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© 2007 American Society for Nutrition J. Nutr. 137:2114-2120, September 2007

Nutritional Epidemiology

Folic Acid and Vitamin B-12 Supplementation Does Not Favorably Influence Uracil Incorporation and Promoter Methylation in Rectal Mucosa DNA of Subjects with Previous Colorectal Adenomas1,2

Maureen van den Donk3, Linette Pellis4, Jimmy W. Crott5, Manon van Engeland6, Pieter Friederich7, Fokko M. Nagengast7, Jeroen D. van Bergeijk8, Sybrand Y. de Boer9, Joel B. Mason5, Frans J. Kok3, Jaap Keijer4 and Ellen Kampman3,*

3 Division of Human Nutrition, Wageningen University, 6700 EV Wageningen, The Netherlands; 4 Food Bioactives Group, RIKILT-Institute of Food Safety, 6700 AE Wageningen, The Netherlands; 5 Vitamins and Carcinogenesis Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02478; 6 The Research Institute GROW, Department of Pathology, University Maastricht, 6200 MD Maastricht, The Netherlands; 7 Department of Gastroenterology and Hepatology, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands; 8 Department of Gastroenterology, Gelderse Vallei Hospital, 6710 HN Ede, The Netherlands; and 9 Department of Gastroenterology, Slingeland Hospital, 7000 AD Doetinchem, The Netherlands

* To whom correspondence should be addressed. E-mail: ellen.kampman{at}wur.nl.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Subjects and Methods
 Results
 Discussion
 LITERATURE CITED
 
Adequate folate availability is necessary to sustain normal DNA synthesis and normal patterns of DNA methylation and these features of DNA can be modified by methylenetetrahydrofolate reductase (MTHFR) C677T genotype. This study investigated the effect of MTHFR C677T genotype and daily supplementation with 5 mg folic acid and 1.25 mg vitamin B-12 on uracil misincorporation into DNA and promoter methylation. Subjects (n = 86) with a history of colorectal adenoma and MTHFR CC or TT genotype were randomly assigned to receive folic acid plus vitamin B-12 or placebo for 6 mo. Uracil misincorporation and promoter methylation of 6 tumor suppressor and DNA repair genes were assessed in DNA from rectal biopsies at baseline and after the intervention. The biomarkers did not differ between the treated group and the placebo group after 6 mo compared with baseline. The uracil concentration of DNA increased in the treated group (5.37 fmol/µg DNA, P = 0.02), whereas it did not change in the placebo group (P = 0.42). The change from baseline of 4.01 fmol uracil/µg DNA tended to differ between the groups (P = 0.16). An increase in promoter methylation tended to occur more often in the intervention group than in the placebo group (OR = 1.67; P = 0.08). This study suggests that supplementation with high doses of folic acid and vitamin B-12 may not favorably influence uracil incorporation and promoter methylation in subjects with previous colorectal adenomas. Because such alterations may potentially increase the risk of neoplastic transformation, more research is needed to fully define the consequences of these molecular alterations.


   Introduction
TOP
 ABSTRACT
 Introduction
Subjects and Methods
 Results
 Discussion
 LITERATURE CITED
 
Folate is an essential vitamin in DNA synthesis and repair. As 5-methyltetrahydrofolate (5-methylTHF),10 folate provides methyl groups for S-adenosylmethionine, the methyl donor for a large number of biological reactions, including the methylation of DNA. Furthermore, 5,10-methylenetetrahydrofolate acts as a cofactor in the conversion of deoxyuridylate to thymidylate, and is thus essential for DNA synthesis (1,2).

Inadequate folate availability in humans has been shown to increase uracil misincorporation into DNA and cause global hypomethylation, 2 factors that may be operative in colorectal carcinogenesis (1). Methylation of the promoter region of tumor suppressor genes is thought to play a role in cancer development through silencing of gene transcription (3).

Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme in folate metabolism that catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methylTHF (1). A common C-to-T substitution in the MTHFR gene at nucleotide 677 converts an alanine to valine and produces diminished enzyme activity in vivo (4). Studies indicate that the TT genotype is associated with global DNA hypomethylation in peripheral blood cells (5–7), probably restricted to those with a low folate intake (6), although 1 study could not demonstrate an association (8). Studies examining the association between MTHFR genotype and promoter methylation show ambiguous results (9–12). A study examining the relationship between MTHFR genotype and uracil misincorporation in human lymphocyte DNA showed a similar uracil concentration for all MTHFR variants (8).

In the Netherlands, the mean folate intake is ~200 µg/d (13), which is below the Dutch recommended daily intake of 300 µg. Enrichment of foods with folic acid and supplement use are not common in the Netherlands. Thus, part of the population may be ingesting insufficient quantities of folate to sustain normal DNA metabolism and integrity.

