the viable approaches to therapy for Alzheimer's disease, cancer, diabetes mellitus, osteoporosis, and bipolar mood disorder. Here, we have investigated the aneugenic potential of two potent and highly specific inhibitors of GSK3 by using an in vitro micronucleus test with human lymphoblastoid TK6 cells. One inhibitor was a newly synthesized maleimide derivative and the other was a previously known aminopyrimidine derivative. Both compounds elicited statistically significant and concentration-dependent increases in micronucleated cells. One hundred micronuclei (MN) of each were analyzed using centromeric DNA staining with fluorescence in situ hybridization. Both the two structurally distinct compounds induced centromere-positive micronuclei (CMN). Calculated from the frequency of MN cells and the percentage of CMN, CMN cell incidence after treatment with the maleimide compound at 1.2muM, 2.4muM, and 4.8muM was 11.6, 27.7, and 56.3 per 1000 cells, respectively; the negative control was 4.5. CMN cell incidence after the treatment with the aminopyrimidine compound at 1.8muM, 3.6muM, and 5.4muM was 6.7, 9.8 and 17.2 per 1000 cells, respectively. Both compounds exhibited concentration-dependent increase in the number of mitotic cells. The frequency of CMN cells correlated well with mitotic cell incidence after treatment with either compound. Furthermore, both inhibitors induced abnormal mitotic cells with asymmetric mitotic spindles and lagging anaphase chromosomes. These results lend further support to the hypothesis that the inhibition of GSK3 activity affects microtubule function and exhibits an aneugenic mode of action.     

Ascorbic acid induced TET2 enzyme activation enhances cancer immunotherapy efficacy in renal cell carcinoma

Exploring the regulatory mechanism of PD-L1 in renal cancer is one of the key strategies to improve the response of renal cancer patients to checkpoint blockade therapy. In this study, the synergistic effect of ascorbic acid (vitamin C) supplementation and the impact of TET2 depletion on anti-PD-L1 therapy were determined in xenograft model experiments. Lymphocyte infiltration and chemokine expression were determined using flow cytometry and qRT-PCR. To determine the downstream targets of TET2, we performed hMeDip-seq and RNA-seq analyses. The molecular mechanism was further confirmed by hMeDip-qPCR, MeDip-qPCR, bisulfite sequencing, Western blotting, qRT-PCR and xenograft model experiments in vitro and in vivo. The present study demonstrated that ascorbic acid enhanced the efficacy of immunotherapy and that the loss of TET2 function enabled renal cancer cells to evade antitumor immunity. Ascorbic acid treatment significantly increased the intratumoral infiltration of T cells and the expression of cytokines and chemokines, while the loss of TET2 impaired the infiltration of T cells and the expression of cytokines and chemokines. TET2 was recruited to IRF1 by IFN-γ-STAT1 signaling, thereby maintaining IRF1 demethylation and ultimately inducing PD-L1 expression. These results suggest a new strategy of stimulating TET activity to improve immunotherapy for renal cell carcinoma.     

Ascorbic acid blunts oxidant stress due to menadione in endothelial cells

Endothelial cells are exposed to potentially damaging reactive oxygen species generated both within the cells and in the bloodstream and underlying vessel wall. In this work, we studied the ability of ascorbic acid to protect cultured human-derived endothelial cells (EA.hy926) from oxidant stress generated by the redox cycling agent menadione. Menadione caused intracellular oxidation of dihydrofluorescein, which required the presence of D-glucose in the incubation medium, and was inhibited by intracellular ascorbate and desferrioxamine. At concentrations of 100 microM and higher, menadione depleted the cells of both GSH and ascorbate, and ascorbate loading partially prevented the decrease in GSH due to menadione. Menadione increased L-arginine uptake by the cells, but inhibited endothelial nitric oxide synthase, an effect that was prevented by acute loading with ascorbate. Ascorbate blunts menadione-induced oxidant stress in EA.hy926 cells, which may help to preserve nitric oxide synthase activity under conditions of excessive oxidant stress.     

Chromosomal aberrations and micronuclei induced in rat and mouse bone marrow cells by sodium nitrate

The genotoxicity of several anthraquinone compounds metabolically related to aflatoxin B1 was examined by means of the hepatocyte primary culture (HPC)/DNA repair test and the Salmonella microsome mutagenesis test, and compared to versicolorins A and B which are potent mutagenic and genotoxic intermediates of the aflatoxin biosynthetic pathway. 6,8-O-Dimethyl-versicolorins A, B and 6-deoxyversicolorin A were found to be strongly mutagenic and genotoxic. Genotoxicity of versicolorin A and 6,8-O-dimethylversicolorin A was stronger than that of versicolorin B and 6,8-O-dimethylversicolorin B, respectively, in the HPC/DNA repair test. Nidurufin and norsolorinic acid, which do not possess a bisfuran ring, exhibited questionable activities for mutagenicity and no genotoxicity. It is suspected that 6,8-O-dimethylversicolorins A, B and 6-deoxyversicolorin A as well as versicolorins A and B are genotoxic carcinogens.     

