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J. Biol. Chem., Vol. 277, Issue 19, 16895-16899, May 10, 2002

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Vitamin C Prevents DNA Mutation Induced by Oxidative Stress^*

Eugene A. Lutsenko, Juan M. Cárcamo, and David W. Golde

From the Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Received for publication, February 4, 2002, and in revised form, March 5, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 H₂O₂. Depletion of glutathione led to cytotoxicity and an increase in H₂O₂-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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

DNA damage caused by reactive oxygen species such as H₂O₂, O, and ^·OH radicals has been implicated in mutagenesis, oncogenesis, and aging (1). Oxidative lesions in DNA include base modifications, sugar damage, strand breaks, and abasic sites. In vitro studies suggest that the hydroxyl radical is highly reactive toward DNA (2). One of the most common oxidized adducts in human cells and tissues is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG)¹ (3). This adduct results from exposure to oxidizing agents as well as from  irradiation of DNA (4), and quantitation of 8-oxo-dG has been used as a marker of DNA damage (5). 8-oxo-dG "mis-pairs" with adenine during replication (6), resulting in G to T transversions in 50% of the replicated DNA (7).

The role of vitamin C in protecting against oxidatively induced DNA mutations is controversial. Although numerous studies demonstrate the antioxidant effects of vitamin C (8, 9), in vitro studies are often confounded by the pro-oxidant effects of ascorbic acid in the presence of free transition metals (10). We circumvented this problem using dehydroascorbic acid to load cells with vitamin C. Vitamin C is transported into most cells in the oxidized form, dehydroascorbic acid (DHA), via facilitative glucose transporters (11, 12) and as ascorbic acid in specialized cells by sodium-dependent ascorbic acid transporters (13). When transported as DHA it is rapidly reduced inside the cells and accumulated as ascorbic acid (14).

We developed a new genetic system to quantify oxidative DNA damage and resulting mutagenesis in human cells to determine directly the role of vitamin C in maintaining genomic integrity. We found that vitamin C markedly decreased mutations induced by H₂O₂. The mutation results correlated with a decrease in total 8-oxo-dG found in genomic DNA of cells that were loaded with vitamin C and oxidatively stressed.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture and Transfections-- Human kidney 293T cells were cultured in Dulbecco's high glucose medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, 1% L-glutamine, and 1% sodium pyruvate. Human myeloid HL-60 cells were cultured in Iscove's modified Dulbecco's medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% L-glutamine. Cells were maintained in an incubator with 5% CO₂ at 37 °C. Cells (293) were transfected by Ca²⁺-phosphate method at 1.5 × 10⁶ cells per 100-mm plate (15). Plates were incubated overnight, and the cells were collected.

Uptake of Ascorbic Acid (AA) and DHA-- Cells were washed with PBS and incubated for 30 min in incubation buffer (15 mM HEPES pH 7.4, 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, and 0.8 mM MgCl₂). 1.5 × 10⁶ cells were added to incubation buffer containing 100 µM ascorbic acid and 0.2 µCi of L-[¹⁴C]ascorbic acid (PerkinElmer Life Sciences) for AA uptake or to a mixture containing ascorbic acid and ascorbate oxidase for DHA uptake. Following incubation samples were washed twice with cold Ca²⁺/Mg²⁺-free PBS. After lysis in 10 mM Tris-HCl (pH 8.0) containing 0.2% SDS, the incorporated radioactivity was determined by liquid scintillation spectrometry.

Cell Volume Determination-- Intracellular volume was estimated as described previously (11, 16) with 30% correction for trapped extracellular radioactivity (17). Briefly, five million cells were incubated for 60 min at room temperature in 200 µl of incubation buffer containing 1 mM 3-oxy-methyl-glucose (OMG) and 5 µCi of ³H-OMG. During incubation equilibrium (zero-trans) was established between intra- and extracellular concentrations of OMG. After incubation 2 µl of 2 mM cytochalasin B was added to the cells to prevent efflux of trapped OMG during washing, and the mixture was incubated at room temperature for 5 min. Cells were then washed three times with cold Ca²⁺/Mg²⁺-free PBS containing 20 µM cytochalasin B to remove unincorporated radioactivity. After lysis in 10 mM Tris-HCl (pH 8.0) containing 0.2% SDS the incorporated radioactivity was determined by liquid scintillation spectrometry. The amount of radioactivity accumulated inside the cells is in direct proportion to the intracellular volume.