Previous studies in subjects with colorectal adenomas indicate that folic acid supplementation (0.4–10 mg/d) can reverse DNA hypomethylation in normal colorectal mucosa (14–17). A study examining the effect of a high-dose folic acid intervention in colorectal adenoma patients showed a nonsignificant reduction in adenoma recurrence (18). The numbers in these studies are relatively small and in most, no distinction is made between different MTHFR genotypes.

To further explore the effects of folate supplementation on relevant molecular events in the colon, we conducted a randomized, placebo-controlled intervention study with a high dose of folic acid and vitamin B-12. Vitamin B-12 was added to prevent the risk of masking a vitamin B-12 deficiency through folic acid and vitamin B-12 also aids folate metabolism, as it is the essential cofactor for methionine synthase. We examined the effect of a 6-mo intervention on uracil misincorporation and promoter hypermethylation of 6 tumor suppressor and DNA repair genes in DNA from rectal mucosa biopsies. The study was conducted in individuals with a history of colorectal adenomas and the possible modulation of effects by the MTHFR C677T genotype was considered.


   Subjects and Methods
TOP
 ABSTRACT
 Introduction
 Subjects and Methods
Results
 Discussion
 LITERATURE CITED
 
    Study population. We recruited participants from patients in a Dutch case-control study (19) who had the MTHFR 677 CC or TT genotype and a history of sporadic colorectal adenomas. The history of sporadic adenomas was verified through colonoscopy and pathology reports. All colorectal adenomas were confirmed histologically. Eligibility criteria were: Dutch speaking; of European origin; aged 18–80 y; no hereditary colorectal cancer syndromes; no chronic inflammatory bowel disease; no history of colorectal cancer; no (partial) bowel resection; not pregnant or lactating; no serious liver or renal disease; and not using anti-epileptic medication, antifolate medication, or supplements containing B vitamins. All eligible patients received a written invitation to the intervention study and, if they did not respond, were also invited by telephone. Patients who were invited but refused to participate in the study were called nonresponders. The participants were recruited between March 2002 and February 2003. We obtained written informed consent from all participants.

The study was conducted in 3 hospitals in the Netherlands: the Radboud University Nijmegen Medical Centre in Nijmegen, the Gelderse Vallei Hospital in Ede, and the Slingeland Hospital in Doetinchem. The study protocol was approved by the ethical committees of all participating centers.

    Design. After stratification by MTHFR C677T genotype, participants were randomly allocated to vitamin or placebo groups at entry into the study. Capsules were produced by Dutch BioFarmaceutics. By analysis, vitamin capsules contained 4.6 mg folic acid (pteroylmonoglutamic acid) and 1.1 mg vitamin B-12 (cyanocobalamin), whereas placebo capsules contained <0.04 µg folic acid and <0.002 µg vitamin B-12. Within each genotype group, treatment was allocated using random permuted blocks with lengths of 4 and 6. A random number table was used to determine block lengths and allocation within each block. All participants and study personnel were unaware of the treatment assignment for the duration of the study. Compliance was judged by pill-return counts and by analyzing plasma homocysteine and erythrocyte folate concentrations before and after the 6-mo intervention period.

    Data collection. Dietary intake was assessed with a semiquantitative FFQ that was originally developed for the Dutch cohort of the European Prospective Investigation into Cancer and Nutrition (20). The participants also completed a general questionnaire on medical history and lifestyle factors. Participants were advised not to alter their diet or lifestyle during the study.

Venous blood samples were obtained before and after the intervention period to measure plasma homocysteine, serum and erythrocyte folate, and serum vitamin B-12 concentrations. Plasma homocysteine was determined with HPLC and fluorimetric detection (21). Serum and erythrocyte folate concentrations and serum vitamin B-12 concentrations were measured by a paramagnetic-particle, chemiluminescent immunoassay on an Access Immunoassay system (Beckman Coulter). Three rectal biopsies of normal-appearing mucosa were obtained by flexible sigmoidoscopy without bowel preparation. Biopsies were immediately snap-frozen and stored in liquid nitrogen until extraction of DNA for determination of uracil misincorporation and promoter methylation. DNA was extracted using TRIZOL (Invitrogen).

The uracil concentration of DNA samples was measured using the method of Blount and Ames (22) with modifications (23,24). A quality control sample was tested in triplicate with each analysis. The mean intra-run CV was 10.1% and the between-day CV was 12.1% (over 12 d). All samples were analyzed in duplicate.

