[Show abstract][Hide abstract] ABSTRACT: High folate intake may increase the risk of cancer, especially in the elderly. The present study examined the effects of ageing and dietary folate on uracil misincorporation into DNA, which has a mutagenic effect, in the mouse colon and liver. Old (18 months; n 42) and young (4 months; n 42) male C57BL/6 mice were pair-fed with four different amino acid-defined diets for 20 weeks: folate deplete (0 mg/kg diet); folate replete (2 mg/kg diet); folate supplemented (8 mg/kg diet); folate deplete (0 mg/kg diet) with thymidine supplementation (1·8 g/kg diet). Thymidylate synthesis from uracil requires folate, but synthesis from thymidine is folate independent. Liver folate concentrations were determined by the Lactobacillus casei assay. Uracil misincorporation into DNA was measured by a GC/MS method. Liver folate concentrations demonstrated a stepwise increase across the spectrum of dietary folate levels in both old (P = 0·003) and young (P < 0·001) mice. Uracil content in colonic DNA was paradoxically increased in parallel with increasing dietary folate among the young mice (P trend = 0·033), but differences were not observed in the old mice. The mean values of uracil in liver DNA, in contrast, decreased with increasing dietary folate among the old mice, but it did not reach a statistically significant level (P < 0·1). Compared with the folate-deplete group, thymidine supplementation reduced uracil misincorporation into the liver DNA of aged mice (P = 0·026). The present study suggests that the effects of folate and thymidine supplementation on uracil misincorporation into DNA differ depending on age and tissue. Further studies are needed to clarify the significance of increased uracil misincorporation into colonic DNA of folate-supplemented young mice.
The British journal of nutrition 03/2011; 105(5):688-93. DOI:10.1017/S0007114510004332 · 3.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Older age, dietary folate and chronic alcohol consumption are important risk factors for the development of colon cancer. The present study examined the effects of ageing, folate and alcohol on genomic and p16-specific DNA methylation, and p16 expression in the murine colon. Old (aged 18 months; n 70) and young (aged 4 months; n 70) male C57BL/6 mice were pair-fed either a Lieber-DeCarli liquid diet with alcohol (18 % of energy), a Lieber-DeCarli diet with alcohol (18 %) and reduced folate (0.25 mg folate/l) or an isoenergetic control diet (0.5 mg folate/l) for 5 or 10 weeks. Genomic DNA methylation, p16 promoter methylation and p16 gene expression were analysed by liquid chromatography-MS, methylation-specific PCR and real-time RT-PCR, respectively. Genomic DNA methylation was lower in the colon of old mice compared with young mice (P < 0.02) at 10 weeks. Alcohol consumption did not alter genomic DNA methylation in the old mouse colon, whereas it tended to decrease genomic DNA methylation in young mice (P = 0.08). p16 Promoter methylation and expression were higher in the old mouse colon compared with the corresponding young groups. There was a positive correlation between p16 promoter methylation and p16 expression in the old mouse colon (P < 0.02). In young mice the combination of alcohol and reduced dietary folate led to significantly decreased p16 expression compared with the control group (P < 0.02). In conclusion, ageing and chronic alcohol consumption alter genomic DNA methylation, p16 promoter methylation and p16 gene expression in the mouse colon, and dietary folate availability can further modify the relationship with alcohol in the young mouse.
The British journal of nutrition 03/2010; 104(1):24-30. DOI:10.1017/S0007114510000322 · 3.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with a decreased risk of colon cancer although it may increase the risk of breast cancer. This polymorphism is associated with changes in intracellular folate cofactors, which may affect DNA methylation and synthesis via altered one-carbon transfer reactions. We investigated the effect of this mutation on DNA methylation and uracil misincorporation and its interaction with exogenous folate in further modulating these biomarkers of one-carbon transfer reactions in an in vitro model of the MTHFR 677T mutation in HCT116 colon and MDA-MB-435 breast adenocarcinoma cells. In HCT116 cells, the MTHFR 677T mutation was associated with significantly increased genomic DNA methylation when folate supply was adequate or high; however, in the setting of folate insufficiency, this mutation was associated with significantly decreased genomic DNA methylation. In contrast, in MDA-MB-435 cells, the MTHFR 677T mutation was associated with significantly decreased genomic DNA methylation when folate supply was adequate or high and with no effect when folate supply was low. The MTHFR 677T mutation was associated with a nonsignificant trend toward decreased and increased uracil misincorporation in HCT116 and MDA-MB-435 cells, respectively. Our data demonstrate for the first time a functional consequence of changes in intracellular folate cofactors resulting from the MTHFR 677T mutation in cells derived from the target organs of interest, thus providing a plausible cellular mechanism that may partly explain the site-specific modification of colon and breast cancer risks associated with the MTHFR C677T mutation.
