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DNA instability (strand breakage, uracil misincorporation, and defective repair) is increased by folic acid depletion in human lymphocytes in vitro

^a Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, United Kingdom

	ABSTRACT

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Folic acid is essential for the synthesis and repair of DNA. We report the effects of folate depletion on DNA stability in normal human lymphocytes in vitro. DNA strand breakage, uracil misincorporation, oxidative DNA base damage, and DNA repair capability were determined using variants of the comet assay (single cell gel electrophoresis). Lymphocyte proliferation was measured as an indicator of normal replication. Lymphocytes isolated from human venous blood were stimulated to grow in either complete medium containing folic acid (1 ng/ml–2 µg/ml) or medium deficient in folic acid for up to 10 days. Cells prepared for comet analysis were treated either with the bacterial DNA repair enzyme endonuclease III to determine the level of oxidized pyrimidines in lymphocyte DNA or with uracil DNA glycosylase, which detects misincorporated uracil. Cell number and viability were measured. Normal human lymphocyte DNA contained detectable amounts of misincorporated uracil (estimated as approximately 1000 per cell). DNA strand breakage and uracil misincorporation increased in a time- and concentration-dependent manner after lymphocytes were cultured with decreasing amounts of folic acid. DNA damage was induced at folic acid concentrations routinely observed in plasma from the human population (1–10 ng/ml). Lymphocytes cultured under folate-deficient conditions failed to grow normally compared with control cells. However, all lymphocytes remained viable as measured by Trypan blue exclusion. Cells deprived of folate were unable to efficiently repair oxidative DNA damage induced by hydrogen peroxide. Inhibition of repair was maximal after 8 days in culture. Folate supply had no effect on the level of oxidized pyrimidines in lymphocyte DNA, even after 10 days in culture, suggesting that folate deficiency increases uracil misincorporation relatively specifically. These in vitro results help to determine the mechanism(s) through which folic acid maintains DNA stability.—Duthie, S. J., Hawdon, A. DNA instability (strand breakage, uracil misincorporation, and defective repair) is increased by folic acid depletion in human lymphocytes in vitro. FASEB J. 12, 1491–1497 (1998)

Key Words: DNA breaks • misincorporated uracil • DNA repair • oxidized pyrimidines • comet assay

	INTRODUCTION

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FOLIC ACID STATUS may be an important determinant in the development of cancer. In epidemiological studies, dietary folate deficiency is associated with an increased risk of several specific malignancies, notably cancer of the cervix, lung, colorectum, and brain (1), whereas supplementation with folic acid reduces the incidence of premalignant lesions such as cervical and bronchial dysplasia in oral contraceptive users and smokers, respectively, and the risk of cancer in patients diagnosed with chronic ulcerative colitis (2–5). In addition, folic acid deficiency is associated with chromosomal damage and gene mutations (6–10).

The mechanisms by which folate deficiency increases the risk of cancer are not known; however, folic acid is involved in both methyl metabolism and in DNA synthesis and repair.

Folic acid is important for the production of S-adenosylmethionine, the primary methyl donor for DNA methylation. Genes methylated at specific locations are either not transcribed or are transcribed at a reduced level of expression. Decreased DNA methylation is associated with an increased risk of some forms of cancer (11). Folate deficiency may deplete cellular S-adenosylmethionine levels, causing DNA hypomethylation and inappropriate activation of proto-oncogenes. Experimental animals deprived of folic acid and other dietary methyl donors have lower S-adenosylmethionine activity, hypomethylated DNA, and an increased rate of tumorigenesis. DNA hypomethylation precedes an increase in mRNA for the proto-oncogenes c-fos, c-myc, and c-Ha-ras (12–14).

Folic acid is crucial for DNA synthesis and repair. The conversion of deoxyuridine monophosphate (dUMP)² to thymidine monophosphate (TMP) requires folic acid in the form 5,10, methylenetetrahydrofolate as methyl donor. Imbalances in deoxyribonucleotide pools, resulting from folate deficiency, negatively affect cell replication and DNA repair and can lead to mutagenesis and malignant transformation. Under conditions of folate depletion, a block in the methylation of dUMP to TMP leads to an increase in the cellular levels of deoxyuridine triphosphate and uracil misincorporation into the DNA molecule in place of thymine. Normal DNA repair processes remove the uracil. However, if conversion of dUMP to thymidine is continually limited by folate availability, uracil is misincorporated and removed in a catastrophic repair cycle, which may ultimately induce double strand breakage, chromosome instability, and cancer (15–18).

