Methylenetetrahydrofolate reductase C677T polymorphism does not alter folic acid deficiency-induced uracil incorporation into primary human lymphocyte DNA in vitro

Jimmy W. Crott1,2, Susan T. Mashiyama3, Bruce N. Ames3 and Michael F. Fenech2,4

1 Department of Physiology, Adelaide University, SA 5005,
2 CSIRO Health Sciences and Nutrition, PO Box 10041, Adelaide BC, SA 5000, Australia and
3 University of California at Berkeley and Children's Hospital Oakland Research Institute, CA, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Methylenetetrahydrofolate reductase (MTHFR) is an enzyme which converts 5,10-methylene tetrahydrofolate (5,10-MnTHF) to 5-methyl tetrahydrofolate. A common C to T transition (C677T) in the MTHFR gene is reported to reduce the risk for colorectal cancer and acute lymphocytic leukemia in homozygotes (TTs). It is hypothesized that because TTs have reduced MTHFR activity, more 5,10-MnTHF is available to provide methyl groups for the conversion of uracil to thymidine. Folic acid deficiency causes the intracellular accumulation of dUMP and the subsequent incorporation of uracil into DNA. The removal of uracil from DNA may result in double-stranded DNA breaks, the accumulation of which is a putative risk factor for cancer. We tested whether human lymphocytes taken from TTs (n = 10) were more able to resist uracil incorporation into DNA than controls (n = 14 CCs and 6 CTs) when cultured in medium containing 12–120 nM folic acid for 9 days. DNA uracil content of these lymphocytes was measured by CG-MS. TTs and controls showed a dose-dependent increase in DNA uracil content during folic acid deficiency (P < 0.0001, R2 = 0.23 for TTs and P < 0.0001, R2 = 0.19 for controls). DNA uracil content was not different between the two groups at any of the folic acid concentrations (two-way ANOVA: media [folic acid], P < 0.0001; genotype, P = 0.4). The results show that, in this in vitro system, the MTHFR C677T polymorphism does not affect the cell's ability to resist uracil incorporation into DNA. Chromosome breakage, as measured by micronuclei, was also shown to correlate with folic acid concentration in a preliminary experiment (P < 0.0001). Although the results appear not to support the hypothesis that a reduced risk for certain cancers in TTs is due to diversion of folic acid to thymidine synthesis, differences between the in vivo and in vitro situation make this conclusion not definitive.

Abbreviations: ALL, acute lymphocytic leukemia; BN, binucleated cell; CBMN, cytokinesis block micronucleus assay; CC/CT/TT, persons nullizygous/heterozygous/homozygous for the C677T polymorphism of MTHFR; DSB, double-stranded (DNA) break; FCS, fetal calf serum; GC-MS, gas chromatography-mass spectrometry; MTHFR, methylenetetrahydrofolate reductase; PHA, phytohaemagglutinin; MNed, micronucleated; MS, methionine synthase; THF, tetrahydrofolate; 5,10-MnTHF/5-MeTHF, 5,10-methylene/5-methyl THF; UDG, uracil DNA glycosylase.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Methylenetetrahydrofolate reductase (MTHFR) is a key folate-metabolizing enzyme which catalyses the conversion of 5,10-methylene tetrahydrofolate (5,10-MnTHF) to 5-methyl tetrahydrofolate (5-MeTHF). This latter folic acid species provides methyl groups for the methionine synthase-mediated remethylation of homocysteine to methionine. 5,10-MnTHF donates methyl groups for the thymidine synthase-mediated conversion of dUMP to dTMP (Figure 1Go).



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Fig. 1. Metabolism of folate.

 
A number of polymorphisms have been identified in the gene encoding MTHFR (15), with the C to T base transition at position 677 of the gene, discovered by Frosst et al. (4), being the most thoroughly researched. The allele frequency of this polymorphism is reported to range from 4.5 to 44.9% depending on the population studied (6). Individuals who are heterozygous (CT) or homozygous (TT) for this polymorphism have an in vitro enzyme activity of 65 and 30% of wild-type (CC), respectively (4).

It is now well established that TTs have elevated plasma homocysteine concentrations (4,7,8), which is thought to be due to the inefficient recycling of homocysteine to methionine. The TT genotype is associated with an elevated risk for cardiovascular disease (8,9), which may be linked to homocysteine. In addition, TTs are reported to have a 2.8-fold higher risk for endometrial cancer (10), a 2.7- and 2.8-fold higher risk for Crohn's disease and ulcerative colitis, respectively (11), and a 2.6–3.2-fold higher risk for Down syndrome in offspring (12,13). Various reports also show that homozygotes have a 1.6-, 1.8- or 2.2-fold higher risk for neural tube defects (1416).

In contrast, various reports document that TTs have a 1.2-, 1.7- and 3.0-fold reduced risk for colorectal cancer (carcinoma) (1719). Despite this evidence relating to carcinomas, the TT genotype does not seem to afford any protection against the formation of colorectal adenomas (20,21) and may even increase risk for adenoma if dietary intakes of folic acid, vitamin B12, vitamin B6 and methionine are low (22). Recently, the TT genotype has also been associated with a 4.3-fold reduction in the risk for developing acute lymphocytic leukemia (23). It is hypothesized that the reduced risk of some cancers afforded by the polymorphism is due to a diversion of folic acid to thymidine synthesis. Reduced MTHFR activity is thought to increase the intracellular concentration of 5,10-MnTHF and, hence, the amount of methyl groups available for the conversion of dUMP to dTMP (Figure 1Go).

It is known that during folic acid deficiency there is a build up of dUMP within the cell which leads to excessive uracil incorporation into DNA (2426). The simultaneous removal of two uracil bases (or other modified bases e.g. 8-oxoguanine) within 12 base pairs of each other on opposite strands may result in the formation of double-stranded DNA breaks (DSB) (27). The induction of DSBs by uracil incorporation is thought to be the molecular mechanism for the formation of chromosome breaks and micronuclei which are observed during folic acid deficiency (24). The mutagenic potential of uracil is highlighted by the fact that, of eight known human glycosylases involved in DNA repair, four remove uracil from the code (28). Promotion of uracil incorporation into DNA and the resulting chromosome breakage could be important because it is now established that the accumulation of chromosome aberrations is a risk factor for cancer (29,30).