International Journal of Cancer 05/2009; 124(9):1999-2005. DOI:10.1002/ijc.24003 · 5.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Folate deficiency may affect gene expression by disrupting DNA methylation patterns or by inducing base substitution, DNA breaks, gene deletions and gene amplification. Changes in expression may explain the inverse relationship observed between folate status and risk of colorectal cancer. Three cell lines derived from the normal human colon, HCEC, NCM356 and NCM460, were grown for 32-34 days in media containing 25, 50, 75 or 150 nM folic acid, and the expression of genes involved in cell-cycle checkpoints, intracellular signaling, folate uptake and cell adhesion and migration was determined. Expression of Folate Receptor 1 was increased with decreasing media folate in all cell lines, as was p53, p21, p16 and beta-catenin. With decreasing folate, the expression of both E-cadherin and SMAD-4 was decreased in NCM356. APC was elevated in NCM356 but unchanged in the other lines. No changes in global methylation were detected. A significant increase in p53 exon 7-8 strand breaks was observed with decreasing folate in NCM460 cells. The changes observed are consistent with DNA damage-induced activation of cell-cycle checkpoints and cellular adaptation to folate depletion. Folate-depletion-induced changes in the Wnt/APC pathway as well as in genes involved in cell adhesion, migration and invasion may underlie observed relationships between folate status and cancer risk.
The Journal of Nutritional Biochemistry 06/2008; 19(5):328-35. DOI:10.1016/j.jnutbio.2007.05.003 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Preclinical and clinical studies suggest that diminished folate status increases the risk of colorectal carcinogenesis. However, many biochemical functions of folate are dependent on the adequate availability of other 1-carbon nutrients, including riboflavin, vitamin B-6, and vitamin B-12. Aberrations in the Wnt pathway are thought to play an important role in human colorectal cancers. This study therefore investigated if mild depletion of folate combined with depletion of riboflavin, vitamin B-6, and vitamin B-12 could induce alterations in the Wnt pathway in the colonic mucosa. Ninety-six mice were pair-fed diets with different combinations of B vitamin depletion for 10 wk. Genomic DNA methylation and uracil misincorporation were measured by LC/MS and GC/MS. Gene-specific methylation, strand breaks, and expressions were measured by real-time PCR and immunoblotting. Proliferation and apoptosis were determined by immunohistochemistry. DNA strand breaks within the Apc mutation cluster region were induced by folate depletion combined with inadequacies of riboflavin, vitamin B-6, and vitamin B-12 (P < 0.05), but such effects were not induced by folate depletion alone. Similarly, minor changes in the expression of Apc, beta-catenin, and cyclin D1 produced by mild folate depletion were significantly magnified by multiple vitamin depletion. Apoptosis, which can be suppressed by increased Wnt-signaling, was attenuated by the combined deficiency state (P < 0.05) but not by singlet or doublet deficiencies. These findings indicate that a mild depletion of folate that is of insufficient magnitude by itself to induce alterations in components of the Wnt pathway may produce such effects when present in conjunction with mild inadequacies of other 1-carbon nutrients.
Journal of Nutrition 12/2007; 137(12):2701-8. · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Older age and inadequate folate intake are strongly implicated as important risk factors for colon cancer and each is associated with altered DNA methylation. This study was designed to determine the effects of aging and dietary folate on select features of DNA methylation in the colon that are relevant to carcinogenesis. Old (18 mo; n = 34) and young (4 mo; n = 32) male C57BL/6 mice were randomly divided into 3 groups and fed diets containing 0, 4.5, or 18 mumol folate/kg (deplete, replete, and supplemented groups, respectively) for 20 wk. Genomic DNA methylation and p16 promoter methylation in the colonic mucosa were analyzed by liquid chromatography/electrospray ionization/MS and methylation-specific PCR, respectively. p16 gene expression was determined by real-time RT-PCR. Old mice had significantly lower genomic DNA methylation compared with young mice at each level of dietary folate (4.5 +/- 0.2, 4.8 +/- 0.1, and 4.9 +/- 0.1 vs. 6.0 +/- 0.1, 5.3 +/- 0.2, and 5.9 +/- 0.2%, in folate-deplete, -replete, and -supplemented groups, respectively, P < 0.05) and markedly higher p16 promoter methylation (61.0 +/- 2.7, 69.7 +/- 6.9, and 87.1 +/- 13.4 vs. 10.8 +/- 3.6, 8.4 +/- 1.8, and 4.9 +/- 1.7%, respectively, P < 0.05). In old mice, genomic and p16 promoter DNA methylation each increased in a manner that was directly related to dietary folate (P(trend) = 0.009). Age-related enhancement of p16 expression occurred in folate-replete (P = 0.001) and folate-supplemented groups (P = 0.041), but not in the folate-deplete group. In conclusion, aging decreases genomic DNA methylation and increases promoter methylation and expression of p16 in mouse colons. This effect is dependent on the level of dietary folate.