Construction of pCAT19-Cm^s-- Tyrosine 33 codon (TAT) within the CAT gene of plasmid pCAT19 was changed to a TAG stop codon by site-directed mutagenesis using the unique restriction site elimination method (18). The change in the sequence was confirmed by sequencing. The following primers were used in mutagenesis: mutagenic primer (amber mutation is underlined), GCT CAA TGT ACC TAG AAC CAG ACC GTT CAG C; selection primer (eliminates AatII site, underlined), GGT TTC TTA GAC Gta AGG TGG CAC TTT TCG.

Depletion of Glutathione-- 293T cells were depleted of glutathione as previously described (19). Briefly, 293T cells (2 × 10⁶/100-mm plate) transfected with 6 µg of pCAT19-Cm^S were incubated with 200 µM DL-buthionine-[S,R]-sulfoximine (Sigma) for 16 h, followed by a 1-h incubation with 1 mM diethyl maleate (Sigma). Cells were washed with PBS and used in the in vivo DNA damage assay as described below.

Enzymatic Digestion of DNA-- Total genomic DNA from 50 × 10⁶ HL-60 cells was isolated using the blood and cell culture kit from Qiagen. The isolated DNA was digested by nuclease P₁ (Sigma) and calf alkaline phosphatase (Roche Molecular Biochemicals). Briefly, 5-15 µg of DNA (200 µl) in 25 mM sodium acetate, 1 mM zinc chloride (pH 4.8) was boiled for 5 min and quickly chilled on ice. Nuclease P₁ (0.1 units/µg of DNA) was added to DNA and incubated at 37 °C for 1 h. 20 µl of 1.5 M Tris-HCl (pH 8) was added, and the solution was briefly vortexed. Calf alkaline phosphatase (0.075 units/µg of DNA) was added, and the solution was incubated for another 30 min at 37 °C. Digested DNA was filtered through a 0.2-µm Nalgene 4-mm nylon syringe filter. Samples were further analyzed by HPLC-ECD.

Determination of 8-oxo-dG by HPLC-ECD-- Digested DNA was injected onto a modified C18 column (catalog no. 70-4106, ESA, Chelmsford, MA). Equal amounts of digested DNA isolated from untreated or treated cells were injected (based on absorbance at 260 nm). The column was equilibrated with the following buffer on the mobile phase (100 mM lithium acetate, 10% methanol, pH 5) at 30 °C. The flow rate was 0.5 ml/min. Under these conditions the retention time of 8-oxo-dG was 4 min. Peak areas for 8-oxo-dG were determined using a dominant potential for 8-oxo-dG of 270 mV. Based on calibration curve (obtained with standard solution of 8-oxo-dG) the amounts of 8-oxo-dG in total genomic DNA were determined.

In Vitro DNA Mutation Assay-- One microgram of plasmid pCAT19-Cm^S was incubated at 37 °C for 1 h in 15 mM potassium phosphate buffer (pH 7.2) in the absence or presence of 0.05 and 5 mM H₂O₂ and Cu²⁺ (25 µM). DNA was ethanol precipitated, resuspended in 5 µl of water, and electroporated into PR195. Bacteria were plated on LB plates supplemented with chloramphenicol (Cm) at 20 µg/ml as well as on LB plates supplemented with carbenicillin (Carb) at 50 µg/ml. Plates were incubated at 37 °C for 16 h. Mutation frequencies were determined as ratios between the number of colonies on Cm plates over the total number of colonies on Carb plates.

In Vivo DNA Mutation Assay-- 293T cells (2 × 10⁶/100-mm plate) transfected with 6 µg of pCAT19-Cm^S were incubated with 500 µM DHA for 1 h and treated with 0.1, 0.5, and 5 mM H₂O₂ with Cu²⁺ (100 µM) or with 10 mM H₂O₂ without Cu²⁺ for 1 h. The plasmid was isolated using the SDS-NaOH lysis method (20) and electroporated into PR195. Mutation frequencies were determined as outlined above.