The genes analyzed for promoter methylation are the DNA repair genes O6-MGMT and hMLH1 and tumor suppressor genes that affect important cellular processes such as the cell cycle (p14ARF, p16INK4A, RASSF1A) and the Wnt signaling pathway (APC). All of these genes are reported to be frequently methylated in colorectal cancer (25,26). We determined DNA methylation of the CpG islands of the promoters of these genes by chemical modification of 500 ng of genomic DNA with sodium bisulfite and subsequent methylation-specific PCR (MSP), according to the method described by Herman et al. (27), with nested PCR (26). This method is based on the use of 2 distinct methylation-specific primer sets for the sequence of interest. The unmethylated primer will only amplify sodium bisulfite-converted DNA in unmethylated condition, whereas the methylated primer is specific for sodium bisulfite-converted methylated DNA. Both the forward and reverse primers contain 1–3 CpG dinucleotides in the 3' region of the primers to accomplish optimal discriminative power between methylated and unmethylated DNA (27). We used MSP primers that were located around the transcription start site and that have been reported, validated, and shown to be associated with gene silencing in cell lines and colorectal cancer (25,28). All PCR were performed with controls for unmethylated alleles (DNA from normal lymphocytes), methylated alleles (normal lymphocyte DNA treated in vitro with SssI methyltransferase; New England Biolabs), and a control without DNA. Primer sequences and PCR conditions are listed elsewhere (26). Ten microliters of each MSP reaction was loaded onto 6% polyacrylamide gels, stained with ethidium bromide, and visualized under UV illumination.

    Statistical analyses. Descriptive statistics of intake and blood concentrations of relevant nutrients and other baseline characteristics were computed for the intervention and placebo groups. Because the nonresponders also participated in the aforementioned case-control study (19), we calculated the descriptive statistics of some baseline characteristics of this group.

The primary endpoints were changes in the uracil concentration and promoter methylation in rectal mucosa DNA. We used paired t tests to assess differences in uracil concentration between baseline and postintervention values in each group. We assessed the differences in response between the intervention and the placebo group with linear regression analysis to be able to adjust for MTHFR genotype. In the total intervention group (n = 36), a change in uracil concentration of 11.6 fmol/µg DNA could be detected with a power of 80% and a 2-sided CI of 0.95 at an assumed SD of 17.8 fmol/µg DNA (23). Within the TT (n = 8) and CC (n = 28) genotypes, the detectable differences were 25.0 and 13.4 fmol/µg DNA, respectively.

We did not have enough power to assess changes in promoter methylation for all genes separately, as methylation frequencies are too low. Because colorectal carcinogenesis is widely held to be the result of a multistep process in which the aberrant expression of several genes collectively creates a milieu that facilitates the progression of carcinogenesis, we considered it appropriate to assess the methylation of all these genes collectively. Furthermore, folate intake, especially in combination with MTHFR TT genotype, was inversely associated with promoter methylation of all 6 genes in cases of the Dutch case-control study from which our participants were recruited (29). For each participant, we calculated the percentage of gene promoters that were not methylated at baseline but were methylated after the intervention period ("upmethylated") and the percentage of gene promoters that were methylated at baseline but not after the intervention period ("downmethylated"). These percentages were converted into logits {ln [p/(1 – p)]} to calculate OR for upmethylation and for downmethylation, taking the placebo group as the reference group. These OR are an estimate for the probability ratio of either upmethylation or downmethylation of any of the 6 genes in the intervention group compared with the placebo group. We used linear regression analysis for these calculations. The OR were weighed for the number of genes that were assessed in each participant to be able to include participants that had missing values for 1 or more of the 6 genes.

We analyzed the data by intention to treat, with complete case analyses where data were missing at random. All tests of significance were 2-sided and the significance level was 5%. We used SAS version 9.1 (SAS Institute) for all analyses.


   Results
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 ABSTRACT
 Introduction
 Subjects and Methods
 Results
Discussion
 LITERATURE CITED
 
Of 245 invited patients, 100 were willing to participate, of whom 14 did not meet the eligibility criteria (Fig. 1). The 86 eligible patients, 67 with the CC genotype and 19 with the TT genotype, were randomly assigned to either the intervention or placebo. After randomization, 5 participants withdrew from the study, 4 with CC genotype (3 in the placebo group and 1 in the intervention group) and 1 with TT genotype in the intervention group. The reasons for withdrawal were: difficulties with taking rectal mucosa biopsies; traffic accident; influenza; pericarditis; and lost to follow-up for an unknown reason. Moreover, there were missing data on either the baseline or the end measurement of the primary endpoints due to insufficient quality or quantity of DNA. As we judged these missing data to be random, we did not include these participants in the analyses. One participant (placebo, CC genotype) stopped taking the capsules after 52 d when developing a peptic ulcer but did not withdraw. This person was included in the analyses.


Figure 1
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  		FIGURE 1  Study flowchart. CC, MTHFR CC genotype; TT, MTHFR TT genotype.

 
Baseline characteristics did not differ between the intervention and the placebo group. Nonresponders were younger than participants (Table 1).


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  		TABLE 1 Baseline characteristics of the study population and nonresponders1

 
We measured participants' plasma homocysteine and erythrocyte folate concentrations as measures of compliance (Table 2). Plasma homocysteine and erythrocyte folate did not change in the placebo group during the study, whereas plasma homocysteine decreased significantly in the intervention group and erythrocyte folate increased significantly in the intervention group. Furthermore, we counted returned pills from the participants. Of 81 participants who completed the study, 5 did not return their remaining pills, 1 stopped taking the pills after 52 d, 7 took between 90 and 95% of the pills, and 68 took >95% of the pills. Based on blood measurements and pill-return counts, we judged that compliance was adequate.


