1 Department of Physiology, University of Adelaide, South Australia 5005, and
2 CSIRO Human Nutrition, PO Box 10041, Gouger Street, Adelaide, SA 5000, Australia
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Abstract |
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Abbreviations: AP, anti-oxidant-poor; BN, binucleate; CBMN, cytokinesis-block micronucleus; cyto B, cytochalasin B; FBS, fetal bovine serum; MNed BN, micronucleated binucleate; PHA, phytohaemagglutinin; RDI, recommended daily intake; ROM, reactive oxygen metabolite; SCE, sister chromatid exchange.
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Introduction |
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Vitamin C is a redox chemical (8). The pro-oxidant properties of vitamin C rely on the presence of transition metal ions, which are normally carefully sequestered in the cell (5). However, DNA is a powerful chelator of transition metals, and oxidative stress can release Fe and Cu ions into forms that are capable of binding to DNA (2). Vitamin C may reduce DNA-bound copper di-anions (Cu2+) and the resulting anions (Cu+) can reduce hydrogen peroxide (H2O2) to form the highly reactive hydroxyl radical (9). Interaction of hydroxyl radicals with DNA may cause breaks in the phosphodiester backbone and modification of DNA bases (e.g. 8-oxoguanine). Increased amounts of such damage are linked to an increased risk of cancer (10). Recent reports suggest that Fe ions may also bind to DNA (11). Vitamin C interacts with Fe3+ in the same way as Cu2+, leading to the formation of hydroxyl radicals via the Fenton reaction (5,8). Vitamin C may also oxidize spontaneously or in the presence of transition metals to form dehydroascorbic acid and H2O2 (12).
The genotoxic effects of vitamin C in vitro are well documented. It is reported to cause dose-dependent (0500 µM) increases in DNA damage (13) and, at high concentrations, to enhance the cytotoxicity of H2O2, to human lymphocytes by increasing hydroxyl radical production (14). Vitamin C (10 µM to 1 mM) has also been shown to induce small increases in the frequency of sister chromatid exchanges (SCEs) in Chinese hamster ovary cells in vitro (15). When cells were exposed to a superoxide-generating system, low dose vitamin C ( 10 µM) reduced, while high dose (
100 µM) vitamin C increased the number of SCEs. Others have found that low dose vitamin C (1 and 10 µM) had no effect on baseline SCE frequency in Chinese hamster ovary cells in vitro. These concentrations of vitamin C reduced ROM generation from H2O2 and lowered H2O2-induced SCE frequency. However, vitamin C increased the frequency of other forms of H2O2-induced chromosomal aberrations by at least 60%. It was suggested that this increase in chromosomal aberrations was caused by hydroxyl radicals, which are unlikely to be scavenged by anti-oxidants and may not be detected in the fluorescence assay used to detect ROMs (16).
Together these experiments have shown that vitamin C is able to cause DNA damage in vitro at concentrations achievable in vivo via supplementation. It is reported that a 2 g supplement can raise plasma vitamin C levels from around 74 to 170 µM (17). It is apparent that the redox properties of vitamin C are concentration dependent. However, it is not known whether the concentrations attainable in vivo are sufficient to facilitate the expression of pro-oxidant properties. There has been considerable debate concerning the health benefits of using vitamin C supplements as it has been suggested that above the recommended daily intake (RDI), vitamin C can promote free radical production by fuelling the Cu- and Fe-dependent generation of hydroxyl radicals (8). Supplements can also increase the availability of Fe for such reactions by liberating Fe2+ from ferritin (18).
Reactive oxygen metabolites may also play an important role in the signal transduction and effector phases of apoptosis (19). Vitamin C (250 µM) has been reported to inhibit chemically-induced DNA fragmentation, a characteristic of apoptosis, in human thymocytes in vitro (20). This effect was abolished by catalase and reproduced with 20 µM H2O2 (20). The finding that chelation of Cu, but not Fe inhibits chemically-induced apoptosis also suggests a critical role for Cu in apoptosis (21). It is possible that vitamin C may influence apoptosis by reducing Cu, thereby initiating ROM generation, or by interacting with ROMs involved in signal transduction or DNA/protein oxidation. Although it is unclear whether apoptosis is affected in vivo, an inhibition of apoptosis would reduce the probability of mutated cells dying, thereby increasing the accumulation of mutant cells in vivo.