Fifty million HL-60 cells (0.3 × 10⁶/ml) were incubated for 2 days prior to the experiment. Cells were washed in PBS, incubated with different amounts of DHA for 1 h, and treated with H₂O₂/Cu²⁺ for 22 h. Total genomic DNA was isolated using the blood and cell culture kit from Qiagen. DNA was enzymatically digested for HPLC-ECD assays as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The chloramphenicol-sensitive allele (Cm^S) of the chloramphenicol acetyl transferase (CAT) gene was used as a marker for the determination of 8-oxo-dG-related mutagenesis in DNA. The wild type sequence of the CAT gene in plasmid pCAT19 was changed by site-directed mutagenesis at codon 33 from TAT (tyrosine) to TAG (stop) as outlined under "Materials and Methods." The resulting plasmid (pCAT19-Cm^S) expresses a truncated chloramphenicol acetyl transferase; therefore, bacterial cells with this allele are sensitive to chloramphenicol. The plasmid pCAT19 also carries the carbenicillin resistance gene (Carb) used for positive selection for the plasmid.

Under oxidative stress 8-oxo-dG can be created throughout the DNA molecule including the mutated codon 33 within the Cm^S allele. Subsequent replication introduced adenine across the 8-oxo-dG. The second round of replication restored the wild type TAT codon in 50% of the replicated DNA (7) (Fig. 1A), and consequently those cells became resistant to Cm. Thus, the degree of oxidative DNA damage was expressed as a function of Cm-resistant colonies that appeared over Carb-resistant colonies. Mutations outside the TAG stop codon in the mutated CAT gene were not detected by this assay. The probability of two mutations occurring in the same plasmid (as two independent events) is approximately 10⁹ and is beyond the detection sensitivity of the assay.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Mutations induced by H2O2 in naked DNA and 293T human kidney cells. A, conversion of 2'-deoxy-guanine to 8-oxo-dG by oxidation. A plasmid containing the CAT-sensitive allele, pCAT19-CmS, was constructed by introducing the amber TAG mutation at codon 33 of the CAT gene. Oxidation of dG into 8-oxo-dG (*) during DNA replication introduces 50% of G to T transversions generating functional CAT gene. B, frequency of mutations induced by hydrogen peroxide in naked DNA. The plasmid pCAT19-CmS was incubated with 5 mM H2O2 or with 50 µM H2O2 and 25 µM Cu2+. The plasmid was transformed into the E. coli strain PR195, and the frequency of mutations was calculated as the ratio of colonies CmR/CarbR. Results are given as fold increase over mutation frequency of untreated DNA. The absolute mutation frequency in untreated DNA was about 4 × 105. Values represent the mean ± standard deviation from triplicate measurements. The data are statistically significant at p < 0.05 using the Student's t test. C, schematic representation of the oxidative DNA damage assay in human cells. D, 293T cells transfected with pCAT19-CmS were either left untreated (c), incubated with H2O2, or incubated with H2O2 and Cu2+ for 1 h. The plasmid was extracted and electroporated into PR195. The frequency of mutations was determined by the ratio of colonies CmR/CarbR. Results are given as fold increase over mutation frequency of DNA isolated from untreated cells.

To test the plasmid for the induction of mutations by oxidative stress, pCAT19-Cm^S was incubated with either 5 mM H₂O₂ or 25 µM Cu²⁺ as well as with 50 µM H₂O₂ in the presence of 25 µM Cu²⁺ for 1 h at 37 °C. Treated DNA was electroporated into Escherichia coli strain PR195 (fpg mutY) along with untreated DNA as a control. The PR195 strain was deficient in the removal of both 8-oxo-dG (fpg) (21) and adenines across 8-oxo-dG (mutY) (22). The latter mutation ensured that all 8-oxo-dG created in human cells were counted. Bacteria were plated on LB plates with chloramphenicol at 20 µg/ml as well as on LB plates with carbenicillin at 50 µg/ml. Mutation frequencies were determined as ratios of the number of colonies on Cm plates to the number of colonies on Carb plates. The absolute mutation frequency in untreated DNA was approximately 4 × 10⁵. DNA treated with 5 mM H₂O₂ showed a 27-fold increase in mutation frequencies, and the addition of Cu²⁺ ions induced further increases in mutation frequencies even at low concentrations of hydrogen peroxide (Fig. 1B). Copper ions alone, however, did not induce mutagenesis (Fig. 1B). These experiments demonstrated that the system was suitable for quantifying the frequency of mutations induced by oxidative stress.