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  		TABLE 2 Effect of a 6-mo intervention with folic acid and vitamin B-12 or placebo on plasma homocysteine and erythrocyte folate concentration among 81 subjects with previous colorectal adenomas1

 
The changes between baseline and 6 mo in the biomarkers measured did not differ significantly between the groups. Within the total intervention group, the concentration of uracil in rectal mucosa DNA increased from 3.19 fmol/µg DNA at baseline to 8.56 fmol/µg DNA after the intervention period (P = 0.02), whereas in the placebo group, rectal mucosa uracil concentration did not change. The increase in the intervention group was 4.01 fmol uracil/µg DNA more than that of the placebo group (P = 0.16). When we stratified the analyses for MTHFR genotype, the effects of the intervention were similar in both the TT and CC group (Table 3).


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  		TABLE 3 Effect of a 6-mo intervention with folic acid and vitamin B-12 or placebo on uracil concentrations in rectal mucosa DNA among 73 subjects with previous colorectal adenomas1

 
The results of the MSP analyses indicated that intervention with folic acid and vitamin B-12 tended to increase the probability of upmethylation (P = 0.08), but not downmethylation. Again, results were similar in both genotype groups (Table 4). When we considered all 6 genes separately, results pointed in the same direction for all genes except APC (data not shown); however, numbers were too small to draw conclusions from these analyses.


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  		TABLE 4 Effect of a 6-mo intervention with folic acid and vitamin B-12 or placebo on the probability ratio of promoter upmethylation and downmethylation in rectal mucosa DNA among 76 subjects with previous colorectal adenomas1

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Subjects and Methods
 Results
 Discussion
LITERATURE CITED
 
In this randomized, placebo-controlled trial, promoter methylation or uracil concentration did not change in rectal mucosa DNA of patients with a history of sporadic colorectal adenomas after supplementation with high doses of folic acid and vitamin B-12 for 6 mo. However, the intervention tended to increase the probability of promoter methylation in rectal mucosa DNA. Moreover, the uracil concentration in rectal mucosa DNA increased to >250% of the initial value as a result of intervention, although this increase was not significantly higher than the mild increase observed among the placebo group. Although the uracil concentration of peripheral blood mononuclear cell DNA has been previously shown to revert to lower, more normal, concentrations with the treatment of folate deficiency (30), our study is, to our knowledge, the first human folic acid intervention study to examine uracil misincorporation and promoter methylation in rectal mucosa DNA as primary endpoints. These endpoints in the colonic mucosa are of particular relevance because: 1) excess uracil incorporation and promoter hypermethylation of critical genes are mechanistically implicated in colorectal carcinogenesis (1,3); 2) the colon appears to be an especially sensitive organ with regard to how folate status might modulate cancer risk (1); and 3) the biochemical and molecular alterations that arise as a result of changes in folate status are known to be highly tissue specific (31).

The results from this study do not support our initial hypotheses. Earlier studies have generally observed that folic acid supplementation of 2–10 mg/d for 3–12 mo alters purported biomarkers of colon cancer in a favorable manner (14–16,32–34). Favorable effects were found on DNA hypomethylation (14,15), colonic mucosal cell proliferation (33,34), loss of heterozygosity of the tumor suppressor gene DELETED IN COLORECTAL CANCER (34), or activity of the proto-oncogene ornithine decarboxylase (32). In a study by Kim et al. (16), 6 mo of folate supplementation increased genomic DNA methylation and decreased p53 strand breaks, although after 12 mo, the effects were the same in the placebo group. Recently, an intervention study utilizing a physiological dose of folic acid (400 µg/d) over 10 wk found increased genomic DNA methylation in rectal mucosa of colorectal adenoma patients (17).

The presence of adenomas is presently considered to be the only intermediary biomarker of colorectal cancer for which firm validation data exist, because removal of such lesions leads to a reduction in the subsequent risk of cancer (35). Unfortunately, a very limited amount of information is available to date from folate intervention trials that have used adenoma recurrence as the primary endpoint. A small study investigating daily supplementation with 1 mg folic acid for 2 y suggested a reduction in adenoma recurrence (18), but the results from a large, placebo-controlled multicenter trial showed no reduction in adenoma recurrence (36). Moreover, subjects in the folic acid group tended to have higher rates of advanced adenomas and multiple adenomas (36). In summary, human folic acid intervention studies to date generally have shown favorable effects on biochemical, molecular, and cytokinetic biomarkers for colorectal cancer, whereas the paucity of data available from adenoma recurrence trials precludes any firm conclusions regarding effects on the latter biomarker.