The mechanism behind reduced cancer risk in TTs compared with CCs is thought to involve lowered dUMP pools and therefore the incorporation of less uracil into DNA. This report describes an experiment which aimed to test this hypothesis. We compared the amount of uracil incorporated into DNA by primary human lymphocytes taken from TTs, CTs and CCs during folic acid repletion or depletion in vitro.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Approval for this study was obtained from CSIRO Health Sciences and Nutrition and Adelaide University Human Ethics committees.

Ten volunteers homozygous for the C677T polymorphism in MTHFR (TT) and 20 age and sex-matched controls [14 wild-types (CC) and six heterozygotes (CT)] were selected from a database of previous study participants. The presence of this polymorphism was determined using the method of Frosst et al. (4). Volunteers were also previously tested for the presence of the A1298C MTHFR and A2756G methionine synthase polymorphisms using the methods of van der Put et al. (31) and Leclerc et al. (32), respectively. Controls were divided into two groups of 10.

The first group (Ctrl 1) consisted entirely of CCs which were optimally matched for age, gender and the MTHFR A1298C and MS A2756G polymorphisms. The second group (Ctrl 2) consisted of a mixture of CCs and CTs matched for age and gender but not as well with respect to the other MTHFR and MS polymorphisms. Volunteer characteristics are listed in Table IGo.


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Table I. Volunteer statistics
 
On one occasion volunteers donated approximately 90 ml blood (lithium-heparin) after an overnight fast and lymphocytes were isolated using Ficol-paque gradients (Pharmacia Biotech, Uppsala, Sweden). Lymphocytes were cultured (5x105 cells/ml) in 10 ml medium in eight 25 ml culture flasks with vented lids (Sarstedt, Adelaide, Australia). Duplicate cultures were established in four different types of media.

Media was custom made (in house) RPMI-1640 which contained 5% dialysed fetal calf serum (FCS) (Trace Biosciences, Victoria, Australia), 10 U/ml interleukin-2 (Roche Diagnostics, Basel, Switzerland) and either 120, 60, 24 or 12 nM folic acid. The amount of vitamin B12 added to media was 700 pM and all other constituents were standard for RPMI-1640 as described by Moore et al. (33) and were all purchased from Sigma (St Louis, MO). The dialysed FCS contained 356 pM vitamin B12 and 9 nM folic acid which equates to a contribution of 17.8 pM B12 and 0.45 nM folic acid in the complete media containing 5% FCS.

A diagrammatic representation of the culture protocol is shown in Figure 2Go. Once cells were placed in culture, mitogenesis was stimulated by the addition of 22 µg/ml phytohaemagglutinin (PHA) (Murex Biotech, Kent, UK) and cells were incubated at 37°C and 5% CO2 in a humidified atmosphere. After 3 days, cell number and viability were determined using a Coulter counter and Trypan blue exclusion, respectively. After gently spinning (100 g) the cell suspension in a 10 ml conical base tube, the supernatant was removed and cells were resuspended in 500–1500 µl of this spent medium at a concentration of 10x106 cells/ml. This volume was calculated so that 5x106 cells could be returned to the flask in 500 µl spent medium. Fresh medium (9.5 ml) was added to each flask and warmed to 37°C prior to returning the cells. This was done to return some of the growth factors from the spent medium to the system. This process of counting and re-culturing cells was repeated 6 days post-PHA treatment.



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Fig. 2. Experimental design.

 
At 8 days post-PHA treatment, a 750 µl aliquot of each culture was removed to 6 ml culture tubes for the cytokinesis block micronucleus assay to give a measure of chromosome damage. Cytochalasin B (4.5 µg/ml; Sigma) was added to each tube and approximately 30 h later cells were harvested onto microscope slides using a cyto-centrifuge (Shandon Southern Products, Cheshire, UK). Slides were then air dried, fixed and stained using Diff-Quik (similar results to Wright-Giemsa stain; LabAids, New South Wales, Australia). Coded slides were scored for micronucleated binucleates (MNed BNs) until 1000 binucleated cells were counted using the scoring criteria described by Fenech (34). These data will be presented elsewhere; however, micronucleus data from a preliminary dose-finding experiment, where media folic acid concentrations of 0, 3, 6, 12 and 120 nM were used, is presented here. In this preliminary experiment, fasted blood was collected from three healthy asymptomatic male volunteers, all 24 years of age. The experiment was performed in duplicate.

Nine days after PHA treatment, cell number and viability were determined before cells were stored at –80°C in approximately 1 ml medium in cryovials (Nalgene, Rochester, NY). Note that the growth data presented in Figure 4Go are a projection of cell growth because only 5x106 cells were returned to each flask in fresh medium on days 3 and 6. The curves were generated by calculating the percentage increase in (viable) cell number and applying it to the number of cells present at the end of the previous 3 day period.



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Fig. 4. Growth curves for primary human lymphocytes grown in media with varying concentrations of folic acid. n = 10 TTs and n = 20 controls (Ctrl, n = 14 CCs, n = 6 CTs). Media changed at days 3 and 6. Cultures initiated with 5x106 cells and 5x106 cells were returned to each flask after refreshing medium on days 3 and 6. Two-way ANOVA at days 3, 6 and 9: [folic acid], P < 0.0001, genotype, P >> 0.05.

 
Genomic DNA was isolated from stored cells by repeated extraction with phenol, 25:24:1 (v/v/v) phenol:chloroform:isoamyl alcohol and 24:1 (v/v) chloroform:isoamyl alcohol followed by precipitation in ethanol and sodium acetate. DNA was washed once in ethanol and resuspended in Milli-Q water (pH 7.5–8) before being quantified by measuring absorbance at 260 nm in a spectrophotometer (Shimadzu UV 160U, Japan). It was assumed that a solution containing 10 µg/ml DNA has an absorbance of 1.0 at 260 nm. DNA was stored at –20°C for a maximum of 3 weeks until uracil content was analysed.