By using the bacterial DNA repair enzyme uracil DNA glycosylase, we have developed a novel variation of the comet assay (single cell gel electrophoresis) to detect uracil misincorporation in human DNA specifically (19). In this study, we investigate the relationship between folic acid, uracil misincorporation, and DNA stability in vitro.

	MATERIALS AND METHODS

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Materials
`Simultrac Radioassay Kit Vitamin B₁₂ [⁵⁷Co]/Folate [¹²⁵I]' for the simultaneous determination of vitamin B₁₂ and folate was supplied by ICN Flow (Irvine, U.K.). Dutch modified RPMI 1640 medium without folic acid was prepared using a recipe from ICN Flow. Fetal calf serum (FCS) was obtained from Globepharm Ltd (Surrey, U.K.). Ultrapure low melting point (LMP) agarose, standard melting point (SMP) agarose, and Nunc sterile plastics were from Gibco Life Technologies Inc. (Paisley, U.K.). LymphoPrep lymphocyte separation medium (LSM) was supplied by Nycomed U.K. (Birmingham, U.K.). Recombinant interleukin (Aldesleukin 18x10⁶ IU) was from EuroCetus U.K. Ltd (Harefield, U.K.), and Murex Diagnostics Ltd (Dartford, U.K.) supplied HA15 phytohemagglutinin. Uracil DNA glycosylase (1 unit/ml) and 4',6-diamidine-2-phenylindole dihydrochloride (DAPI) were provided by Boehringer-Mannheim (Lewes, U.K.). The Escherichia coli strain overproducing endonuclease III was a generous gift from Dr. R. Cunningham (Department of Biological Sciences, State University of New York, Albany, N.Y.). Richardson Supply Co. (London, U.K.) provided the frosted microscope slides. Bovine serum albumin (BSA, fraction V), folic acid (pteroylglutamic acid), L-glutamine, hydrogen peroxide (30%), penicillin G, sodium pyruvate, and streptomycin sulfate were from Sigma (Poole, U.K.).

Isolation of human peripheral blood lymphocytes
Venous blood samples (50 ml) were collected from healthy normal male volunteers (20–55 years of age). Plasma and red blood cells were prepared from one aliquot of blood (10 ml), immediately snap-frozen using liquid nitrogen, and stored at -80°C before folate levels were determined using a commercially available kit. The remaining blood (40 ml) was diluted 1:1 with RPMI medium, layered onto an equal volume of LymphoPrep LSM, and centrifuged at 700 x g for 30 min. The lymphocyte-containing buffy coat was removed into a fresh tube, washed using RPMI, and spun for 20 min under the same conditions. The supernatant was decanted and the remaining cell pellet was washed in medium and spun as before for 15 min. All steps were carried out at room temperature. The lymphocytes were then resuspended in RPMI containing 100 µg/ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine, 100 µg/ml pyruvic acid, and 10% (v/v) heat-inactivated FCS (complete medium).

In some experiments, human lymphocytes were obtained from a finger prick sample. Whole blood (30 µl) was resuspended in 1 ml RPMI 1640 medium containing 10% (v/v) FCS, underlaid with LymphoPrep (100 µl), and centrifuged at 200 x g for 3 min at 4°C. The buffy coat (100 µl) was washed once in PBS, pH 7.4, centrifuged as before, and resuspended in LMP agarose for comet analysis.

Stimulation of human lymphocytes for growth studies and comet analysis
Lymphocytes isolated from venous blood were resuspended at 1 x 10⁵ cells/ml and stimulated to divide in medium containing interleukin (100 units/ml) and phytohemagglutinin (0.5%). All cell cultures were carried out at 37°C in a humidified atmosphere of 95% air/5% CO₂.

The lymphocytes were allowed to grow in either complete culture medium containing folic acid (2 mg/l) or in folic acid-deficient medium for up to 10 days. Folate was measurable in deficient culture medium at a concentration of approximately 0.1 ng/ml. In certain experiments, cells were cultured in either folate-deficient medium or in medium containing 1, 10, or 100 ng/ml folate.