Journal of Nutrition 07/2007; 137(7):1713-7. · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although oestrogen replacement therapy (ERT), which can affect the risk of major cancers, has been known to reduce total plasma homocysteine concentrations in postmenopausal women, the mechanisms and subsequent molecular changes have not yet been defined. To investigate the effect of ERT on homocysteine metabolism, thirteen healthy postmenopausal women were enrolled in a double-blind, placebo-controlled, randomized, cross-over study consisting of two 8-week long phases, placebo and conjugated equine oestrogen (CEE; 0.625 mg/d). Concentrations of total plasma homocysteine, vitamin B6 and serum folate and vitamin B12 were measured by conventional methods. Genomic DNA methylation was measured by a new liquid chromatography/MS method and promoter methylation status of the oestrogen receptor (ER)alpha, ERbeta and p16 genes was analysed by methylation-specific PCR after bisulfite treatment. The CEE phase demonstrated a significantly decreased mean of total plasma homocysteine concentrations compared with the placebo phase (8.08 micromol/l (6.82-9.39) v. 9.29 (7.53-11.35), P < 0.05) but there was no difference in the blood concentrations of the three B vitamins. The CEE phase also showed a significantly increased genomic DNA methylation in peripheral mononuclear cells compared with the placebo phase (2.85 (SD 0.12) ng methylcytosine/microg DNA v. 2.40 +/- (SD 0.15) P < 0.05). However, there was no difference in promoter methylation in the ERalpha, ERbeta and p16 genes. This study demonstrates that decreased homocysteinaemia by CEE therapy parallels with increased genomic DNA methylation, suggesting a potential new candidate mechanism by which ERT affects the risk of cancers and a possible new candidate biomarker for the oestrogen-related carcinogenesis through folate-related one-carbon metabolism.
British Journal Of Nutrition 04/2007; 97(4):617-21. DOI:10.1017/S0007114507433013 · 3.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Smoking causes genetic damage in buccal cells and increases the risk of oral cancer. Because folate is instrumental in DNA synthesis and repair, it is a determinant of genetic stability and therefore might attenuate the genotoxic effects of smoking.
Our aim was to compare the presence of folate metabolites and select indicators of genetic damage in the mouths of chronic smokers and nonsmokers.
Dietary, biochemical, and molecular correlates of folate status were measured in healthy smoker (n = 35) and nonsmoker (n = 21) groups of comparable age, sex, and body mass indexes.
After correction for dietary intake, the smokers displayed lower plasma, erythrocyte, and buccal mucosal cell (BMC) folate (20%, 32%, and 50% lower, respectively; P < 0.05) and lower plasma vitamin B-12 and pyridoxal 5-phosphate (P < 0.05) than did nonsmokers. Folate in the BMCs of smokers comprised significantly greater proportions of pteroylmonoglutamate, formyltetrahydrofolate, and 5,10-methenyltetrahyrofolate than did folate in the BMCs of nonsmokers. Although the degree of genomic methylation and uracil incorporation in the buccal cells of the 2 groups were not significantly different, the BMC micronucleus index, a cytologic indicator of genetic damage, in the smokers was 2-fold that of the nonsmokers (9.57 compared with 4.44 micronuclei/1000 cells; P < 0.0001). Neither systemic nor oral folate status was an independent predictor of micronuclei.
Chronic smoking is associated with a lower systemic status of several B vitamins, reduced oral folate, and changes in folate form distribution in the mouth. However, the cytologic damage that is evident in the mouths of smokers does not correlate with oral folate status.
American Journal of Clinical Nutrition 05/2006; 83(4):835-41. · 6.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Folate is among the most strongly implicated dietary components to convey protection against colon cancer, and diminished folate status is associated with an enhanced risk of colon cancer. Age is also regarded as one of the most important risk factors for colonic carcinogenesis. It is therefore of considerable interest to determine whether the process of aging influences folate metabolism in the colon and whether folate supplementation might prevent the procarcinogenic effects associated with aging. Recent studies in our laboratory demonstrated that the colonic mucosa of elder rats is more susceptible to folate depletion than that of young rats. Depletion of folate results in a shift in the forms of folate in the colon as well as increased uracil incorporation into DNA, a purported mechanism for colonic carcinogenesis. However, modest folate supplementation eliminates evidence of inadequate folate status in the colons of elder rats, suggesting that the relation between age and folate status in the colon might be one mechanism by which aging modulates colorectal cancer risk. Interactions between folate and aging also affect a spectrum of epigenetic and genetic phenomena such as uracil misincorporation, DNA methylation, protein methylation, mitochondrial deletion, and critical gene expression, which could be related to carcinogenesis. Aging and inadequate dietary folate may interact and collectively induce derangements in folate metabolism, thereby provoking subsequent molecular aberrations, which may enhance carcinogenesis. However, folate supplementation appears to reverse these adverse effects of aging, which is potentially of substantial import because the latter is an unmodifiable risk factor.
Journal of Nutrition 01/2006; 135(12 Suppl):2967S-2971S. · 4.23 Impact Factor