Joel B. Mason

A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status

DNA methylation, an essential epigenetic feature of DNA that modulates gene expression and genomic integrity, is catalyzed by methyltransferases that use the universal methyl donor S-adenosyl-l-methionine. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for synthesis of methionine from homocysteine and precursor of S-adenosyl-l-methionine. In the present study we sought to determine the effect of folate status on genomic DNA methylation with an emphasis on the interaction with the common C677T mutation in the MTHFR gene. A liquid chromatography/MS method for the analysis of nucleotide bases was used to assess genomic DNA methylation in peripheral blood mononuclear cell DNA from 105 subjects homozygous for this mutation (T/T) and 187 homozygous for the wild-type (C/C) MTHFR genotype. The results show that genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels (P < 0.01). T/T genotypes had a diminished level of DNA methylation compared with those with the C/C wild-type (32.23 vs.62.24 ng 5-methylcytosine/μg DNA, P < 0.0001). When analyzed according to folate status, however, only the T/T subjects with low levels of folate accounted for the diminished DNA methylation (P < 0.0001). Moreover, in T/T subjects DNA methylation status correlated with the methylated proportion of red blood cell folate and was inversely related to the formylated proportion of red blood cell folates (P < 0.03) that is known to be solely represented in those individuals. These results indicate that the MTHFR C677T polymorphism influences DNA methylation status through an interaction with folate status.

A Temporal Association between Folic Acid Fortification and an Increase in Colorectal Cancer Rates May Be Illuminating Important Biological Principles: A Hypothesis

Nationwide fortification of enriched uncooked cereal grains with folic acid began in the United States and Canada in 1996 and 1997, respectively, and became mandatory in 1998. The rationale was to reduce the number of births complicated by neural tube defects. Concurrently, the United States and Canada experienced abrupt reversals of the downward trend in colorectal cancer (CRC) incidence that the two countries had enjoyed in the preceding decade: absolute rates of CRC began to increase in 1996 (United States) and 1998 (Canada), peaked in 1998 (United States) and 2000 (Canada), and have continued to exceed the pre-1996/1997 trends by 4 to 6 additional cases per 100,000 individuals. In each country, the increase in CRC incidence from the prefortification trend falls significantly outside of the downward linear fit based on nonparametric 95% confidence intervals. The statistically significant increase in rates is also evident when the data for each country are analyzed separately for men and women. Changes in the rate of colorectal endoscopic procedures do not seem to account for this increase in CRC incidence. These observations alone do not prove causality but are consistent with the known effects of folate on existing neoplasms, as shown in both preclinical and clinical studies. We therefore hypothesize that the institution of folic acid fortification may have been wholly or partly responsible for the observed increase in CRC rates in the mid-1990s. Further work is needed to definitively establish the nature of this relationship. In the meantime, deliberations about the institution or enhancement of fortification programs should be undertaken with these considerations in mind. (Cancer Epidemiol Biomarkers Prev 2007;16(7):1325–9)

Folate Status: Effects on Pathways of Colorectal Carcinogenesis

Many epidemiologic, animal and human studies suggest that folate status modulates carcinogenesis. Although these observations have been made in a number of tissues, the data are clearly most compelling for the colorectum. The mechanism(s) by which this modulation is mediated remains ill defined. Alterations in either genome-wide or gene-specific DNA methylation and/or alterations in DNA stability, resulting from DNA strand breaks or uracil misincorporation, are leading candidates in this regard. Folate has a central role in biological methylation and nucleotide synthesis, and therefore it is not surprising that folate depletion has been observed to alter DNA methylation and diminish DNA stability. The hypothesis that these two pathways are the means by which folate modulates cancer risk is also supported by the epidemiological observation that a common polymorphism in the methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) gene differentially affects the relative risk of colon cancer depending on folate status, because MTHFR catalyzes the reaction that determines whether cellular folate is diverted into biological methylation or nucleotide synthesis. This phenomenon suggests that it is an imbalance between biological methylation and nucleotide synthesis that is responsible for folate-related carcinogenesis. The control of cell proliferation, which also is related to DNA methylation, is another candidate mechanism by which folate status modulates carcinogenesis. In cell culture studies, folate supplementation has been observed to suppress excessive cell proliferation. Understanding the mechanisms by which folate status modulates carcinogenesis is important for advancing insight into cancer biology and for facilitating those efforts to translate research in folate and carcinogenesis into effective and safe public health initiatives.

Dietary folate protects against the development of macroscopic colonic neoplasia in a dose responsive manner in rats.