There is a clear need to determine what effect vitamin C intake, at above RDI, may have on baseline or oxidative stress-induced DNA damage, and apoptosis in human cells under physiological conditions. Through the use of a human intervention trial, this study aimed to determine (i) whether vitamin C supplementation alters baseline levels of chromosome damage, necrosis and apoptosis, and (ii) whether vitamin C supplementation protects against or exacerbates the genotoxic and cytotoxic effects of H2O2 ex vivo. The work we present also emphasizes the practicality and importance of integrating apoptosis and necrosis indices within the cytokinesis-block micronucleus assay.
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Materials and methods |
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Cytokinesis-block micronucleus technique, including apoptosis and necrosis indices
Chromosome damage was assessed using the cytokinesis-block micronucleus (CBMN) technique in isolated lymphocyte cultures as previously described (22). Micronuclei are formed due to chromosome breakage and whole chromosome loss events, and were scored using the established criteria (22). Cells with well-defined cytoplasmic boundaries, exhibiting chromatin condensation and/or nuclear fragmentation were classified as apoptotic. Cells exhibiting loss of well-defined cytoplasmic boundaries, loss of cytoplasm, vacuolization, without nuclear fragmentation or chromatin condensation, were classified as necrotic. Chromosome damage rates are expressed as the number of micronucleated binucleates (MNed BNs) per 1000 binucleated cells (BNs). Cell division frequency is shown as the percentage of viable (non-apoptotic, non-necrotic) cells that are binucleated (% BNs). Apoptosis and necrosis are expressed as percentages of total cell numbers. The total cell number counted included mononuclear cells, BNs, other multinucleated cells, apoptotic cells and necrotic cells (unless otherwise indicated). All assays were performed in duplicate or higher multiples.
H2O2 doseresponse in vitro
A H2O2 doseresponse experiment was performed in order to select a concentration that effectively induces a significant increase in micronucleus formation without causing excessive reduction in the number of cells completing nuclear division as assessed by the BN cell ratio. This dose was then used in subsequent experiments examining the capacity of plasma to protect against H2O2-induced damage.
Fasted blood samples were collected from two healthy non-smoking male volunteers, aged 20 and 40 years, using lithium heparin-coated vacuettes. Plasma was isolated and incubated at 37°C in a humidified atmosphere with 5% CO2. Blood was then reconstituted with saline solution (0.89% NaCl) and lymphocytes were isolated using Ficoll-Paque gradients before being washed in HBSS. Lymphocyte cultures were prepared in quadruplicate in their corresponding plasma at a concentration of 1.0x106 cells/ml in a 750 µl volume, and incubated for 30 min before and after the addition of 0, 128, 256, 512 and 1024 µM H2O2. Lymphocytes were then washed, and replicate cultures prepared in RPMI 1640 medium containing 10% FBS, streptomycin (100 mg/l) and penicillin-G (60 mg/l); the cell concentration was 1.0x106/ml and the culture volume was 750 µl. Mitogenesis was stimulated by phytohaemagglutinin (PHA, 20 µg/ml), followed 44 h later by the addition of cyto B (4.5 µg/ml) to inhibit cytokinesis and accumulate BNs. Cells were harvested 70 h post-PHA and slides were prepared using a cyto-centrifuge (Shandon Southern Products, Cheshire, UK), air dried, then fixed and stained using Diff-Quik. Coded slides were scored for MNed BNs and 500 BNs were scored from each of the four replicate cultures for each treatment. Apoptotic and necrotic cells were not scored in this initial experiment.
Vitamin C doseresponse in vitro
Fasted blood samples were collected from the same two volunteers who donated blood for the H2O2 in vitro doseresponse described above. Cultures were prepared as described above, but were `spiked' with 0, 0.57, 1.13 and 2.27 mM (0, 10, 20 and 40 mg/100 ml) of vitamin C and incubated for 30 min. Extra `spiked' samples were prepared and stored frozen (80°C) for HPLC analysis (electrochemical detection) of vitamin C concentrations. Cells suspended in plasma (spiked with vitamin C) were challenged with 512 µM H2O2 or the vehicle as described above, and incubated for a further 30 min before being washed, resuspended and stimulated for the CBMN assay. MNed BNs and apoptotic cells, but not necrotic cells, were scored until 500 BNs were counted per culture. Experiments for each volunteer were done in quadruplicate.