The effect of folic acid depletion on hydrogen peroxide (H₂O₂) -induced DNA damage and repair was investigated in lymphocytes cultured for up to 10 days either in the presence or absence of folic acid . To induce DNA damage, human lymphocytes were washed once in PBS, pH 7.4, before exposure to H₂O₂ (200 µM) on ice for 5 min. The cells were either resuspended immediately in LMP agarose for comet analysis or incubated at 37°C in 95% air/5% CO₂ in complete culture medium (in the presence or absence of folic acid, as appropriate) for up to 8 h after H₂O₂ treatment to determine repair of oxidative damage. Cells were counted using a Neubauer Improved Hemocytometer. Viability was determined by Trypan blue exclusion.

Single cell gel electrophoresis
Isolated human lymphocytes were suspended in 80 µl of a 1% (w/v) solution of LMP agarose in PBS, pH 7.4, at 37°C and immediately pipetted onto a frosted glass microscope slide precoated with 1% (w/v) SMP agarose in PBS. The agarose was allowed to set for 10 min at 4°C and the slide was incubated in lysis solution [2.5 M NaCl, 10 mM Tris, 100 mM Na₂ EDTA, NaOH to pH 10.0, and 1% (v/v) Triton X-100] at 4°C to remove cellular protein. This leaves the DNA as distinct nucleoids. After lysis, the slides were washed three times for 5 min each in uracil DNA glycosylase buffer (60 mM Tris-HCl, 1 mM EDTA, 0.1 mg/ml BSA, pH 8.0) and blotted dry with tissue paper; the agarose gel was covered with 50 µl of either uracil DNA glycosylase (0.1 unit/gel) or buffer and sealed with a glass coverslip. The slides were then incubated in a moist atmosphere at 37°C for 1 h.

In some experiments slides were treated with the bacterial DNA repair enzyme endonuclease III to determine the effects of folate depletion on oxidative DNA damage in human lymphocytes. After lysis, the slides were washed as described above in endonuclease III buffer (40 mM HEPES-KOH, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml BSA, pH 8.0), blotted dry, and incubated with either endonuclease III (1 µg/ml) or buffer for 45 min.

After enzyme treatment, the slides were aligned in a 260 mm wide horizontal electrophoresis tank containing buffer (1 mM Na₂ EDTA and 0.3 M NaOH, pH 12.7) for 40 min before electrophoresis at 25 V for 30 min at 4°C (temperature of the running buffer, approximately 15°C). The slides were then washed three times for 5 min each at 4°C in neutralizing buffer (0.4M Tris-HCl, pH 7.5) and stained with 20 µl DAPI (5 µg/ml).

Quantitation of the comet assay
DAPI stained nucleoids were scored visually as described previously (20). One hundred images per slide were classified according to the intensity of the fluorescence in the comet tail and given a value of either 0, 1, 2, 3, or 4 (from undamaged class 0 to maximally damaged class 4). In this way, the total score per slide could range from 0 to 400. This method of visual classification has been extensively validated by comparison with comets selected using computerized image analysis. Briefly, representative images of comet classes were analyzed (Komet 3.0, Kinetic Imaging Ltd., Liverpool, U.K.) and the percentage of fluorescence in the comet tail (representing the fraction of DNA in the tail) was plotted against the total score for 100 comets in that class. There is a clear linear relationship (R=0.987) between visual classification and the percentage of DNA measured in the tail ( Fig. 1).

View larger version (8K): [in this window] [in a new window]   Figure 1. Relationship between visual scoring and computerized image analysis of human lymphocyte comets. Results are mean ± SEM (n=50)

	RESULTS

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Human lymphocytes showed increased DNA strand breakage after incubation with the bacterial DNA repair enzyme uracil DNA glycosylase compared with cells incubated with buffer alone ( Fig. 2). This enzyme specifically removes uracil from DNA and introduces breaks at the resulting AP sites. Plasma and red blood cell folate levels from these subjects were within accepted values [6.78 ± 0.73 ng/ml and 281.27 ± 28.39 ng/ml (n=9), respectively]. Thus, normal human lymphocyte DNA contains detectable levels of misincorporated uracil.

View larger version (11K): [in this window] [in a new window]   Figure 2. DNA strand breakage and misincorporated uracil in human lymphocytes. Lymphocytes were incubated with uracil DNA glycosylase in buffer () or in buffer alone (). Results are mean ± SEM (n=9).

After stimulation with interleukin and phytohemagglutinin in the presence of folic acid (2 mg/l), cell number increased by sixfold over an 8-day culture period. Lymphocytes incubated in folate-deficient medium did not grow ( Fig. 3). DNA strand breakage and misincorporated uracil increased correspondingly in lymphocytes cultured for 5–8 days in folate-deficient medium ( Fig. 4). Folate supply had no effect prior to this. Cell viability, measured at the end of the experiment, was comparable for the two groups [98.3% ± 0.25% (n=8) in folate-replete compared with 97.0% ± 1.47% (n=8) in folate-deficient lymphocytes].