Uracil content of DNA was measured using the method of Blount and Ames (35). DNA (10 µg) was dried in a speed-vac (Savant, SC110A-120 fitted with a Savant refrigerated vapor trap, RVT400; Farmingdale, NY) and resuspended in 40 µl TE buffer. Meanwhile, 1 µl (1 U) uracil DNA glycosylase (UDG; Epicenter Technologies, Madison, WI) was filtered per sample in a Microcon® centrifugal filter unit (YM-10; Millipore, Bedford, MA) and eluted in TE to give a concentration of 1 U/10 µl. Filtered UDG (10 µl) was added to the DNA and tubes were incubated at 37°C for 1 h in a water bath. After incubation, 100 pg internal standard, labeled uracil (13C4H4O215N2; Cambridge Isotope Laboratories, Andover, MA) was added and tubes were dried in a speed-vac. Uracil and internal standard were then derivatized by adding 61 µl 1:50:10 (v/v/v) 3,5-bis(trifluoromethyl) benzyl bromide (BTFMBzBr; Aldrich, Milwaukee, WI):acetonitrile:triethyl amine and incubating at 30°C in a shaker (G24 Environmental Incubator Shaker, New Brunswick Scientific, New Brunswick, NJ) for 30 min. Milli-Q water (50 µl) was then added to each tube and the derivatized uracil and internal standard were extracted into 100 µl isooctane. Tubes were vortexed for at least 30 s and a maximum of 70 µl isooctane was removed into an auto-sampler vial which was capped immediately.

One µl of this isooctane was then injected into a Hewlett-Packard 5890-Series II Gas chromatograph using a Hewlett-Packard 7673 auto-sampler. The injection port was maintained at 280°C. Separation was achieved using a Hewlett-Packard HP-5ms column (30 mx0.25 mm internal diameterx0.25 µm film thickness) kept at 100°C for 1 min then ramped to 280°C at 25°C/min and held for 5 min. The GC-MS interface temperature was 300°C. A Hewlett-Packard 5989A mass spectrometer (quadrupole temperature, 100°C; electron energy, 200 eV; ion source temperature, 280°C; negative chemical ionization mode) was used in single ion monitoring mode to detect uracil and internal standard peaks at 337 and 343 m/z, respectively. Methane was maintained at 2 Torr.

The concentration of uracil (pg/10 µg DNA) in samples was determined by comparing the ratio of abundances at 337 and 343 m/z to a standard curve of 337/343 ratios generated from standards containing 0, 2.5, 5, 25, 50 or 100 pg uracil. R2 values for the curve were 0.94 or higher (data not shown). One unit of heat denatured UDG and 12.5 µg human placenta DNA (Sigma) was added to make standards as similar to samples as possible.

Statistics
One-way ANOVA was used to compare results of different media types. Two-way ANOVA was used to determine the effect of media type and genotype on uracil levels. Unpaired t-tests were used in addition to two-way ANOVA to compare levels of uracil between groups at each folic acid concentration. Pearson correlation coefficients were determined to analyse relationships between variables. Significance was accepted at P < 0.05. All calculations were performed using GraphPad Prism ver. 2.01 (GraphPad, San Diego, CA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Results from the preliminary dose-finding experiment are shown in Figure 3Go. Folic acid deficiency caused a dose-dependent increase in the number of micronucleated (MNed) cells (ANOVA, P < 0.05; Pearson correlation, P < 0.0001, R2 = 0.6794).



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Fig. 3. Effect of medium folic acid concentration on micronucleus frequency and DNA uracil content in primary human lymphocytes in vitro. (A) MNed BNs/1000 BNs, micronucleated binucleates per 1000 binucleates. *P < 0.001 versus 120 nM (Tukeys). n = 3 in duplicate. (B) ANOVA, P > 0.05. Pearson correlation, P = 0.013, R2 = 0.26. n = 3 in duplicate.

 
Folic acid concentrations of 12–120 nM were selected for the main experiment on the basis that 120 nM gave a baseline frequency of MNed cells (8.13 ± 1.19/1000 BNs); similar to those routinely observed in whole blood and short-term micronucleus cultures, while 12 nM induced a 2.5-fold increase in the micronucleus index (20.05 ± 0.72) whilst still being within a physiologically relevant range (Figure 3AGo).

Figure 3BGo shows the uracil data from the same preliminary experiment. Note that uracil analysis for cells from these cultures was performed at a later date and with samples from the main experiment. Although the ANOVA result did not reach significance (P = 0.27), there was a significant negative correlation between folic acid concentration and DNA uracil content (P = 0.013, R2 = 0.26). MNed cell frequency and DNA uracil content were significantly and positively correlated with each other (Pearson correlation, P = 0.035, R2 = 0.32).

Figure 4Go shows the cell growth in media with various concentrations of folic acid. There was a steady increase in cell number over a 9 day period with the maximum cell growth being achieved in medium with 120 nM folic acid. Two-way ANOVAs were performed to determine the effect of genotype (TT versus controls) and folic acid concentration on cell number at days 3, 6 and 9. At each time point folic acid was a significant determinant of cell number (P < 0.0001) while genotype was not (P = 0.9, 0.51 and 0.71 for days 3, 6 and 9, respectively).

Age did not correlate with DNA uracil content at any folic acid concentration (P = 0.2–0.86). The gender of volunteers also did not affect DNA uracil content (two-way ANOVA; gender, P = 0.12; [folic acid], P < 0.0001).