Human intervention trial
A double-blind placebo-controlled cross-over intervention design was used to study the effects of vitamin C supplementation on chromosome damage, apoptosis and necrosis ex vivo. This design has previously been used to assess the effects of red and white wine consumption on baseline and H2O2-induced MNed BNs (23).
Eleven healthy, non-smoking male volunteers, aged between 21 and 44 years, were recruited. Each gave their informed consent, and approval was obtained from the University of Adelaide and CSIRO human ethics committees. Volunteers donated a fasted blood sample before being placed on an anti-oxidant-poor (AP) diet for 2 days (Table I). The purpose of the AP diet was to lower the volunteers' plasma anti-oxidant levels, thereby maximizing the probability of observing any effects of vitamin C. Forty-eight hours after commencing the AP diet, a second fasted blood sample was taken. Ten minutes later, subjects were randomly assigned to consume 2 g vitamin C or placebo as a drink. The vitamin C drink consisted of 2 g vitamin C and 2 g sugar dissolved in 200 ml of spring water, while the placebo contained only sugar in water. Further blood samples were taken 2 and 4 h after the drink, while still on the AP diet. Volunteers then returned to their normal diets and after an 11 day break the intervention was repeated with a cross-over in the type of drink taken (Figure 1
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Plasma iron concentrations
Subjects with plasma Fe levels exceeding 2.0 mg/ml were excluded from taking the vitamin C supplement because it has been suggested that individuals with high plasma iron levels, such as those seen in haemochromatosis, may experience complications arising from the vitamin C-mediated release of Fe2+ from ferritin (8,18). Total Fe levels in haemochromatosis patients range from 2.25 to 3.25 mg/ml with transferrin being 80100% saturated at these levels (24). At the start of the trial, an extra 9 ml of blood (lithium heparin) was collected and total plasma Fe levels were determined using Atomic Absorption Spectroscopy (Varian Spectra AA-400, Varian Techtron, Victoria, Australia).
Whole blood cultures
Whole blood cultures were prepared, in duplicate, from the 4 h post-drink blood samples for the CBMN assay to test for an in vivo genotoxic affect of high dose vitamin C. Blood samples (lithium heparin), with plasma removed, were reconstituted with HBSS and 500 µl of the suspension was added to 5 ml of media. PHA was then added (281 µg/ml), followed 44 h later by cyto B (6 µg/ml). Seventy hours post-PHA, lymphocytes were isolated using Ficoll-Paque gradients, washed and resuspended in culture medium. Slides from each culture were prepared by centrifugation and scored for MNed BNs, apoptotic and necrotic cells until 500 BNs were counted for each of the two replicate cultures.
Ex vivo H2O2 challenge in plasma collected during the intervention
After the intervention, volunteers returned within 8 weeks to provide a final fasted blood sample from which lymphocytes were isolated. Lymphocyte suspensions were prepared in quadruplicate with plasma collected at each of the eight intervention time points. Plasma temperature was 37°C when cells were added. Cell suspensions were incubated for 30 min before and after the addition of 512 µM H2O2 or vehicle. Lymphocytes were then washed and resuspended in culture medium, before the addition of PHA and completion of the CBMN assay, and scoring of slides as described above.
Statistical analysis
Friedman non-parametric repeated measure ANOVA tests with Dunn's multiple comparison post-tests were used to analyse doseresponse and time response data from in vitro and ex vivo experiments. Baseline and H2O2-induced data were analysed separately. Whole blood culture data was analysed using a paired non-parametric Wilcoxon test. Friedman and Dunn's tests were also used to compare plasma vitamin C concentrations at each intervention time point. Relationships between variables were assessed using a Spearman rank correlation. For vitamin C doseresponse and intervention trials difference of difference data was also analysed whereby results were standardised against 0 mM vitamin C and t = 0 h results (by subtraction), respectively. Data are expressed as mean ± SEM. Significance was accepted as P < 0.05. Statistical calculations were performed using Instat Version 2.0 (GraphPad, CA).