We used the 293T human kidney cell line to study mutagenesis induced by hydrogen peroxide because of the high transfection frequency of these cells (80-90%). This made them suitable for the study of DNA damage under non-replicative conditions (15). The plasmid pCAT19-Cm^S has no mammalian origin of replication and therefore cannot be replicated inside 293T cells. Cells were transfected with pCAT19-Cm^S and were treated with H₂O₂ 24 h later (Fig. 1C). Plasmid DNA was extracted from the cells, purified and electroporated into PR195, and analyzed for mutation frequencies (ratio of Cm^R to Carb^R colonies). We found a roughly linear increase in mutation frequency with increasing concentrations of H₂O₂ in the presence of 100 µM Cu²⁺ (Fig. 1D). The addition of Cu²⁺ ions presumably led to increased oxidation and G to T transversion mutations in the plasmid due to ^·OH generation via the Fenton reaction. Cells treated with 5 mM H₂O₂ and 100 µM Cu²⁺ showed a 6-fold increase in mutation frequency compared with control. However, cells incubated with Cu²⁺ ions alone evidenced no increase in mutation frequencies (Fig. 1D). Hydrogen peroxide without added copper did not cause a significant increase in mutations under these conditions even at concentrations of 10 mM (Fig. 1D). We therefore used 5 mM H₂O₂ and 100 µM Cu²⁺ to study the role of vitamin C in the prevention of oxidative DNA damage in 293T cells.

Previously we showed that vitamin C is transported into the cells preferentially in the oxidized form as DHA through facilitative glucose transporters (11). Once inside the cell DHA is rapidly reduced to ascorbic acid (14). We found that 293T cells incubated with 500 µM DHA for 60 min accumulated 7 nmol of ascorbic acid/10⁶ cells (Fig. 2A). We estimated the internal volume of 293T cells as 1.0 µl/10⁶ cells from tritiated methylglucose equilibrium studies (see "Materials and Methods"). Based on this internal volume 293T cells incubated with 500 µM DHA for 60 min accumulated 7 mM ascorbic acid. However, cells incubated with 500 µM AA for 60 min accumulated only 0.7 mM ascorbic acid (Fig. 2A).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Vitamin C protects DNA from mutations induced by oxidation in normal and glutathione-depleted cells. A, uptake of AA and DHA by human 293T kidney cells. Cells were incubated with either radiolabeled AA or radiolabeled DHA for the periods indicated. Cells were washed, and the incorporated radioactivity was determined. Results are shown as accumulation in nmol of vitamin C/106 cells. B, frequency of mutations in human 293T kidney cells. Cells transfected with pCAT19-CmS were incubated with 500 µM DHA for 60 min followed by treatment with 5 mM H2O2 and 100 µM Cu2+ for 1 h. The mutation frequencies were resolved in E. coli as outlined in the legend to Fig. 1C. Results are shown as fold increase over mutation frequency of DNA isolated from untreated cells. Values represent the mean ± standard deviation from triplicate measurements. Results are statistically significant at p < 0.05 using the Student's t test. C, 293T cells transfected with pCAT19-CmS were depleted of glutathione and incubated with 100 µM Cu2+ and H2O2 for 1 h. The mutation frequencies were resolved in E. coli as outlined in the legend to Fig. 1C. Results are shown as fold increase over mutation frequency of DNA isolated from H2O2 untreated cells (closed circles represent cells depleted of glutathione, and open circles represent cells with glutathione). D, frequency of mutations in glutathione-depleted human 293T kidney cells preloaded with DHA. The transfected cells were pre-incubated with 500 µM DHA for 60 min followed by treatment with 250 µM H2O2 and 100 µM Cu2+ for 1 h. The mutation frequencies were resolved in E. coli as outlined in the legend to Fig. 1C. Results are shown as fold increase over mutation frequency of DNA isolated from untreated cells. Values represent the mean ± standard deviation from triplicate measurements. Results are statistically significant at p < 0.05 using the Student's t test.