Uracil data from the main experiment are shown in Figure 5Go. When data from all volunteers were combined (n = 29), there was a highly significant negative correlation between DNA uracil content and folic acid concentration (Pearson correlation, R2 = 0.192, P < 0.0001). Furthermore, as reported in Table IIGo, uracil content at 12 and 24 nM folic acid was significantly higher than at both 60 and 120 nM folic acid (ANOVA, P < 0.0001, post-test P < 0.05). There was no difference in the DNA uracil content between TTs and the two control groups (two-way ANOVA; genotype/group, P = 0.4; [folic acid], P < 0.0001). Furthermore, when data from TTs and CTs were combined (n = 14), there was no difference in DNA uracil content to the CCs (n = 15) (Table IGo; two-way ANOVA; genotype, P = 0.752; [folic acid], P < 0.0001). When TTs, CTs and CCs were considered separately, there was also no difference in DNA uracil content (two-way ANOVA; genotype, P = 0.12; [folic acid], P < 0.0001). No difference between genotypes was detected when genotypes were compared two at a time or at each folic acid concentration separately by one-way ANOVA.



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Fig. 5. Effect of medium folic acid concentration and MTHFR C677T polymorphism on DNA uracil content of primary human lymphocytes in vitro. Two-way ANOVA: [folic acid], P < 0.0001; genotype/group, P = 0.4.

 

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Table II. Effect of MTHFR and MS polymorphisms on DNA uracil content of primary human lymphocytes in vitro
 
When baseline uracil levels (120 nM results) were subtracted from the remaining three data sets (to give a change from baseline result) there was also no significant difference between TTs and controls (two-way ANOVA; genotype, P = 0.125; [folic acid], P = 0.007).

The effect of MTHFR A1298C and MS A2756G polymorphisms on DNA uracil content was also determined (Table IIGo). When considering the MTHFR A1298C polymorphism, data from 11 heterozygotes (AC) was compared with 18 wild-types (AA). Two-way ANOVA revealed no differences due to the polymorphism (genotype, P = 0.362; [folic acid], P < 0.0001); however, when data from each concentration was compared separately using an unpaired t-test, ACs exhibited a larger DNA uracil content than wild-types in deficient medium containing 12 nM folic acid (51.5 ± 9.4 and 31.9 ± 4.1 pg/10 µg DNA, respectively; P = 0.038).

Similarly, two-way ANOVA revealed no differences in DNA uracil content between volunteers heterozygous for the MS A2756G polymorphism (AG; n = 6) and wild-types (AA; n = 23) (Table IIGo). However, when treatments were compared separately using an unpaired t-test, AGs were found to have a significantly higher DNA uracil content than AAs when their cells were grown in replete medium containing 120 nM folic acid (28.8 ± 10.0 and 10.8 ± 3.0 pg/10 µg DNA, respectively; P = 0.025).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of this study provide further evidence that folic acid deficiency causes an imbalance of intracellular nucleotide pools which facilitates the excessive incorporation of uracil into DNA. The results also indicate a positive correlation between uracil content and MNed cell frequency which is in agreement with the results of Blount et al. (24).

The medium folic acid concentrations chosen for the main experiment were selected on the basis of micronucleus induction in a preliminary experiment. The frequency of MNed cells in the `replete' medium (120 nM folic acid) after long-term culture was 8.13 ± 1.19/1000 BNs, which is similar to previously reported baseline values in men of the same age obtained using the conventional short-term culture (3 days) CBMN assay (6.35 ± 0.39 MNed BNs/1000 BNs, n = 49, age = 25.5 ± 0.64 years) (36). Because the frequency of MNed cells seen in replete medium in the current long-term assay closely matches those of the short-term assay, we deduce that the levels of uracil detected in these same cells may be a good indication of baseline uracil levels in vivo for folic acid replete individuals. The lowest folic acid concentration used was 12 nM which corresponded with an approximately 2.5-fold elevation in micronucleus index (20.05 ± 0.72 MNed BNs/1000 BNs). Although a further elevation in MNed cell frequency can be achieved by adding no folic acid to the medium (25.35 ± 1.66, P < 0.05 versus 12 nM), this increase is relatively small (Figure 3Go). Furthermore, cell proliferation in this deficient medium is very poor and compromises the lymphocyte yield after 9 days, leaving insufficient DNA for uracil analysis.

The three lower concentrations of folic acid used in this experiment (12–60 nM) closely reflect those seen in vivo. For example, Jacob et al. (37) report that baseline plasma folic acid concentrations (in postmenopausal women) were 19.5 ± 4.2 nM and fell to 9.3 ± 1.8 nM after a 5 week folic acid depletion period. Furthermore, plasma concentrations are reported to rise to almost 50 nM with 8 weeks of folic acid supplementation (400 µg/day) (38). Data from this laboratory also show that baseline plasma folic acid concentrations range from approximately 21 to 36 nM depending on age, gender and diet (39,40). It is unclear whether in vivo plasma concentrations of 120 nM could be sustainably achieved by supplementation.

The amount of uracil detected in the DNA samples from this study ranged from approximately 10–48 pg/10 µg DNA or approximately 34 600–167 400 uracils/diploid cell. These figures are in good agreement with 7–30 pg/10 µg DNA observed in rat liver DNA (35) and 62 220–256 200 uracils/diploid cell (17–70 uracils/106 thymidines) observed for cultured primary murine erythroblasts (41). However, these values are considerably lower than the 500 000–4 000 000 uracils/diploid cell reported by Blount et al. (24) in blood DNA from folic acid replete and deficient splenectomized humans, respectively.

One factor that may help explain this large difference in uracil values is the length of folate deficiency involved. The two animal studies (35,41), as well as this current study of cultured human cells, involved short-term folate deficiency of approximately 1–4 weeks. In contrast, the study of human splenectomized patients by Blount et al. (24) used samples from individuals who were often severely anaemic, with marked macrocytosis and grossly megaloblastic erythropoiesis, which is indicative of chronic, extended folate deficiency. Furthermore, micronuclei were shown to increase significantly with age and the folate deficient group tended to be elderly (mean = 60.4 ± 13.4 years) (B.Blount, personal communication). These factors could have interacted with a long-term, poor diet to exacerbate uracil incorporation into DNA. It is interesting to note that there was great inter-individual variation of uracil between folate-deficient individuals (approaching 50-fold). This indicates the possibility of underlying complex genetic differences in folate metabolism that could cause such highly variable responses to folate deficiency. Further human studies should be done to clarify this issue, and differences in age as well as length of folate deficiency should be considered in the comparative analyses. We have recently made improvements to the uracil assay and are also currently examining whether methodological factors may have contributed to the differences in values obtained in the different studies mentioned above.

