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
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Abstract |
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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.
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Introduction |
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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 2.63.2-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 1).
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.
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Materials and methods |
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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 I.
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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 2. 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 5001500 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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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 4 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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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).
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Results |
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Figure 3B 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 4 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 = 0.20.86). 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 5. 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 II
, 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 I
; 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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The effect of MTHFR A1298C and MS A2756G polymorphisms on DNA uracil content was also determined (Table II). 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 II). 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).
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Discussion |
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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 3). 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 (1260 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 1048 pg/10 µg DNA or approximately 34 600167 400 uracils/diploid cell. These figures are in good agreement with 730 pg/10 µg DNA observed in rat liver DNA (35) and 62 220256 200 uracils/diploid cell (1770 uracils/106 thymidines) observed for cultured primary murine erythroblasts (41). However, these values are considerably lower than the 500 0004 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 14 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 II 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 II, 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 1213 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 2030 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).
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Notes |
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Acknowledgments |
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References |
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