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Results |
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Intervention trial
Plasma iron concentrations. Plasma Fe levels ranged from 0.608 to 2.691 µg/ml. One volunteer had an Fe level exceeding the 2.0 µg/ml cut off (2.691 µg/ml). This person was excluded from taking the vitamin C supplement, but was able to complete the placebo phase of the intervention.
Plasma vitamin C concentrations. The AP diet reduced plasma vitamin C levels from approximately 6163 µM at 48 h to 5253 µM at 0 h, a 15% reduction (P < 0.05). Compared with 0 h, the vitamin C supplement raised plasma levels by approximately 116 and 125%, after 2 and 4 h, respectively (P < 0.05; Table IV).
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To assess the effect of the 2 g vitamin C supplement relative to placebo, we compared the difference in toxicity measurements at 2 and 4 h relative to the results at 0 h. The results of this difference of difference analysis (Table VI) show that plasma collected post-vitamin C ingestion did not significantly alter micronucleus frequency, BN ratio or apoptosis but it moderately increased necrosis only at the 2 h time-point in control (P < 0.04) and H2O2-treated (P < 0.08) lymphocytes.
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Data from the placebo and vitamin C arms of the intervention showed that plasma vitamin C was significantly correlated with (i) % BNs (r = 0.41, P = 0.0001) in cells that were not treated with H2O2, and (ii) the % apoptosis (r = 0.33, P = 0.0031) and the % necrosis (r = 0.36, P = 0.0015) in cells that were treated with H2O2. Combining all data revealed that the % necrosis correlated positively with MNed BN frequency and negatively with the % apoptosis (r = 0.66, P < 0.0001 and r = 0.81, P < 0.0001, respectively), and the % apoptosis correlated negatively with the frequency of MNed BNs (r = 0.67, P < 0.0001).
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Discussion |
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Ex vivo experiments
Results from the ex vivo whole blood and plasma cultures show that vitamin C, at well above average concentrations, does not increase the micronucleus index in the absence of oxidative challenge. Therefore, it appears that a 2 g vitamin C supplement is relatively harmless, as assessed by this index, in the absence of oxidative stress. Although plasma vitamin C levels increased by approximately 125% to 120 µM 4 h after supplementation, this concentration was substantially lower than the 170 µM peak reported by Melethil and colleagues in a similar intervention (17).
The ex vivo plasma experiments, in accordance with in vitro experiments, show that a brief incubation of lymphocytes in plasma with varying levels of vitamin C does not affect the frequency of apoptotic cells observed after culture. Our experiments were not optimally designed to study the influence of vitamin C on the apoptotic process because lymphocytes were exposed to high dose vitamin C only in G0, but by the time the cells arrived at the G1/S and G2/M cell cycle checkpoints they were in culture medium with the normal levels of vitamin C. It is usually at these checkpoints that apoptosis is initiated or the process of cell division allowed to continue (25).
Though post-vitamin C plasma did not alter the % BNs, a measure of mitogenic and immune responsiveness, there was a significant negative correlation between plasma vitamin C concentrations and the % BNs when all data were combined (r = 0.407, P = 0.0001). This finding parallels those of Ramirez et al. (26) who showed that millimolar concentrations of vitamin C induced dose-dependent reductions in DNA synthesis in vitro. Inhibition of DNA synthesis was greatest when vitamin C was added at the initiation of the culture, suggesting that vitamin C may influence essential early cell membrane events, such as molecular transport, phospholipid metabolism, and sodium, potassium and calcium fluxes (26).
Plasma collected after vitamin C ingestion did not seem to influence H2O2-induced micronucleus formation. This agrees with Green et al. (27) who showed that ingestion of 500 mg vitamin C with breakfast did not alter the frequency of baseline or -irradiation-induced MNed BNs ex vivo; however, they reported a reduction of single strand DNA breakage in control and irradiated samples detected by the single cell gel electrophoresis assay. Anderson et al. (28) also report that vitamin C supplementation (60 mg and 6 g per day for 14 days) did not alter chromosome baseline damage as measured using the single cell gel electrophoresis assay and metaphase analysis of chromosome aberrations.