In this study, we used a high dosage of 5 mg/d folic acid, which is 12.5 times as high as the Recommended Dietary Allowance in the United States (400 µg/d). This high dosage is feasibly responsible, at least in part, for the counterintuitive nature of our results. This hampers generalizability to lower doses. In animal models of colon cancer, folate supplementation has been shown to be protective under most conditions, but if it is given in very high doses or at a stage of carcinogenesis where neoplastic transformation has already firmly been established, it instead enhances the development of neoplasms (37–39).

We used the synthetic, fully oxidized form of folate (pteroylmonoglutamic acid), which is normally fully metabolized by the intestine before it is released into the plasma as 5-methylTHF; consequently, the latter form is the sole circulating form of folate under normal conditions. However, studies show that this absorption and biotransformation process is saturated at doses in the region of 400 µg folic acid or less (40). At higher doses, synthetic folic acid is also transported into the blood and may enter in large quantities. Compelling data about possible antagonistic activities of this fully oxidized form of folate in tissues is lacking, although occasional concern has been voiced about this possibility (41). A study in postmenopausal women showed that unmetabolized folic acid in plasma, which was detected in 78% of participants, was associated with decreased natural killer cell cytotoxicity (42). Natural killer cell cytotoxicity is important in immune surveillance against tumor cells (43). In a human intervention study on the effect of folic acid on neural tube defects, a higher all-cause mortality and mortality from breast cancer was found in participants in 2 intervention groups compared with the placebo group (44). As this was not a prespecified hypothesis, these results have to be interpreted cautiously but still deserve attention. Consistent with this are prospective analyses in a screening trial that showed a positive association between total folate and supplemental folic acid intake and postmenopausal breast cancer (45). Two trials investigating the effect of treatment with B vitamins on prevention of cardiovascular events reported a nonsignificant increase in the risk of total cancer (relative risk 1.22, 95% CI 0.88,1.70) (46) or colon cancer (relative risk 1.36, 95% CI 0.89,2.08) (47).

A relative lack in knowledge about the specific pathways by which uracil incorporation and promoter methylation effect a greater risk of cancer might also explain our counterintuitive observations. Although seeming procarcinogenic, the observations of increased DNA uracil and promoter methylation represent only a fraction of the plethora of possible molecular and genetic aberrations that are known to determine our risk for carcinogenesis. For example, it is assumed that global DNA hypomethylation is an early event in carcinogenesis, which is followed by hypermethylation of the promoter region of certain tumor suppressor genes, which may accelerate the carcinogenic process (48). Studies to date showed that folic acid supplementation can reverse DNA hypomethylation in normal colorectal mucosa (14–17) and limited data showed that folate depletion seemed associated with promoter hypermethylation (26). However, it also is possible that folic acid supplementation, together with an effect on overall DNA methylation, directly leads to promoter methylation, thereby simultaneously exerting both a favorable and an unfavorable effect. Clearly, more research is needed to unravel such a mechanism.

A limitation of our study might be the measurement of biomarkers in rectal mucosa, whereas rectal cancer has not been associated with folate deficiency to the same extent as colon cancer. However, due to the acceptability of the biopsy procedure for the participants, it was not possible to take colon mucosa biopsies in this study. Furthermore, other folic acid intervention studies that used intermediate biomarkers as endpoints and that did show favorable effects also used rectal mucosa biopsies (14,15,17,33,34), so we think that this limitation is not crucial for the interpretation of the results.

Folate is not the only vitamin that is needed for DNA synthesis and DNA methylation. Riboflavin and vitamins B-6 and B-12 also play a prominent role in the 1-carbon metabolism. FAD, a metabolite of riboflavin, serves as a cofactor for MTHFR (49). Vitamin B-6 is a cofactor for serine hydroxymethyltransferase, which catalyses the conversion of tetrahydrofolate to 5,10-methylTHF (50). Although the mean intakes of riboflavin and vitamin B-6 in this study are in the range of the Dutch recommended dietary intake, it is possible that some people had a suboptimal intake. Vitamin B-6 and especially riboflavin intakes are lower in the Netherlands compared with the United States. We reported that riboflavin intake may be important in the association between folate intake and colorectal adenoma risk (19). Unfortunately, we lacked statistical power to conduct subgroup analyses according to intake of riboflavin or vitamin B-6 in this intervention study.

The results of this study suggest that, in individuals with a history of colorectal adenomas, high doses of supplemental folic acid and vitamin B-12 do not favorably influence promoter methylation of selected genes in the colonic mucosa. Instead, there was a tendency toward a small change in promoter methylation that is usually perceived as procarcinogenic. Parallel changes in uracil content were observed, although these also were not significant. Our results underscore the importance of further studies needed to determine whether or not folate supplementation is an effective cancer chemopreventive agent and, if so, what the appropriate dose, timing, form of folate, and subject may be for such an intervention.


   ACKNOWLEDGMENTS
 
We thank Jenneke Poortman for her help with the DNA isolation, Sandra van den Bosch for her support in the methylation assessment, and Pieter van 't Veer and Jan Burema for methodological advice and statistical assistance.