We investigated the effect of vitamin C loading by exposure to DHA in preventing mutations induced by oxidative stress in the 293T cells. Hydrogen peroxide with copper increased mutation frequency by 8-fold compared with the control (Fig. 2B). Cells incubated with 500 µM DHA for 60 min prior to H₂O₂/Cu²⁺ treatment showed a markedly reduced mutation frequency similar to the control level (Fig. 2B). This result indicated that vitamin C inhibits mutagenesis induced by oxidative stress in vitro. Under these conditions, cells with an intracellular concentration of 7 mM vitamin C were resistant to mutagenesis when treated with 5 mM H₂O₂ and 100 µM Cu²⁺.

Glutathione and vitamin C are the most abundant natural antioxidants in human cells, and their functions are partially overlapping (23). We investigated whether vitamin C could protect cells depleted of glutathione under oxidative stress. 293T cells were depleted of glutathione as described (19, 23) and treated with H₂O₂. Depletion of glutathione substantially increased the toxicity of H₂O₂. Glutathione-depleted cells did not survive treatment with 5 mM H₂O₂ and 100 µM Cu²⁺ for 1 h (data not shown). Cells depleted of glutathione showed a 2- to 3-fold increase in frequency of mutations as compared with unmodified cells (Fig. 2C). To study the role of vitamin C in the prevention of mutagenesis in glutathione-depleted cells, the concentration of H₂O₂ was lowered to the micromolar level. At 250 µM H₂O₂ and 100 µM Cu²⁺ there was a 7-fold increase in mutation frequency over control (Fig. 2D). Under these conditions a prominent antimutagenic effect of vitamin C was observed in glutathione-depleted cells. Cells incubated with 500 µM DHA for 60 min prior to oxidative stress had mutation frequencies similar to those of untreated cells (Fig. 2D). As evidenced in the results shown in Fig. 2, antimutagenic effects of vitamin C could be observed in both unmodified and glutathione-depleted cells. Similar results were obtained when the glutathione-depleted cells were treated with 500 µM H₂O₂ 100 µM Cu²⁺ (data not shown).

Because generation of 8-oxo-dG on the plasmid DNA induces G to T transversions, we sought to determine whether vitamin C directly inhibits formation of 8-oxo-dG in genomic DNA. A time course analysis demonstrated that to generate 8-oxo-dG in genomic DNA by oxidative stress, prolonged incubation for at least 20 h with H₂O₂ and Cu²⁺ was necessary (data not shown). We chose HL-60 for these studies because 293T cells could not survive prolonged incubation with H₂O₂ and Cu²⁺. HL-60 cells were incubated with DHA for 1 h and treated with 100 µM H₂O₂/100 µM Cu²⁺ for 22 h. Cells treated under these conditions expressed ~75% viability (Fig. 3A).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Vitamin C protects genomic DNA from oxidation. A, HL-60 cells were treated with different concentrations of H2O2 in the presence of 100 µM Cu2+ for 22 h, and the cell number was determined. Cell viability is presented as a percentage of survivors over control untreated cells. B, HL-60 cells were incubated with different concentrations of DHA for 1 h and treated with 0.1 mM H2O2.in the presence of 100 µM Cu2+ for 22 h. The total genomic DNA was isolated and digested with nuclease P1 and calf alkaline phosphatase. Samples were separated by HPLC, and areas of the peaks corresponding to 8-oxo-dG were determined by coulometric detection. Results are shown as fold increase over the basic amount of 8-oxo-dG in control DNA isolated from untreated HL-60. The absolute level of 8-oxo-dG was approximately 50 µmol of 8-oxo-dG/mol of G. Values represent the mean ± standard deviation from triplicate measurements. The results are statistically significant at p < 0.05 using the Student's t test.