View larger version (10K): [in this window] [in a new window]   Figure 3. The effect of folate depletion on human lymphocyte growth. Lymphocytes were grown for up to 8 days in either folate-replete () or folate-deficient medium (). Cells were counted and the results expressed per milliliter. Results are mean ± SEM (n=8). *P < 0.01, where P values refer to differences between cells grown in the presence or absence of folic acid.

View larger version (15K): [in this window] [in a new window]   Figure 4. The effect of folate depletion on strand breakage and misincorporated uracil in human lymphocyte DNA. Lymphocytes grown for 1 (A), 3 (B), 5 (C), or 8 (D) days in either folate-replete () or folate-deficient medium () were incubated either with uracil DNA glycosylase (enzyme) or in buffer alone (buffer). Results are mean ± SEM (n=8). *P < 0.01, where P values refer to differences in strand breakage; +P < 0.05, where P values refer to differences in uracil misincorporation in cells grown in the presence or absence of folic acid.

The effects of folate deficiency were concentration dependent. Lymphocytes incubated with 100 ng/ml folic acid grew normally. Growth was retarded after culture in medium supplemented with 10 ng/ml folic acid and was completely abolished at 1 ng/ml ( Fig. 5). Similarly, DNA strand breakage and misincorporated uracil increased with decreasing folate concentration within the medium ( Fig. 6).

View larger version (11K): [in this window] [in a new window]   Figure 5. The effect of folic acid concentration on human lymphocyte growth. Lymphocytes were grown for up to 8 days in either folate-deficient medium () or in medium supplemented with 1 ng/ml (), 10 ng/ml (), or 100 ng/ml () folic acid. Cell number is expressed per milliliter. Results are mean ± SEM (n=10). *P < 0.01, where P values refer to differences in cell number between cells grown in the presence or absence of folic acid; +P < 0.05, where P values refer to differences between lymphocytes cultured in 10 or 100 ng/ml folic acid.

View larger version (13K): [in this window] [in a new window]   Figure 6. The effect of folic acid concentration on strand breakage and misincorporated uracil in human lymphocyte DNA. Lymphocytes grown for 8 days in either folate-deficient medium or in medium supplemented with increasing concentrations of folic acid were incubated either with uracil DNA glycosylase in buffer () or buffer alone (). Results are mean ± SEM (n=10). *P < 0.01, where P values refer to differences in DNA strand breakage and misincorporated uracil in the presence or absence of folic acid; +P < 0.05, where P values refer to differences in strand breaks in lymphocytes cultured in 10 or 100 ng/ml folic acid.

Lymphocytes cultured for 5 days in folate-deficient medium were unable to repair oxidant-induced DNA strand breakage efficiently ( Fig. 7). After 8 days, cells grown under folate-deprived conditions contained more endogenous strand breaks (untreated) than control cells, as described previously ( Fig. 4), and were slow to recover over an 8 h incubation period ( Fig. 7). Lymphocytes cultured in the presence of folic acid were able to remove most of the damage caused by hydrogen peroxide compared with folate-depleted cells [88.8% ± 2.3% (n=8) in control cells vs. 17.9% ± 4.5% (n=8) in folate-deficient cells].

View larger version (13K): [in this window] [in a new window]   Figure 7. The effect of folic acid depletion on human lymphocyte DNA repair capacity. Lymphocytes were grown for 1 (A), 3 (B), 5 (C), or 8 days (D) in either folate-deficient () or replete () medium. DNA strand breakage was measured immediately, 4 h, and 8 h after exposure to hydrogen peroxide. Results are mean ± SEM (n=8). *P < 0.01, where P values refer to differences between lymphocytes cultured in the presence or absence of folic acid.

Lymphocyte DNA contained a measurable amount of oxidized pyrimidines after incubation with the bacterial DNA repair enzyme endonuclease III. The level of oxidized bases was unaffected by folate status ( Fig. 8).

View larger version (14K): [in this window] [in a new window]   Figure 8. The effect of folate depletion on oxidized pyrimidines in human lymphocyte DNA. Lymphocytes, grown for up to 10 days either in folate-deficient (triangles) or folate-replete (circles) medium, were incubated with either endonuclease III in buffer (filled) or in buffer alone (open). Results are mean ± SEM (n=4).