   FOOTNOTES
 
1 Supported by the Netherlands Organization for Health Research and Development (ZonMw), grant 980-10-020, and by the Netherlands Foundation for Digestive Diseases (MLDS), grant WS 99-72. JWC and JBM were supported in part by the USDA Agricultural Research Service, under agreement no. 581950-9-001 and by the NIH grants K05 CA100048 and U54 CA100971. Any opinions, findings, conclusions, or recommendations expressed in this publication do not necessarily reflect the view of the USDA. Back

2 Author disclosures: M. van den Donk, L. Pellis, J. W. Crott, M. van Engeland, P. Friederich, F. M. Nagengast, J. D. van Bergeijk, S. Y. de Boer, J. B. Mason, F. J. Kok, J. Keijer, and E. Kampman, no conflicts of interest. Back

10 Abbreviations used: 5-methylTHF, 5-methyltetrahydrofolate; MSP, methylation-specific PCR; MTHFR, methylenetetrahydrofolate reductase. Back

Manuscript received 12 February 2007. Initial review completed 15 March 2007. Revision accepted 25 June 2007.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Subjects and Methods
 Results
 Discussion
 LITERATURE CITED
 

1. Choi SW, Mason JB. Folate and carcinogenesis: an integrated scheme. J Nutr. 2000;130:129–32.[Abstract/Free Full Text]

2. Kim YI. Folate and carcinogenesis: evidence, mechanisms, and implications. J Nutr Biochem. 1999;10:66–88.[Medline]

3. Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med. 2003;349:2042–54.[Free Full Text]

4. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ, den Heijer M, Kluijtmans LA, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet. 1995;10:111–3.[Medline]

5. Stern LL, Mason JB, Selhub J, Choi SW. 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. 2000;9:849–53.[Abstract/Free Full Text]

6. Friso S, Choi SW, Girelli D, Mason JB, Dolnikowski GG, Bagley PJ, Olivieri O, Jacques PF, Rosenberg IH, et al. A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status. Proc Natl Acad Sci USA. 2002;99:5606–11.[Abstract/Free Full Text]

7. Castro R, Rivera I, Ravasco P, Camilo ME, Jakobs C, Blom HJ, de Almeida I. 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C->T and 1298A->C mutations are associated with DNA hypomethylation. J Med Genet. 2004;41:454–8.[Free Full Text]

8. Narayanan S, McConnell J, Little J, Sharp L, Piyathilake CJ, Powers H, Basten G, Duthie SJ. Associations between two common variants C677T and A1298C in the methylenetetrahydrofolate reductase gene and measures of folate metabolism and DNA stability (strand breaks, misincorporated uracil, and DNA methylation status) in human lymphocytes in vivo. Cancer Epidemiol Biomarkers Prev. 2004;13:1436–43.[Abstract/Free Full Text]

9. Oyama K, Kawakami K, Maeda K, Ishiguro K, Watanabe G. The association between methylenetetrahydrofolate reductase polymorphism and promoter methylation in proximal colon cancer. Anticancer Res. 2004;24:649–54.[Abstract/Free Full Text]

10. Paz MF, Avila S, Fraga MF, Pollan M, Capella G, Peinado MA, Sanchez-Cespedes M, Herman JG, Esteller M. Germ-line variants in methyl-group metabolism genes and susceptibility to DNA methylation in normal tissues and human primary tumors. Cancer Res. 2002;62:4519–24.[Abstract/Free Full Text]

11. Clarizia AD, Bastos-Rodrigues L, Pena HB, Anacleto C, Rossi B, Soares FA, Lopes A, Rocha JC, Caballero O, et al. Relationship of the methylenetetrahydrofolate reductase C677T polymorphism with microsatellite instability and promoter hypermethylation in sporadic colorectal cancer. Genet Mol Res. 2006;5:315–22.[Medline]

12. Kawakami K, Ruszkiewicz A, Bennett G, Moore J, Grieu F, Watanabe G, Iacopetta B. DNA hypermethylation in the normal colonic mucosa of patients with colorectal cancer. Br J Cancer. 2006;94:593–8.[Medline]

13. de Bree A, Verschuren WM, Blom HJ, Kromhout D. Association between B vitamin intake and plasma homocysteine concentration in the general Dutch population aged 20–65 y. Am J Clin Nutr. 2001;73:1027–33.[Abstract/Free Full Text]

14. Cravo M, Fidalgo P, Pereira AD, Gouveia-Oliveira A, Chaves P, Selhub J, Mason JB, Mira FC, Leitao CN. DNA methylation as an intermediate biomarker in colorectal cancer: modulation by folic acid supplementation. Eur J Cancer Prev. 1994;3:473–9.[Medline]

15. Cravo ML, Pinto AG, Chaves P, Cruz JA, Lage P, Nobre Leitao C, Costa Mira F. Effect of folate supplementation on DNA methylation of rectal mucosa in patients with colonic adenomas: correlation with nutrient intake. Clin Nutr. 1998;17:45–9.[Medline]