The results of this experiment do not support the hypothesis that the C677T polymorphism protects against uracil incorporation into DNA, though we argue also that this result is not definitive due to differences between the in vitro and in vivo conditions. It may also be possible that changes in DNA methylation, as a result of the polymorphism, may be an alternative explanation for the impact of folate deficiency on cancer risk. There is now preliminary evidence (42) that the C677T polymorphism causes global DNA hypomethylation in normal somatic cells. However, global DNA hypomethylation is believed to be associated with increased cancer risk (43) and cancer progression (44,45). One plausible explanation is that the MTHFR mutation may specifically protect against the hypermethylation-induced silencing of tumor suppressor genes. Hypermethylation of tumor suppressor genes is a common event in many cancers (46).

The C677T polymorphism has been associated with a reduced risk for colorectal cancer and acute lymphocytic leukemia (ALL) (1719,23). It is, however, probable that the polymorphism has different effects in different types of cells. In contrast to reports of a reduced risk for cancer, the polymorphism has no apparent benefit with respect to acute myeloid leukemia (23) and has been associated with a 2.8-fold increased risk of endometrial cancer (10). In this instance, the authors suggest that compromised DNA methylation, as a result of reduced MTHFR activity, was responsible for the elevated risk. It is possible that in the case of endometrial cancer, hypomethylation may activate proto-oncogenes to increase cancer risk, whereas in the case of colorectal cancer and ALL, C677T induced hypomethylation may prevent the silencing of tumour-suppressor genes and lower the risk for cancer. It is important that future experiments test the effects of MTHFR polymorphisms and folic acid deficiency on various different types of cells, in particular stem cells originating from the colon and bone marrow that have the potential of becoming cancer cells in vivo.

In the study by Skibola et al. (23) which shows a reduced risk for ALL associated with the C677T polymorphism, it is reported that people heterozygous for the A1298C polymorphism also have a 3-fold reduced risk for ALL. The preliminary data presented in Table IIGo do not support the hypothesis that reduced uracil incorporation into DNA is the mechanism responsible. In fact, at the deficiency concentration of 12 nM folic acid, heterozygotes (AC) incorporated significantly more uracil into DNA than wild-types (51.5 ± 9.4 and 31.9 ± 4.1 pg/10 µg DNA, respectively; t-test, P = 0.038).

Also, as shown in Table IIGo, heterozygotes for the MS A2756G polymorphism incorporated approximately 3-fold more uracil into DNA at the 120 nM concentration than wild-type (28.8 ± 10.0 and 10.8 ± 3.0, respectively; t-test, P = 0.025). It is unlikely that this observed increase in uracil incorporation is due to methyl folic acid trapping and an increase in the ratio of dUMP to dTMP, because there is no evidence that this polymorphism is associated with reduced MS activity. Rather, two reports describe significantly lower plasma homocysteine levels in people homozygous for the mutation (47,48). These data do not support the hypothesis that altered uracil incorporation into DNA is a main causative factor of the altered cancer risk in those with common polymorphisms in MTHFR and MS.

Another factor that may cause differences in uracil misincorporation is the presence of other polymorphisms in folic acid metabolizing genes. To date, five polymorphisms in the MTHFR gene have been identified (15). Coupling these with polymorphisms in genes coding for other folic acid metabolizing enzymes, such as methionine synthase, methionine synthase reductase and thymidine synthase, gives a large combination of polymorphisms, all of which could conceivably influence uracil incorporation into DNA

The C677T mutation is known to reduce enzyme activity in vitro (4) and, by virtue of the fact that TTs have elevated plasma homocysteine levels, in vivo. It is possible that in this cell culture system the enzyme activity may be modulated by factors other than the polymorphism and folic acid concentrations in the medium. The C677T polymorphism is thought to decrease the binding affinity of flavin adenine dinucleotide (FAD) to MTHFR and may increase the rate of dissociation of FAD from the enzyme (49). Recent evidence suggests that riboflavin (vitamin B2), the immediate precursor to FAD, is an independent determinant of plasma homocysteine levels, but only in individuals with the C677T polymorphism (50). It is possible that an abundance of riboflavin may correct or improve MTHFR activity in people with the polymorphism. This may serve to negate or lessen any influence that the C677T polymorphism may have on uracil incorporation into DNA. Although not a definitive indicator of intracellular concentrations, the riboflavin concentration of RPMI-1640 medium is 530 nM, which is considerably higher than the 12–13 nM found in plasma (50) and may therefore raise intracellular FAD levels. We therefore suggest that high levels of riboflavin in the culture medium, which increases FAD concentrations, may cause our in vitro results to differ from the normal situation in humans in vivo (B.N.Ames, in preparation). Further research is clearly needed to investigate the interaction of riboflavin, folic acid and MTHFR polymorphisms on uracil misincorporation into DNA in vivo.

The concentration of methionine in the medium may also influence MTHFR activity in this system. The concentration of L-methionine in RPMI (not considering FBS) was 100 µM, which is more than the 20–30 µM normally found in vivo (51). It is possible that as a result of high methionine levels, intracellular levels of the next metabolite in the methionine cycle, S-adenosyl methionine (SAM), may have become elevated. SAM is a known inhibitor of MTHFR (52) and it is possible that this effect may have lowered the MTHFR activity of control cells to a level similar to that of TT cells.

In conclusion, we have shown that folic acid deficiency causes a dose-dependent increase in uracil incorporation into lymphocyte DNA in vitro which is not influenced by the C677T polymorphism in this system. These results suggest that the hypothesis that reduced cancer risks associated with MTHFR polymorphisms are due to a diversion of folic acid to thymidine synthesis may be an over-simplification of real events. Clearly, further research is needed to define the effect of this polymorphism in combination with folic acid, riboflavin and methionine deficiency on DNA uracil incorporation in a variety of cell types, particularly those originating from tissue in which there is evidence for a change in the risk of pathology (e.g. colon, endometrium, cervix, breast and leukocyte stem cells).