In oxidatively stressed cells there was a 10-fold increase in 8-oxo-dG over the control. Cells incubated with either 500 µM or 1 mM DHA for 60 min prior to oxidative stress showed a complete inhibition in formation of 8-oxo-dG (Fig. 3B). This protective effect of vitamin C loading was dose-dependent. 100 µM DHA reduced 8-oxo-dG by ~50% (Fig. 3B). Cells incubated with 0.1, 0.5, or 1.0 mM DHA showed no increase in 8-oxo-dG (Fig. 3B). Although dead cells are not excluded by the assay, and vitamin C could protect cells from death caused by oxidative challenge, such an effect would not change the conclusions based on 8-oxo-dG measurements.

The viability of the HL-60 cells treated with 1 mM DHA for 60 min was greater then 95%, as measured by trypan blue exclusion. We determined previously that in HL-60 cells uptake of DHA proceeds in a near linear fashion for about 30 min with no saturation by 60 min (14). The lack of toxicity to DHA may relate to its rapid uptake and conversion intracellularly to ascorbic acid. Quantification of 8-oxo-dG in genomic DNA revealed that cells treated with H₂O₂ contained 0.53 mmol of 8-oxo-dG/mol of G. Background levels of 8-oxo-dG in HL-60 cells were 0.053 mmol of 8-oxo-dG/mol of G (or 1 8-oxo-dG/19,000 G), which is higher than previously reported (1). The degree of protection against oxidative stress in HL-60 cells correlated to the quantity of vitamin C present inside the cells. We determined the intracellular volume of HL-60 as 0.6 µl per million of cells (see "Materials and Methods"). Based on DHA uptake studies (24) we found that HL-60 cells that were incubated with 100 µM, 500 µM, and 1 mM DHA for 60 min accumulated 3, 8, and 12 mM ascorbic acid, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

There is considerable lay and scientific controversy regarding the role of vitamin C in preventing oxidative DNA damage. Numerous in vitro and in vivo studies have reported on the antioxidant (25-27) and antimutagenic (28, 29) effects of ascorbic acid. Conversely, other studies have showed that under certain conditions, vitamin C functions as a pro-oxidant and can increase DNA damage (30-34). It is well known that ascorbic acid acts as a pro-oxidant in the presence of free transition metals (Cu²⁺, Fe³⁺) (10). Many in vitro experiments with cells have been performed with ascorbic acid with confounding results caused by the generation of H₂O₂ and subsequently ^·OH via the Fenton reaction (35). Although free transition metals (iron, for example) are essential, iron-dependent reactions are controlled in vivo through sequestration of iron in non-catalytic protein-bound forms (35). Thus, it is generally believed that the antioxidant properties of ascorbate outweigh any pro-oxidant properties in vivo.

Podmore et al. (36) suggested that vitamin C exhibits simultaneous pro-oxidant and antioxidant properties based on in vivo studies. He administered vitamin C as a dietary supplement to healthy volunteers and found that the level of 8-oxo-dG in peripheral blood lymphocytes decreased with supplementation relative to placebo; however, there was a significant increase in 8-oxo-dA levels. These contradictory results are difficult to rationalize. On the other hand, Fraga et al. (25) showed that decreasing the dietary uptake of ascorbate elevated the level of 8-oxo-dG in human sperm DNA as measured by HPLC. In another study, Brennan et al. (37) found that oral supplementation with vitamin C in human volunteers decreased H₂O₂-induced DNA damage in isolated human lymphocytes but had no effect on endogenous levels of DNA damage. A recent study by Lee et al. (38) pointed to potential pro-oxidant properties of ascorbic acid based on the decomposition of lipid hydroperoxides induced by vitamin C in vitro independent of free transition metals. Under controlled and extracellular conditions they found that vitamin C can generate 4,5-epoxy-2(E)-decenal (4,5-EDE) from lipid hydroperoxides. Because 4,5-EDE can lead to the generation of etheno-2'-deoxyadenosine, they concluded that vitamin C could cause mutations. These studies, however, did not address the question of whether this reaction occurs inside cells, and limited studies in human volunteers did not support the notion of vitamin C-induced lipid peroxidation (39).