16. Kim YI, Baik HW, Fawaz K, Knox T, Lee YM, Norton R, Libby E, Mason JB. Effects of folate supplementation on two provisional molecular markers of colon cancer: a prospective, randomized trial. Am J Gastroenterol. 2001;96:184–95.[Medline]

17. Pufulete M, Al-Ghnaniem R, Khushal A, Appleby P, Harris N, Gout S, Emery PW, Sanders TA. Effect of folic acid supplementation on genomic DNA methylation in patients with colorectal adenoma. Gut. 2005;54:648–53.[Abstract/Free Full Text]

18. Paspatis GA, Karamanolis DG. Folate supplementation and adenomatous colonic polyps. Dis Colon Rectum. 1994;37:1340–1.[Medline]

19. van den Donk M, Buijsse B, van den Berg SW, Ocké MC, Harryvan JL, Nagengast FM, Kok FJ, Kampman E. Dietary intake of folate and riboflavin, MTHFR C677T genotype, and colorectal adenoma risk: a Dutch case-control study. Cancer Epidemiol Biomarkers Prev. 2005;14:1562–6.[Abstract/Free Full Text]

20. Ocké MC, Bueno-de-Mesquita HB, Goddijn HE, Jansen A, Pols MA, van Staveren WA, Kromhout D. The Dutch EPIC food frequency questionnaire. I. Description of the questionnaire, and relative validity and reproducibility for food groups. Int J Epidemiol. 1997;26 Suppl 1:S37–48.[Abstract/Free Full Text]

21. Ubbink JB, Hayward Vermaak WJ, Bissbort S. Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum. J Chromatogr. 1991;565:441–6.[Medline]

22. Blount BC, Ames BN. Analysis of uracil in DNA by gas chromatography-mass spectrometry. Anal Biochem. 1994;219:195–200.[Medline]

23. Crott JW, Mashiyama ST, Ames BN, Fenech MF. Methylenetetrahydrofolate reductase C677T polymorphism does not alter folic acid deficiency-induced uracil incorporation into primary human lymphocyte DNA in vitro. Carcinogenesis. 2001;22:1019–25.[Abstract/Free Full Text]

24. Mashiyama ST, Courtemanche C, Elson-Schwab I, Crott J, Lee BL, Ong CN, Fenech M, Ames BN. Uracil in DNA, determined by an improved assay, is increased when deoxynucleosides are added to folate-deficient cultured human lymphocytes. Anal Biochem. 2004;330:58–69.[Medline]

25. Esteller M, Corn PG, Baylin SB, Herman JG. A gene hypermethylation profile of human cancer. Cancer Res. 2001;61:3225–9.[Abstract/Free Full Text]

26. van Engeland M, Weijenberg MP, Roemen GM, Brink M, de Bruïne AP, Goldbohm RA, van den Brandt PA, Baylin SB, de Goeij AF, et al. Effects of dietary folate and alcohol intake on promoter methylation in sporadic colorectal cancer: the Netherlands cohort study on diet and cancer. Cancer Res. 2003;63:3133–7.[Abstract/Free Full Text]

27. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. 1996;93:9821–6.[Abstract/Free Full Text]

28. van Engeland M, Roemen GM, Brink M, Pachen MM, Weijenberg MP, de Bruine AP, Arends JW, van den Brandt PA, de Goeij AF, et al. K-ras mutations and RASSF1A promoter methylation in colorectal cancer. Oncogene. 2002;21:3792–5.[Medline]

29. van den Donk M, van Engeland M, Pellis L, Witteman BJ, Kok FJ, Keijer J, Kampman E. Dietary folate intake in combination with MTHFR C677T genotype and promoter methylation of tumor suppressor and DNA repair genes in sporadic colorectal adenomas. Cancer Epidemiol Biomarkers Prev. 2007;16:327–33.[Abstract/Free Full Text]

30. Blount BC, Mack MM, Wehr CM, MacGregor JT, Hiatt RA, Wang G, Wickramasinghe SN, Everson RB, Ames BN. Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage. Proc Natl Acad Sci USA. 1997;94:3290–5.[Abstract/Free Full Text]

31. Choi SW, Friso S, Dolnikowski GG, Bagley PJ, Edmondson AN, Smith DE, Mason JB. Biochemical and molecular aberrations in the rat colon due to folate depletion are age-specific. J Nutr. 2003;133:1206–12.[Abstract/Free Full Text]

32. Bukin YUV, Draudin-Krylenko VA, Levchuk AA, Poddubniy BK, Mazurov ST. The effect of high doses of folic acid on the overexpression of ornithine decarboxylase and S-adenosylmethionine content in human colon adenomatous polyps. Ann N Y Acad Sci. 2001;952:175–6.[Medline]

33. Khosraviani K, Weir HP, Hamilton P, Moorehead J, Williamson K. Effect of folate supplementation on mucosal cell proliferation in high risk patients for colon cancer. Gut. 2002;51:195–9.[Abstract/Free Full Text]