    Notes
 
4 To whom correspondence should be addressed Email: michael.fenech{at}hsn.csiro.com.au Back


    Acknowledgments
 
Many thanks are due to all the members of the Ames laboratory for their exceptional hospitality during J.C.'s visit. Special thanks to Patrick Walter and Esther Roitman for their invaluable help with the GC-MS. We are indebted to Philip Thomas at CSIRO for performing the genotyping; all the CSIRO clinic staff for help with the collecting of blood samples and to the Endocrine and Metabolic Unit at the Institute for Medical and Veterinary Science in Adelaide for measuring the folic acid and vitamin B12 content of the FCS. The Australian Nutrition Trust Fund, Adelaide University-Scholarships Branch and the Department of Physiology provided monetary support for travel to the Ames laboratory.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Goyette,P., Sumner,J.S., Milos,R., Duncan,A.M., Rosenblatt,D.S., Matthews,R.G. and Rozen,R. (1994) Human methylenetetrahydrofolate reductase: isolation of cDNA, mapping and mutation identification. Nature Genet., 7, 195–200.[ISI][Medline]
  2. Goyette,P., Frosst,P., Rosenblatt,D.S. and Rozen,R. (1995) Seven novel mutations in the methylenetetrahydrofolate reductase gene and genotype/phenotype correlations in severe methylenetetrahydrofolate reductase deficiency. Am. J. Hum. Genet., 56, 1052–1059.[ISI][Medline]
  3. Goyette,P., Christensen,B., Rosenblatt,D.S. and Rozen,R. (1996) Severe and mild mutations in cis for the methylenetetrahydrofolate reductase (MTHFR) gene and description of five novel mutations in MTHFR. Am. J. Hum. Genet., 59, 1268–1275.[ISI][Medline]
  4. Frosst,P., Blom,H.J., Milos,R., Goyette,P., Sheppard,C.A., Matthews,R.G., Boers,G.J., den Heijer,M., Kluijtmans,L.A., van den Heuvel,L.P. and Rozen,R. (1995) A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase [letter]. Nature Genet., 10, 111–113.[ISI][Medline]
  5. Kluijtmans,L.A., Wendel,U., Stevens,E.M., van den Heuvel,L.P., Trijbels,F.J. and Blom,H.J. (1998) Identification of four novel mutations in severe methylenetetrahydrofolate reductase deficiency. Eur. J. Hum. Genet., 6, 257–265.[ISI][Medline]
  6. Schneider,J.A., Rees,D.C., Liu,Y.T. and Clegg,J.B. (1998) Worldwide distribution of a common methylenetetrahydrofolate reductase mutation [letter]. Am. J. Hum. Genet., 62, 1258–1260.[ISI][Medline]
  7. Zittoun,J., Tonetti,C., Bories,D., Pignon,J.M. and Tulliez,M. (1998) Plasma homocysteine levels related to interactions between folic acid status and methylenetetrahydrofolate reductase: a study in 52 healthy subjects. Metabolism, 47, 1413–1418.[ISI][Medline]
  8. Kluijtmans,L.A., Kastelein,J.J., Lindemans,J., Boers,G.H., Heil,S.G., Bruschke,A.V., Jukema,J.W., van den Heuvel,L.P., Trijbels,F.J., Boerma,G.J., Verheugt,F.W., Willems,F. and Blom,H.J. (1997) Thermolabile methylenetetrahydrofolate reductase in coronary artery disease. Circulation, 96, 2573–2577.[Abstract/Free Full Text]
  9. Gallagher,P.M., Meleady,R., Shields,D.C., Tan,K.S., McMaster,D., Rozen,R., Evans,A., Graham,I.M. and Whitehead,A.S. (1996) Homocysteine and risk of premature coronary heart disease. Evidence for a common gene mutation. Circulation, 94, 2154–2158.[Abstract/Free Full Text]
  10. Esteller,M., Garcia,A., Martinez-Palones,J.M., Xercavins,J. and Reventos,J. (1997) Germ line polymorphisms in cytochrome-P450 1A1 (C4887 CYP1A1) and methylenetetrahydrofolate reductase (MTHFR) genes and endometrial cancer susceptibility. Carcinogenesis, 18, 2307–2311.[Abstract]
  11. Mahmud,N., Molloy,A., McPartlin,J., Corbally,R., Whitehead,A.S., Scott,J.M. and Weir,D.G. (1999) Increased prevalence of methylenetetrahydrofolate reductase C677T variant in patients with inflammatory bowel disease and its clinical implications. Gut, 45, 389–394.[Abstract/Free Full Text]
  12. Hobbs,C.A., Sherman,S.L., Yi,P., Hopkins,S.E., Torfs,C.P., Hine,R.J., Pogribna,M., Rozen,R. and James,S.J. (2000) Polymorphisms in genes involved in folic acid metabolism as maternal risk factors for Down syndrome. Am. J. Hum. Genet., 67, 623–630.[ISI][Medline]
  13. James,S.J., Pogribna,M., Pogribny,I.P., Melnyk,S., Hine,R.J., Gibson,J.B., Yi,P., Tafoya,D.L., Swenson,D.H., Wilson,V.L. and Gaylor,D.W. (1999) Abnormal folic acid metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome. Am. J. Clin. Nutr., 70, 495–501.[Abstract/Free Full Text]
  14. Shields,D.C., Kirke,P.N., Mills,J.L., Ramsbottom,D., Molloy,A.M., Burke,H., Weir,D.G., Scott,J.M. and Whitehead,A.S. (1999) The `thermolabile' variant of methylenetetrahydrofolate reductase and neural tube defects: An evaluation of genetic risk and the relative importance of the genotypes of the embryo and the mother. Am. J. Hum. Genet., 64, 1045–1055.[ISI][Medline]