BACKGROUND AND AIMS: Diminished folate status is associated with enhanced colorectal carcinogenesis. This study investigated the potential chemopreventive role of dietary folate in the dimethylhydrazine colorectal cancer model. SUBJECTS AND METHODS: Sprague-Dawley rats were fed diets containing either 0, 2 (daily dietary requirement), 8 or 40 mg folate/kg diet for 20 weeks. After five weeks of diet, rats were injected with dimethyl-hydrazine (44 mg/kg) weekly for 15 weeks. Fifteen weeks after the first injection of dimethylhydrazine, all rats were killed. Folate status was determined, and the entire colorectum from each rat was analysed for macroscopic and microscopic neoplasms. RESULTS: Plasma and colonic folate concentrations correlated directly with dietary folate levels (p < 0.005). The incidence of microscopic neoplasms was similar among the four groups. However, the incidence and the average number of macroscopic tumours per rat decreased progressively with increasing dietary folate levels up to 8 mg/kg diet (p < 0.05). In the strongly procarcinogenic milieu used in this study, folate supplementation at 20 times the basal requirement was associated with rates of macroscopic tumour development that were intermediate, and not statistically distinct, from rates observed at either 0 or 8 mg/kg diet. CONCLUSIONS: These data indicate that in this rat model, (a) increasing dietary folate up to four times the basal requirement leads to a progressive reduction in the evolution of macroscopic neoplasms from microscopic foci; and (b) folate supplementation beyond four times the requirement does not convey further benefit.

Folate depletion impairs DNA excision repair in the colon of the rat.

Background/Aims—Diminished folate status appears to promote colonic carcinogenesis by, as of yet, undefined mechanisms. Impaired DNA repair plays a significant role in the evolution of many colon cancers. Since folate is essential for thede novo synthesis of nucleotides and since folate depletion has previously been associated with excessive DNA strand breaks, it was hypothesised that folate depletion may impair DNA repair. Studies were therefore performed to examine whether folate depletion affects the two major categories of DNA repair. Methods—Study 1: eight weanling male Sprague-Dawley rats were fed on diets containing either 0 or 8 mg folate/kg diet with 1% succinylsulphathiazole for four weeks. After viable colonocytes had been harvested, DNA excision repair was evaluated by a single cell gel electrophoresis assay. Study 2: eighteen animals were fed on similar diets for five weeks. Also in study 2, 18 additional rats were fed on the same defined diet without succinylsulphathiazole for 15 weeks. Weekly injections with the procarcinogen, 1,2-dimethylhydrazine (20 mg base/kg), were administered to the latter group of animals. Five microsatellite loci from different chromosomes were investigated for instability in hepatic and colonic DNA. Results—In study 1, a significantly retarded rate of DNA excision repair was observed in the folate deficient colonocytes compared with controls (p<0.05). In study 2, there was no evidence of instability at the five microsatellite loci associated with either short or long term folate depletion. Conclusions—Folate deficiency impairs DNA excision repair in rat colonic mucosa; a similar degree of deficiency, even when administered in conjunction with a colonic carcinogen, did not produce evidence of a widespread defect in mismatch repair.

Effects of folate supplementation on two provisional molecular markers of colon cancer: a prospective, randomized trial.

OBJECTIVES:Dietary folate intake is inversely associated with the risk of colorectal cancer. This study investigated the effect of folate supplementation on genomic DNA methylation and DNA strand breaks in exons 5–8 of the p53 gene of the colonic mucosa, two provisional biomarkers of colon cancer.METHODS:Twenty subjects with adenomas were randomized to receive either folate (5 mg/day) or placebo for 1 yr after polypectomy. At baseline, 6 months and 1 yr, systemic and colonic measures of folate status were determined, as were the biomarkers mentioned earlier.RESULTS:Folate supplementation increased serum, red blood cell and colonic mucosal folate concentrations (p < 0.02). Folate supplementation also increased the extent of genomic DNA methylation at 6 months and 1 yr (p = 0.001), whereas placebo administration was associated with an increase in the extent of genomic DNA methylation only at 1 yr. Similarly, folate supplementation decreased the extent of p53 strand breaks in exons 5–8 at 6 months and 1 yr (p < 0.02), whereas placebo administration was associated with a decrease in the extent of p53 strand breaks only at 1 yr.CONCLUSIONS:Both of these provisional biomarkers of colon cancer underwent accelerated improvement at 6 months with folate supplementation. However, these markers also improved with placebo at 1 yr. Therefore, potential confounding factors that seem to modulate these biomarkers need to be identified and corrected in order for these markers to serve as suitable surrogate endpoints in folate chemoprevention trials.

Too much folate: a risk factor for cancer and cardiovascular disease?