The results from the in vitro experiment and to a lesser extent the ex vivo data suggested that vitamin C in plasma, at a concentration greater than 100 µM, may increase H2O2-induced necrosis. This result is in agreement with Nowak et al. (14), who showed that 100 µM vitamin C significantly increased the cytotoxicity of 600 µM H2O2 to human mononuclear cells in vitro and increased the production of hydroxyl radicals from H2O2 (600 µM to 12 mM) in saline solution. Possible mechanisms include the reduction of Fe and Cu ions into forms able to participate in the Fenton reaction (8,18) and the oxidation of vitamin C to yield H2O2 (11). Thus, vitamin C may have increased H2O2-induced necrosis by enhancing the production of ROMs. Alternatively, cells that have been damaged by high concentrations of H2O2 may be more susceptible to the potential cytotoxic effects of high dose ascorbate.
Our studies suggest that high concentrations of vitamin C enhance the cytotoxicity of H2O2 to human lymphocytes in plasma. Whether the levels of H2O2 used in our challenge experiments are relevant in situations of oxidative stress in vivo, such as heavy exercise, infection and autoimmune/inflammatory diseases, remains to be clarified. Recent studies by Schiffl and associates (29) indicated that two consecutive bouts of exhaustive sprinting caused a statistically significant increase of approximately 19 MNed BNs/3000 BN lymphocytes compared with pre-exercise values. This suggests that physiological changes that occur during aerobic exercise are sufficient to induce DNA damage at the chromosomal level. In light of the possible synergistic effects of vitamin C and H2O2 it is therefore important to determine whether excessive intake of vitamin C prior to or during exhaustive exercise may further increase exercise-induced DNA strand breakage and necrosis.
Combined results for control and H2O2-treated cells from the current study show that there is a strong negative correlation between the % apoptosis and necrosis (r = 0.81, P < 0.0001). This may be the result of fewer cells being able to undergo apoptosis due to a shift towards necrosis. This explanation is supported by a recent report that shows an inhibition of caspase activity by H2O2, at concentrations above 50 µM, which coincided with an inhibition of apoptosis (caspases are proteases involved in cellular degradation during apoptosis; ref. 30). Analysis of the data from our studies also revealed a significant negative correlation between the frequency of MNed BNs and the % apoptosis (r = 0.67, P < 0.0001). There are a number of possible explanations for this relationship. First, this may be a consequence of the positive correlation between the % necrosis and MNed BN frequency, with apoptosis decreasing due to a swing towards necrotic cell death. Alternatively, this could be a real effect, where damaged cells undergo apoptosis, rather than express micronuclei, thus cells with a low propensity to undergo apoptosis are more likely to survive and express micronuclei, and vice versa. It is also possible that increases in the frequency of MNed BNs are a marker of whole cell damage, where the key genes and proteins involved in apoptosis are also disabled.
The potentially harmful interactions of vitamin C with Fe have been discussed by Herbert (18), and have recently been highlighted by Anderson et al. (28), who found that although vitamin C supplementation (60 mg/day or 6 g/day for 14 days) increased the anti-oxidant capacity of human plasma, it increased the number of bleomycin-induced chromosome aberrations. Bleomycin forms complexes with Fe2+ and DNA, thereby promoting the formation of hydroxyl radicals (Fenton reaction) extremely close to DNA. Anderson et al. (28) suggested that vitamin C enhanced the effects of bleomycin by increasing the availability of Fe2+ through its liberation from ferritin. Results from our study (data not shown) revealed a positive correlation between total plasma Fe levels and baseline MNed BN frequency in cultures prepared with 48 h plasma (before placebo, r = 0.8, P = 0.005; before vitamin C, r = 0.71, P = 0.03), suggesting that iron levels may be an important factor to consider in future studies.
In conclusion, the ingestion of 2 g vitamin C was found to have no effect on spontaneous levels of MNed BNs, BNs, apoptosis and necrosis in people with normal Fe levels. This may indicate that vitamin C supplementation is not likely to cause adverse health effects in the absence of excessive oxidative stress in vivo. It is evident from our studies that vitamin C supplementation may not provide protection against the toxic effects of H2O2-challenge ex vivo under the chosen experimental conditions. The in vivo effect of high dose vitamin C on cell toxicity under conditions of abnormally high oxidative stress requires further detailed investigation.
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Acknowledgments |
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Notes |
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References |
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