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The Effects of Vitamin B-6 Status on Homocysteine, Oxidative Stress, One-carbon Metabolism and Methylation: Cross-section, Case-control, Intervention and Follow-up Studies in Colorectal Cancer

ABSTRACT: Vitamin B-6, folate and homocysteine may play a critical role in the colorectal cancer progression. This is a 4-y study and the specific aims are: 1) to compare vitamin B-6, folate and homocysteine status, oxidative stress, antioxidant activities and DNA methylation between subjects with colorectal polyps and colorectal cancer; 2) to study the effects of vitamin B-6, folate and homocysteine status on oxidative stress, antioxidant activities and DNA methylation in colorectal cancer patients; 3) to evaluate whether folic acid and/or pyridoxine supplementation had a beneficial effect on oxidative stress, antioxidant activities and DNA methylation in patients with colorectal; 4) to compare vitamin B-6, folate and homocysteine status, oxidative stress, antioxidant activities and DNA methylation between colorectal cancer patients and age-, sex-matched healthy subjects; 5) to evaluate the effect of vitamin B-6, folate and homocysteine status, oxidative stress, antioxidant activities and DNA methylation on the risk of colorectal cancer (odds ratio); 6) to follow the effects of vitamin B-6, folate and homocysteine status, oxidative stress, antioxidant activities and DNA methylation on the occurrence of colorectal cancer; 7) to follow the effects of pyridoxine and/or folic acid supplementation on the occurrence of colorectal cancer. This protocol is designed as a hospital-based cross-sectional, case-control, double blinded randomized placebo-controlled intervention and follow-up trial. Three hundred colorectal cancer patients and 300 age-, sex-matched controls who meet the inclusion criteria will be recruited from Taichung General Veterans Hospital. If cancer patients’ plasma pyridoxal 5’-phosphate (PLP) level less than 30 nmol/L will be asked to participate the intervention study and will be blinded and randomly assigned to either the 1) control (vitamin C, 100 mg/d); 2) 50 mg vitamin B-6; 3) 400 microgram/d folic acid; or 4) vitamin B-6 (50 mg/d) plus folic acid (400 microgram/d) for 1 year. Data on demography, anthropometry, medical history, food frequency questionnaire and 24-h diet recall will be collected. Cancer patients will have fasting blood drawn before surgery or receiving chemotherapy. Additionally, fasting blood samples will be obtained at month 0, 3, 6, 9 and 12 during intervention period and at month 6, 12, 18, 24, 30, 36, 42 and 48 during follow-up. Hematological, plasma and erythrocyte PLP, plasma pyridoxal and 4-pyridoxic acid, serum and erythrocyte folate, serum vitamin B-12, homocysteine, SAM, SAH, lipid peroxidation indicators (TBARS, oxidized LDL), glutathione, antioxidant enzymatic activities and DNA methylation and MTHFR 677C>T polymorphism will be measured.

DISEASE(S): Colorectal Cancer,Colorectal Neoplasms

PROVIDER: 2112226 | ecrin-mdr-crc |

REPOSITORIES: ECRIN MDR

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Project description:Folate, and its synthetic form folic acid, function as donor of one-carbon units and have been, together with other B-vitamins, implicated in programming of epigenetic processes such as DNA methylation during early development. To what extent regulation of DNA methylation can be altered via B-vitamins later in life, and how this relates to health and disease, is not exactly known. The aim of this study was to identify effects of long-term supplementation with folic acid and vitamin B12 on genome-wide DNA methylation in elderly subjects. This project was part of a randomized, placebo-controlled trial on effects of supplemental intake of folic acid and vitamin B12 on bone fracture incidence (B-PROOF study). Participants with mildly elevated homocysteine levels, aged 65-75 years, were randomly assigned to take 400 µg folic acid and 500 µg vitamin B12 per day or a placebo during an intervention period of two years. DNA was isolated from buffy coats, collected before and after intervention, and genome-wide DNA methylation was determined in 87 participants (n=44 folic acid/vitamin B12, n=43 placebo) using the Infinium HumanMethylation450 BeadChip. After intervention with folic acid and vitamin B12, 162 (versus 14 in the placebo group) of the 431,312 positions were differentially methylated as compared to baseline. Comparisons of the DNA methylation changes in the participants receiving folic acid and vitamin B12 versus placebo, revealed one single differentially methylated position (cg19380919) with a borderline statistical significance. However, based on the analyses of differentially methylated regions (DMRs) consisting of multiple positions, we identified 6 regions that differed statistically significantly between the intervention and placebo group. Pronounced changes were found for regions in the DIRAS3, ARMC8 and NODAL genes, implicated in carcinogenesis and early embryonic development. Furthermore, serum levels of folate and vitamin B12 or plasma homocysteine were related to DNA methylation of 173, 425 and 11 regions, respectively. Interestingly, for several members of the developmental HOX genes, DNA methylation was related to serum levels of folate. Long-term supplementation with folic acid and vitamin B12 in elderly subjects resulted in effects on DNA methylation of several genes, among which genes implicated in developmental processes.