34. Nagothu KK, Jaszewski R, Moragoda L, Rishi AK, Finkenauer R, Tobi M, Naumoff JA, Dhar R, Ehrinpreis M, et al. Folic acid mediated attenuation of loss of heterozygosity of DCC tumor suppressor gene in the colonic mucosa of patients with colorectal adenomas. Cancer Detect Prev. 2003;27:297–304.[Medline]

35. Winawer SJ, Zauber AG, Ho MN, O'Brien MJ, Gottlieb LS, Sternberg SS, Waye JD, Schapiro M, Bond JH, et al. Prevention of colorectal cancer by colonoscopic polypectomy. The National Polyp Study Workgroup. N Engl J Med. 1993;329:1977–81.[Abstract/Free Full Text]

36. Cole BF, Baron JA, Sandler RS, Haile RW, Ahnen DJ, Bresalier RS, Keown-Eyssen G, Summers RW, Rothstein RI, et al. Folic acid for the prevention of colorectal adenomas: a randomized clinical trial. JAMA. 2007;297:2351–9.[Abstract/Free Full Text]

37. Bashir O, FitzGerald AJ, Goodlad RA. Both suboptimal and elevated vitamin intake increase intestinal neoplasia and alter crypt fission in the ApcMin/+ mouse. Carcinogenesis. 2004;25:1507–15.[Abstract/Free Full Text]

38. Song J, Medline A, Mason JB, Gallinger S, Kim YI. Effects of dietary folate on intestinal tumorigenesis in the apcMin mouse. Cancer Res. 2000;60:5434–40.[Abstract/Free Full Text]

39. Kim YI, Salomon RN, Graeme-Cook F, Choi SW, Smith DE, Dallal GE, Mason JB. Dietary folate protects against the development of macroscopic colonic neoplasia in a dose responsive manner in rats. Gut. 1996;39:732–40.[Abstract/Free Full Text]

40. Kelly P, McPartlin J, Goggins M, Weir DG, Scott JM. Unmetabolized folic acid in serum: acute studies in subjects consuming fortified food and supplements. Am J Clin Nutr. 1997;65:1790–5.[Abstract/Free Full Text]

41. Pentieva K, McNulty H, Reichert R, Ward M, Strain JJ, McKillop DJ, McPartlin JM, Connolly E, Molloy A, et al. The short-term bioavailabilities of [6S]-5-methyltetrahydrofolate and folic acid are equivalent in men. J Nutr. 2004;134:580–5.[Abstract/Free Full Text]

42. Troen AM, Mitchell B, Sorensen B, Wener MH, Johnston A, Wood B, Selhub J, McTiernan A, Yasui Y, et al. Unmetabolized folic acid in plasma is associated with reduced natural killer cell cytotoxicity among postmenopausal women. J Nutr. 2006;136:189–94.[Abstract/Free Full Text]

43. Tartter PI, Steinberg B, Barron DM, Martinelli G. The prognostic significance of natural killer cytotoxicity in patients with colorectal cancer. Arch Surg. 1987;122:1264–8.[Abstract/Free Full Text]

44. Charles D, Ness AR, Campbell D, Davey Smith G, Hall MH. Taking folate in pregnancy and risk of maternal breast cancer. BMJ. 2004;329:1375–6.[Free Full Text]

45. Stolzenberg-Solomon RZ, Chang SC, Leitzmann MF, Johnson KA, Johnson C, Buys SS, Hoover RN, Ziegler RG. Folate intake, alcohol use, and postmenopausal breast cancer risk in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Am J Clin Nutr. 2006;83:895–904.[Abstract/Free Full Text]

46. Bonaa KH, Njolstad I, Ueland PM, Schirmer H, Tverdal A, Steigen T, Wang H, Nordrehaug JE, Arnesen E, et al. Homocysteine lowering and cardiovascular events after acute myocardial infarction. N Engl J Med. 2006;354:1578–88.[Abstract/Free Full Text]

47. Lonn E, Yusuf S, Arnold MJ, Sheridan P, Pogue J, Micks M, McQueen MJ, Probstfield J, Fodor G, et al. Homocysteine lowering with folic acid and B vitamins in vascular disease. N Engl J Med. 2006;354:1567–77.[Abstract/Free Full Text]

48. Duthie SJ, Narayanan S, Sharp L, Little J, Basten G, Powers H. Folate, DNA stability and colo-rectal neoplasia. Proc Nutr Soc. 2004;63:571–8.[Medline]

49. Yamada K, Chen Z, Rozen R, Matthews RG. Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. Proc Natl Acad Sci USA. 2001;98:14853–8.[Abstract/Free Full Text]

50. Selhub J, Miller JW. The pathogenesis of homocysteinemia: interruption of the coordinate regulation by S-adenosylmethionine of the remethylation and transsulfuration of homocysteine. Am J Clin Nutr. 1992;55:131–8.[Abstract/Free Full Text]





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