The genotoxic and cytotoxic effects of ribavirin in rat bone marrow

The genotoxic and cytotoxic effects of the antiviral drug, ribavirin, was studied in rat bone marrow by employing the micronucleus assay. Ribavirin in doses of 10, 15, 20, 30, 50, 75, 100 and 200 mg/kg, and cyclophosphamide (CP) 40 mg/kg (only for sex-difference study) were injected intraperitoneally. Bone marrow was collected at 24 h and 48 h following the injection. To evaluate the recovery, the bone marrow was also sampled at 72 h from 20, 100 and 200 mg/kg treated rats. The micronucleus assay was conducted according to the standard procedure. Ribavirin elevated the incidence of micronuclei (except 10 mg/kg) in erythrocytes (P<0.01). The micronucleated polychromatic erythrocytes showed the initial steep increase at 15 and 20 mg/kg dose level, then with the gradual increase, possibly due to the limited metabolism and action of higher doses. The incidence of micronucleated normochromatic erythrocytes was not dose dependent. The effect was more at 48 h than 24 h due to prolonged toxicity of the drug or its metabolites, and by 72 h, recovery was observed eventhough the genotoxicity was significant. The PCE% decreased as the dose was increased up to 75 mg/kg, then without much difference between two higher doses. Only 100 mg/kg ribavirin and CP showed more toxicity on male rats. Cytotoxicity was seen due to hindered erythropoiesis or cell destruction. Our findings suggest that ribavirin is genotoxic and cytotoxic agent for rat bone marrow.     

Effect of inactivated viral vaccines (human) on frequency of micronuclei in bone marrow erythrocytes of mice.

Cytogenetic effects of the two inactivated viral vaccines (polio and antirabies) were studied in adult male mice by the micronucleus test. Polio salk vaccine did not induce micronuclei formation at both human (0.5 ml) and 1/5th human doses. Antirabies vaccine induced micronuclei in poly and total erythrocytes only at human dose of 2 ml. Beta-propiolactone (BPL) induced micronuclei at higher dose of 5.7 mg, but not at 0.57 mg (approximate concentration present in 2 ml of rabies vaccine). The P/N ratio was not affected in vaccinated and BPL inoculated animals. Antirabies vaccine induced micronuclei percentage was more than the BPL value.     

Vinblastine treatment induces dose-dependent increases in the frequency of micronuclei in mouse bone marrow.

The effect of various doses (0.005-2.0 mg/kg b.w.) of vinblastine sulfate (VBL) was studied on the induction of micronuclei in polychromatic and normochromatic erythrocytes (PCE, NCE) of the bone marrow of female BALB/c mice. VBL treatment resulted in a dose-dependent increase in the frequency of micronucleated PCE and NCE, while the PCE/NCE ratio and mitotic index declined with increasing drug dose. The dose-effect curves were linear quadratic for all the parameters studied.     

The time-course of micronucleated polychromatic erythrocytes in mouse bone marrow and peripheral blood

The time-course of micronucleated polychromatic erythrocytes (MPCE) in mouse bone marrow and peripheral blood, induced by an acute 0.1 Gy dose of X-rays, was determined using flow cytometric analysis, which made frequent sampling possible and allowed use of a dose low enough not to affect erythroid cell proliferation. The frequency of MPCE (fMPCE) began to increase in the bone marrow at 10 h after irradiation and reached a maximum at 28 h after irradiation. In the peripheral blood fMPCE began to increase at 20 h after irradiation and peaked at about 40 h after irradiation. The time-course found is discussed on the basis of data on the differentiation of erythroid cells. The results indicate that the micronuclei registered in polychromatic erythrocytes may originate from lesions induced not only during the last cell cycle but also during earlier ones. After an acute dose of 1.0 Gy of X-rays the maximum fMPCE was delayed both in bone marrow and peripheral blood reflecting an effect on the cell cycle progression of erythroblasts.     

Ascorbic acid prevents acute myocardial infarction induced by isoproterenol in rats: role of inducible nitric oxide synthase production

The goal of this study was to investigate whether sub-chronic anti-oxidant treatment with ascorbic acid (Vit C) is able to protect the heart against myocardial infarction. The effects of Vit C treatment on the histopatological changes and immunohistochemistry for p53, COX-2 and iNOS were evaluated in rats submitted to acute myocardial infarction induced by isoproterenol (ISO). Male Wistar rats (n = 32) were divided into four groups: group 1, control; group 2, ISO treated; group 3, Vit C treated; group 4, ISO + Vit C treated. An amount of 150 mg/kg of isoproterenol was administered for two consecutive days. The rats were treated with Vit C once a day (150 mg/kg, orally) for seven consecutive days. In the day 5 and 6 the rats from group ISO + Vit C were submitted to acute administration of ISO third minutes after Vit C treatment. The results pointed out that treatment with Vit C showed mild degenerative changes of myocardial tissue in ISO group. Also, the antioxidant was able to decrease the iNOS expression in rats treated with Vit C. Taken together, our results suggest that chronic Vit C administration was able to prevent the myocardial infarction induced by ISO as a result of iNOS downregulation. Certainly, this finding offers new insights into the mechanisms underlying the relation between oxidative stress and cardiac mortality after myocardial infarction.