Purpose of reviewThe intent of this evidence-based review is to analyze the role of folate in chronic diseases, focusing on cancer and cardiovascular disease. Recent findingsLow folate status has been shown to be a risk factor for cancer and cardiovascular disease. Although epidemiological data suggest an inverse association between folate status and disease risk, intervention studies give equivocal results, suggesting the response to folate intake does not follow a linear continuum. Moreover, recent folate intervention trials raise concern about possible adverse effects of folate supplementation and suggest that too much folate in inopportune settings may be potentially harmful in individuals at higher risk for cardiovascular disease and cancer. SummaryAlthough folate intake at sufficient levels appears to be an effective cancer chemopreventive strategy, high-dose supplementation of folate has generally not been effective in reducing recurrence of cardiovascular events or colorectal adenomas in clinical intervention trials. Although controversial, high folate status achieved through folate fortification or supplementation may increase the risk of certain chronic diseases among certain individuals, possibly by interfering with the homeostasis of one-carbon metabolism. Further research is urgently needed to accurately define the relationship between supraphysiological intake of folate and chronic diseases.

Folate supplementation increases genomic DNA methylation in the liver of elder rats

The availability of folate is implicated as a determinant of DNA methylation, a functionally important feature of DNA. Nevertheless, when this phenomenon has been examined in the rodent model, the effect has not always been observed. Several reasons have been postulated for the inconsistency between studies: the rodent is less dependent on folate as a methyl source than man; juvenile animals, which most studies use, are more resistant to folate depletion than old animals; methods to measure genomic DNA methylation might not be sensitive enough to detect differences. We therefore examined the relationship between folate and genomic DNA methylation in an elder rat model with a newly developed method that can measure genomic DNA methylation sensitively and precisely. Thirty-nine 1-year-old rats were divided into three groups and fed a diet containing 0, 4.5 or 18 mumol folate/kg (folate-deplete, -replete and -supplemented groups, respectively). Rats were killed at 8 and 20 weeks. At both time points, mean liver folate concentrations increased incrementally between the folate-deplete, -replete and -supplemented rats (P for trend <0.001) and by 20 weeks hepatic DNA methylation also increased incrementally between the folate-deplete, -replete and -supplemented rats (P for trend=0.025). At both time points folate-supplemented rats had significantly increased levels of DNA methylation compared with folate-deplete rats (P<0.05). There was a strong correlation between hepatic folate concentration and genomic DNA methylation in the liver (r 0.48, P=0.004). In the liver of this animal model, dietary folate over a wide range of intakes modulates genomic DNA methylation.

Folate, cancer risk, and the Greek god, Proteus: a tale of two chameleons

Evidence indicates that an abundant intake of foodstuffs rich in folate conveys protection against the development of colorectal cancer, and perhaps some other common cancers as well. The issue is complex, however, since some observations in animal and human studies demonstrate that an overly abundant intake of folate among those who harbor existing foci of neoplasia might instead produce a paradoxical promotion of tumorigenesis. The pharmaceutical form of the vitamin, folic acid, might affect the process in a manner that is distinct from natural forms of the vitamin, although this remains a speculative concept. Our limited understanding of this complex relationship is impeding efforts to move ahead with widespread folic acid fortification, but this delay may be necessary to ensure that such programs are instituted in a safe manner.

Diet-induced obesity elevates colonic TNF-α in mice and is accompanied by an activation of Wnt signaling: a mechanism for obesity-associated colorectal cancer

Inflammation associated with obesity may play a role in colorectal carcinogenesis, but the underlying mechanism remains unclear. This study investigated whether the Wnt pathway, an intracellular signaling cascade that plays a critical role in colorectal carcinogenesis, is activated by obesity-induced elevation of the inflammatory cytokine tumor necrosis factor-alpha (TNF-α). Animal studies were conducted on C57BL/6 mice, and obesity was induced by utilizing a high-fat diet (60% kcal). An inflammation-specific microarray was performed, and results were confirmed with real-time polymerase chain reaction. The array revealed that diet-induced obesity increased the expression of TNF-α in the colon by 72% (P=.004) and that of interleukin-18 by 41% (P=.023). The concentration of colonic TNF-α protein, determined by ex vivo culture assay, was nearly doubled in the obese animals (P=.002). The phosphorylation of glycogen synthase kinase 3 beta (GSK3β), an important intermediary inhibitor of Wnt signaling and a potential target of TNF-α, was quantitated by immunohistochemistry. The inactivated (phosphorylated) form of GSK3β was elevated in the colonic mucosa of obese mice (P<.02). Moreover, β-catenin, the key effector of canonical Wnt signaling, was elevated in the colons of obese mice (P<.05), as was the expression of a downstream target gene, c-myc (P<.05). These data demonstrate that diet-induced obesity produces an elevation in colonic TNF-α and instigates a number of alterations of key components within the Wnt signaling pathway that are protransformational in nature. Thus, these observations offer evidence for a biologically plausible avenue, the Wnt pathway, by which obesity increases the risk of colorectal cancer.