  15. van der Put,N.M., Eskes,T.K. and Blom,H.J. (1997) Is the common 677C->T mutation in the methylenetetrahydrofolate reductase gene a risk factor for neural tube defects? A meta-analysis. QJM, 90, 111–115.[Abstract]
  16. Christensen,B., Arbour,L., Tran,P., Leclerc,D., Sabbaghian,N., Platt,R., Gilfix,B.M., Rosenblatt,D.S., Gravel,R.A., Forbes,P. and Rozen,R. (1999) Genetic polymorphisms in methylenetetrahydrofolate reductase and methionine synthase, folic acid levels in red blood cells and risk of neural tube defects. Am. J. Med. Genet., 84, 151–157.[ISI][Medline]
  17. Slattery,M.L., Potter,J.D., Samowitz,W., Schaffer,D. and Leppert,M. (1999) Methylenetetrahydrofolate reductase, diet and risk of colon cancer. Cancer Epidemiol. Biomarkers. Prev., 8, 513–518.[Abstract/Free Full Text]
  18. Chen,J., Giovannucci,E., Kelsey,K., Rimm,E.B., Stampfer,M.J., Colditz,G.A., Spiegelman,D., Willett,W.C. and Hunter,D.J. (1996) A methylenetetrahydrofolate reductase polymorphism and the risk of colorectal cancer. Cancer Res., 56, 4862–4864.[Abstract]
  19. Ma,J., Stampfer,M.J., Giovannucci,E., Artigas,C., Hunter,D.J., Fuchs,C., Willett,W.C., Selhub,J., Hennekens,C.H. and Rozen,R. (1997) Methylenetetrahydrofolate reductase polymorphism, dietary interactions and risk of colorectal cancer. Cancer Res., 57, 1098–1102.[Abstract]
  20. Chen,J., Giovannucci,E., Hankinson,S.E., Ma,J., Willett,W.C., Spiegelman,D., Kelsey,K.T. and Hunter,D.J. (1998) A prospective study of methylenetetrahydrofolate reductase and methionine synthase gene polymorphisms and risk of colorectal adenoma. Carcinogenesis, 19, 2129–2132.[Abstract]
  21. Marugame,T., Tsuji,E., Inoue,H., Shinomiya,S., Kiyohara,C., Onuma,K., Hamada,H., Koga,H., Handa,K., Hayabuchi,H. and Kono,S. (2000) Methylenetetrahydrofolate reductase polymorphism and risk of colorectal adenomas. Cancer Lett., 151, 181–186.[ISI][Medline]
  22. Ulrich,C.M., Kampman,E., Bigler,J., Schwartz,S.M., Chen,C., Bostick,R., Fosdick,L., Beresford,A.A., Yasui,Y. and Potter,J.D. (1999) Colorectal adenomas and the C677T MTHFR polymorphism: evidence for gene–environment interaction? Cancer Epidemiol. Biomarkers. Prev., 8, 659–668.[Abstract/Free Full Text]
  23. Skibola,C.F., Smith,M.T., Kane,E., Roman,E., Rollinson,S., Cartwright,R.A. and Morgan,G. (1999) Polymorphisms in the methylenetetrahydrofolate reductase gene are associated with susceptibility to acute leukemia in adults. Proc. Natl Acad. Sci. USA, 96, 12810–12815.[Abstract/Free Full Text]
  24. Blount,B.C., Mack,M.M., Wehr,C.M., MacGregor,J.T., Hiatt,R.A., Wang,G., Wickramasinghe,S.N., Everson,R.B. and Ames,B.N. (1997) Folic acid deficiency causes uracil misincorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage. Proc. Natl Acad. Sci. USA, 94, 3290–3295.[Abstract/Free Full Text]
  25. Duthie,S.J. and Hawdon,A. (1998) 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.[Abstract/Free Full Text]
  26. Melnyk,S., Pogribna,M., Miller,B.J., Basnakian,A.G., Pogribny,I.P. and James,S.J. (1999) Uracil misincorporation, DNA strand breaks and gene amplification are associated with tumorigenic cell transformation in folic acid deficient/repleted Chinese hamster ovary cells. Cancer Lett., 146, 35–44.[ISI][Medline]
  27. Dianov,G.L., Timchenko,T.V., Sinitsina,O.I., Kuzminov,A.V., Medvedev,O.A. and Salganik,R.I. (1991) Repair of uracil residues closely spaced on the opposite strands of plasmid DNA results in double-strand break and deletion formation. Mol. Gen. Genet., 225, 448–452.[ISI][Medline]
  28. Lindahl,T. and Wood,R.D. (1999) Quality control by DNA repair. Science, 286, 1897–1905.[Abstract/Free Full Text]
  29. Hagmar,L., Bonassi,S., Stromberg,U., Brogger,A., Knudsen,L.E., Norppa,H. and Reuterwall,C. (1998) Chromosomal aberrations in lymphocytes predict human cancer: a report from the European Study Group on Cytogenetic Biomarkers and Health (ESCH). Cancer Res., 58, 4117–4121.[Abstract]
  30. Bonassi,S., Hagmar,L., Stromberg,U. et al. (2000) Chromosomal aberrations in lymphocytes predict human cancer independently of exposure to carcinogens. European Study Group on Cytogenetic Biomarkers and Health. Cancer Res., 60, 1619–1625.[Abstract/Free Full Text]
  31. van der Put,N.M., Gabreels,F., Stevens,E.M., Smeitink,J.A., Trijbels,F.J., Eskes,T.K., van den Heuvel,L.P. and Blom,H.J. (1998) A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am. J. Hum. Genet., 62, 1044–1051.[ISI][Medline]
  32. Leclerc,D., Campeau,E., Goyette,P., Adjalla,C.E., Christensen,B., Ross,M., Eydoux,P., Rosenblatt,D.S., Rozen,R. and Gravel,R.A. (1996) Human methionine synthase: cDNA cloning and identification of mutations in patients of the cblG complementation group of folic acid/cobalamin disorders. Hum. Mol. Genet., 5, 1867–1874.[Abstract/Free Full Text]