Measurement of 8-oxo-dG is one of the most common methods of assessing DNA damage, but there is no consensus on the actual levels in human DNA. Halliwell (40) states that a common artifact in measuring 8-oxo-dG levels in DNA is artificial oxidation. We directly addressed the role of vitamin C in mutagenesis by developing a quantitative plasmid-based genetic system that allowed for the quantification of oxidatively induced mutations in human cells in vitro. The effect of extracellular ascorbic acid, generating reactive oxygen species in conjunction with free transition metals, was circumvented by loading cells with vitamin C by incubation with DHA (11, 14). Our results directly support the hypothesis that vitamin C protects against oxidative DNA damage in human cells under oxidative stress. This was true for unmodified cells as well as those depleted of glutathione, and the protection was dose-dependent. The prevention of oxidative damage by vitamin C was general in nature, inhibiting the creation of 8-oxo-dG in plasmid DNA as well as genomic DNA. Thus, direct measurement of 8-oxo-dG in genomic DNA confirmed the results of the genetic assay.

Under physiological conditions vitamin C circulates in the blood in its reduced form, ascorbic acid, at ~30-50 µM. There is a wide range of intracellular concentrations of vitamin C in human cells and tissues. Levine et al. (39) found that lymphocytes accumulate 3.5 mM ascorbic acid at oral intakes of 1,000 mg of vitamin C daily (41). Under similar conditions mononuclear leukocytes were reported to accumulate 3.5-6 mM ascorbic acid (39, 41, 42). Our data show that high intracellular concentrations of ascorbic acid reduce mutations caused by oxidative stress in human cells in vitro and point to a role for vitamin C in preventing DNA mutagenesis in humans.

	ACKNOWLEDGEMENTS

The PR195 strain was provided by Dr. Pablo Radicella (Centre National de la Recherche Scientifique, Fontenay-aux-Roses, France). The plasmid pCAT19 was provided by W. Claiborne Fuqua (Indiana University). We thank Dr. Jerard Hurwitz for a critical review of the manuscript.

	FOOTNOTES

^* This work was supported by grants from the National Institutes of Health and New York State.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021. Tel.: 212-639-8483; E-mail: d-golde@ski.mskcc.org.

Published, JBC Papers in Press, March 7, 2002, DOI 10.1074/jbc.M201151200

	ABBREVIATIONS

The abbreviations used are: 8-oxo-dG, 8-oxo-7,8-dihydro-2'-deoxyguanosine; DHA, dehydroascorbic acid; AA, ascorbic acid; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; ECD, electrochemical detection; Cm, chloramphenicol; Carb, carbenicillin; Cm^s, Cm-sensitive allele; CAT, Cm acetyltransferase; OMG, 3-oxy-methyl-glucose.

	REFERENCES

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				J. M. Carcamo, A. Pedraza, O. Borquez-Ojeda, B. Zhang, R. Sanchez, and D. W. Golde Vitamin C Is a Kinase Inhibitor: Dehydroascorbic Acid Inhibits I{kappa}B{alpha} Kinase {beta} Mol. Cell. Biol., August 1, 2004; 24(15): 6645 - 6652. [Abstract] [Full Text] [PDF]

				E. A. Lutsenko, J. M. Carcamo, and D. W. Golde A Human Sodium-Dependent Vitamin C Transporter 2 Isoform Acts as a Dominant-Negative Inhibitor of Ascorbic Acid Transport Mol. Cell. Biol., April 15, 2004; 24(8): 3150 - 3156. [Abstract] [Full Text] [PDF]

				K. W. Lee, H. J. Lee, Y.-J. Surh, and C. Y. Lee Vitamin C and cancer chemoprevention: reappraisal Am. J. Clinical Nutrition, December 1, 2003; 78(6): 1074 - 1078. [Abstract] [Full Text] [PDF]

				D. W. Golde Vitamin C in Cancer Integr Cancer Ther, June 1, 2003; 2(2): 158 - 159. [PDF]

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