	DISCUSSION

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Folic acid deficiency promotes instability in human DNA, causing damage as increased micronuclei in erythrocytes and lymphocytes, increased chromosomal abnormalities, and elevated mutant frequencies in the hypoxanthine phosphoribosyltransferase gene. Supplementation with folic acid can decrease or reverse these effects (6–9, 21).

In this study, folate deficiency potentiated DNA strand breakage, inhibited cell growth, and increased uracil misincorporation in cultured human lymphocytes. DNA instability was inversely related to the concentration of folic acid available to the cells. This concentration-dependent effect demonstrates both the sensitivity and specificity of the modified comet assay and indicates that an intake of folic acid adequate for the prevention of clinical deficiency may not be optimal for maintaining DNA stability. In support of this, whereas clinical folate deficiency is associated with increased uracil misincorporation, micronuclei formation, and genetic mutations in the human population, mild or marginal deficiency also decreases chromosome stability (6, 7, 9).

Diets that include a large intake of fruits and vegetables are associated with a reduced risk of cancer (22, 23). This may be due to the presence of antioxidants such as vitamin C, vitamin E, and the carotenoids within foods that protect DNA from endogenous and exogenous oxygen free radical attack (24, 25). Oxidized pyrimidines are determined in a modified comet assay (26) using the bacterial DNA repair enzyme endonuclease III, which preferentially incises damaged DNA at sites of oxidized pyrimidines (27). Normal human lymphocytes contain detectable amounts of oxidized pyrimidines that are decreased after supplementation with dietary antioxidants (28). However, lymphocytes grown either in the presence or absence of folic acid for up to 10 days contained similar amounts of oxidized pyrimidines. This suggests that folate depletion has no effect on oxidative DNA damage per se and further indicates that uracil misincorporation is relatively specific for this particular deficiency.

Although oxidative DNA damage itself was not increased by folate depletion, folate deficiency did potentiate the damaging effects of hydrogen peroxide by inhibiting DNA repair. Lymphocytes grown under folate-deficient conditions were unable to repair DNA damage efficiently, as indicated by the persistence of DNA breaks after oxidative attack. Initially, the rate of repair of hydrogen peroxide-induced strand breakage was the same for cells grown in the presence or absence of folic acid. After a few days, however, folate deficiency increased DNA breakage and prevented repair. Endogenous DNA repair activity is inhibited in Chinese hamster ovary (CHO) cells grown without folic acid (29). Similarly, folate-deficient cells are less able to repair radiation-induced damage than folate-replete CHO cells (30). Efficient DNA repair is dependent on the availability and balance of deoxynucleotide precursors. Rats deprived of folic acid show decreased thymidylate and purine pools and reduced DNA repair capacity (31). Diets lacking in fresh fruit and vegetables will be low in both folic acid and antioxidants. Increased exposure to endogenous oxygen free radicals as a result of lower antioxidant defense, combined with increased strand breakage, uracil misincorporation, and defective repair resulting from folate deficiency, thus may potentiate DNA instability.

In summary, poor folic acid status in human lymphocytes in vitro is associated with increased DNA strand breakage, misincorporated uracil, and reduced DNA repair efficiency. Although adequate for preventing clinical deficiency, currently recommended levels of dietary folic acid intake (resulting in folate plasma concentrations >2.5 ng/ml) may not be optimal for maintaining DNA stability. Oxidative DNA base damage is not influenced by folate status per se. However, oxidative stress associated with a diet lacking in antioxidant micronutrients may potentiate the negative effect of folate deficiency. These in vitro results help to determine the mechanism(s) through which folic acid maintains DNA stability.

	ACKNOWLEDGMENTS

 
This work was funded by the World Cancer Research Fund and the Scottish Office Agriculture, Environment and Fisheries Department. The authors would like to thank Mrs. Sharon Wood for expert assistance.

	FOOTNOTES

 
¹ Correspondence: Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, U.K. E-mail sd{at}rri.sari.ac.uk

² Abbreviations: BSA, bovine serum albumin; DAPI, 4',6-diamidine-2-phenylindole dihydrochloride; dUMP, deoxyuridine monophosphate; FCS, fetal calf serum; LMP, low melting point; LSM, lymphocyte separation medium; SMP, standard melting point; TMP, thymidine monophosphate.

Received for publication February 4, 1998. Revision received June 15, 1998.

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