  33. Moore,G.E. and Woods,L.K. (1976) Culture Media for Human Cells—RPMI 1603, RPMI 1634, RPMI 1640 and GEM 1717. Tissue Culture Association Manual, Vol. 3. Rockville, MD, pp. 503–508.
  34. Fenech,M. (2000) The in vitro micronucleus technique. Mutat. Res., 455, 81–95.[ISI][Medline]
  35. Blount,B.C. and Ames,B.N. (1994) Analysis of uracil in DNA by gas chromatography–mass spectrometry. Anal. Biochem., 219, 195–200.[ISI][Medline]
  36. Fenech,M., Aitken,C. and Rinaldi,J. (1998) Folic acid, vitamin B12, homocysteine status and DNA damage in young Australian adults. Carcinogenesis, 19, 1163–1171.[Abstract]
  37. Jacob,R.A., Gretz,D.M., Taylor,P.C., James,S.J., Pogribny,I.P., Miller,B.J., Henning,S.M. and Swendseid,M.E. (1998) Moderate folic acid depletion increases plasma homocysteine and decreases lymphocyte DNA methylation in postmenopausal women. J. Nutr., 128, 1204–1212.[Abstract/Free Full Text]
  38. Bronstrup,A., Hages,M., Prinz-Langenohl,R. and Pietrzik,K. (1998) Effects of folic acid and combinations of folic acid and vitamin B12 on plasma homocysteine concentrations in healthy, young women. Am. J. Clin. Nutr., 68, 1104–1110.[Abstract]
  39. Fenech,M. and Rinaldi,J. (1994) The relationship between micronuclei in human lymphocytes and plasma levels of vitamin C, vitamin E, vitamin B12 and folic acid. Carcinogenesis, 15, 1405–1411.[Abstract]
  40. Fenech,M. and Rinaldi,J. (1995) A comparison of lymphocyte micronuclei and plasma micronutrients in vegetarians and non-vegetarians. Carcinogenesis, 16, 223–230.[Abstract]
  41. Koury,M.J., Horne,D.W., Brown,Z.A., Pietenpol,J.A., Blount,B.C., Ames,B.N., Hard,R. and Koury,S.T. (1997) Apoptosis of late-stage erythroblasts in megaloblastic anemia: association with DNA damage and macrocyte production. Blood, 89, 4617–4623.[Abstract/Free Full Text]
  42. Stern,L.L., Mason,J.B., Selhub,J. and Choi,S.W. (2000) Genomic DNA hypomethylation, a characteristic of most cancers, is present in peripheral leukocytes of individuals who are homozygous for the C677T polymorphism in the methylenetetrahydrofolate reductase gene. Cancer Epidemiol. Biomarkers. Prev., 9, 49–53.[Abstract/Free Full Text]
  43. Laird,P.W. and Jaenisch,R. (1994) DNA methylation and cancer. Hum. Mol. Genet., 3, 1487–1495.[Abstract]
  44. Cravo,M., Fidalgo,P., Pereira,A.D., Gouveia-Oliveira,A., Chaves,P., Selhub,J., Mason,J.B., Mira,F.C. and Leitao,C.N. (1994) DNA methylation as an intermediate biomarker in colorectal cancer: modulation by folic acid supplementation. Eur. J. Cancer Prev., 3, 473–479.[Medline]
  45. Kim,Y.I., Giuliano,A., Hatch,K.D., Schneider,A., Nour,M.A., Dallal,G.E., Selhub,J. and Mason,J.B. (1994) Global DNA hypomethylation increases progressively in cervical dysplasia and carcinoma. Cancer, 74, 893–899.[ISI][Medline]
  46. Robertson,K.D. and Jones,P.A. (2000) DNA methylation: past, present and future directions. Carcinogenesis, 21, 461–467.[Abstract/Free Full Text]
  47. Harmon,D.L., Shields,D.C., Woodside,J.V., McMaster,D., Yarnell,J.W., Young,I.S., Peng,K., Shane,B., Evans,A.E. and Whitehead,A.S. (1999) Methionine synthase D919G polymorphism is a significant but modest determinant of circulating homocysteine concentrations. Genet. Epidemiol., 17, 298–309.[ISI][Medline]
  48. Tsai,M.Y., Bignell,M., Yang,F., Welge,B.G., Graham,K.J. and Hanson,N.Q. (2000) Polygenic influence on plasma homocysteine: association of two prevalent mutations, the 844ins68 of cystathionine ß-synthase and A(2756)G of methionine synthase, with lowered plasma homocysteine levels. Atherosclerosis, 149, 131–137.[ISI][Medline]
  49. Guenther,B.D., Sheppard,C.A., Tran,P., Rozen,R., Matthews,R.G. and Ludwig,M.L. (1999) The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folic acid ameliorates human hyperhomocysteinemia. Nature Struct. Biol., 6, 359–365.[ISI][Medline]
  50. Hustad,S., Ueland,P.M., Vollset,S.E., Zhang,Y., Bjorke-Monsen,A.L. and Schneede,J. (2000) Riboflavin as a determinant of plasma total homocysteine: effect modification by the methylenetetrahydrofolate reductase C677T polymorphism. Clin. Chem., 46, 1065–1071.[Abstract/Free Full Text]
  51. Ubbink,J.B., van der Merwe,A., Delport,R., Allen,R.H., Stabler,S.P., Riezler,R. and Vermaak,W.J. (1996) The effect of a subnormal vitamin B6 status on homocysteine metabolism. J. Clin. Invest., 98, 177–184.[Abstract/Free Full Text]
  52. Kutzbach,C. and Stokstad,E.L. (1971) Mammalian methylenetetrahydrofolate reductase. Partial purification, properties and inhibition by S-adenosylmethionine. Biochim. Biophys. Acta, 250, 459–477.[ISI][Medline]
Received March 15, 2001; revised April 23, 2001; accepted April 24, 2001.