Project description:Defects in homocysteine and folate metabolism are associated with increased risks for neural tube and congenital heart defects, cardiovascular disease and stroke, cancers, and neurodegeneration. In many but not all cases, dietary supplementation with folate significantly reduces the severity and incidence of these conditions. Common polymorphisms modulate these metabolic pathways and disease risks, but do not fully account for the particular birth defects and adult diseases that occur in at-risk individuals. To test whether other pathways contribute to disease pathogenesis, we analyzed global and pathway-specific changes in gene expression and levels of selected metabolites after depletion and repletion of dietary folate in two genetically distinct inbred strains of mice. Compared to the C57BL/6J strain, A/J showed greater homeostatic response to folate perturbation by retaining a higher serum folate level and minimizing global gene expression changes. Remarkably, folate perturbation led to systematic strain-specific differences only in the expression profile of the cholesterol biosynthesis pathway and translated to changes in levels of serum and liver total cholesterol. By genetically increasing serum and liver total cholesterol levels in APOE deficient mice, we modestly but significantly improved folate retention during folate depletion, suggesting an interplay between homocysteine and folate metabolism and cholesterol metabolism. Absence of measurable changes in global methylation patterns or amelioration of effects with supplementation with an alternative methyl donor suggest that dietary folate perturbations do not act through large-scale or general changes in methylation. These results suggest that homeostatic responses in cholesterol metabolism contribute to the beneficial effects of dietary folate supplementation. Keywords: time course, stress response, diet, genetic, homeostasis Six-week old female A/J and C57BL/6J mice were purchased from the Jackson Laboratory. All mice were raised on a control diet containing four ppm folic acid (Basal Diet 5755, TestDiet) for one week before the start of studies. Selected mice were then placed on folic acid deficient diet (58C3, TestDiet) containing 1% succinylsulfathiazole, a non-absorbable antibiotic commonly used to suppress folate production by bacteria in the intestine. We had nine different treatment plans per strain with eight replicate mice per treatment. There were four folic acid depletion treatment in which mice were placed on folic acid deficient diet for 1, 2, 7, or 14 days. There were two folic acid repletion treatment in which mice were placed on folic acid deficient diet for 14 days followed by 1 day on control diet and another set of mice on 14 days of folic acid deficient diet followed by 7 days of control diet. There were three control time points in which mice were placed on the control diet for 0, 9, or 22 days. Eight biological replicate liver tissue from each treatment was pooled and total RNA from each pool and total RNA from Universal Mouse Reference RNA (Stratagene) were aminoallyl labeled with Cy3 and Cy5 in duplicate, with reversing of dyes.

Project description:Background & Aims: Previous studies have suggested that dietary folic acid (FA) can protect against certain types of cancers. However, the findings have varied and the mechanisms by which this vitamin exerts chemopreventive effects remain to be clarified. We examined the effects of FA supplementation on DNA methylation, gene expression and gastric dysplasia in a transgenic mouse model that is etiologically and histologically well matched with human gastric cancers. Methods: Hypergastrinemic mice (INS-GAS) infected with Helicobacter felis were studied at multiple stages of gastric dysplasia and early cancer, with FA supplementation initiated both at weaning and later in life. Global DNA methylation was assessed by a methylation-sensitive cytosine incorporation assay, bisulfite pyrosequencing of B1 repetitive elements and immunohistochemistry (IHC) with anti-5-methylcytosine. We also profiled gene expression in the same tissues. Results: We found a decrease in global DNA methylation and tissue folate and an increase in serum homocysteine with progression of gastric dysplasia. FA supplementation prevented this loss of global DNA methylation and markedly reduced gastric dysplasia and mucosal inflammation. FA protected against the loss of global DNA methylation both in the dysplastic gastric epithelial cells and in gastric stromal myofibroblasts. In addition, FA supplementation had an anti-inflammatory effect, as indicated by expression profiling and IHC for lymphocyte markers. Conclusions: We conclude that FA supplementation is chemopreventive in this model of Helicobacter-associated gastric cancer. The beneficial effect of FA is likely due to its ability to reverse global loss of methylation and suppress inflammation. Study the difference in gene expression between transgenic hypergastrinemic (INS-GAS) mice fed a folate-supplemented diet and a diet with normal folate content. Because folate had a substantial protective effect, we matched the animals not only by folate status but also by dysplasia score (DYS). The degree of dysplasia was scored on a 1-4 scale. True replicates are not included. The first five samples are from folate supplemented animals, and the latter five are from controls on regular chow.

Project description:Study question: Do short-term and long-term exposures to low dose folic acid supplementation alter DNA methylation in sperm? Summary answer: No alterations in sperm DNA methylation patterns were found following the administration of low dose folic acid supplements of 400 μg/day for 90 days (short-term exposure) or when pre-fortification of food with folic acid and post-fortification sperm samples (long-term exposure) were compared. What is known already: Excess dietary folate may be detrimental to health and DNA methylation profiles due to folate’s role in one carbon metabolism and the formation of S-adenosyl methionine, the universal methyl donor. DNA methylation patterns are established in developing male germ cells and have been suggested to be affected by high dose (5 mg/day) folic acid supplementation. Participants/materials, setting, methods: Blood and seminal plasma folate levels were measured in participants before and following the 90-day treatment with placebo or supplement. Sperm DNA methylation was assessed using the whole genome and genome-wide techniques, MassArray epityper, restriction landmark genomic scanning, methyl-CpG immunoprecipitation and Illumina HumanMethylation450 Bead Array. Main results and the role of chance: Following treatment, supplemented individuals had significantly higher levels of blood and seminal plasma folates compared to placebo. Initial first generation genome-wide analyses of sperm DNA methylation showed little evidence of changes when comparing pre- and post-treatment samples. With Illumina HumanMethylation450 BeadChip arrays, no significant changes were observed in individual probes following low-level supplementation; when compared with those of the post-fortification cohort, there were also few differences in methylation despite exposure to years of fortified foods. Limitations, reasons for caution: This study was limited to the number of participants available in each cohort, in particular those who were not exposed to early (pre-1998) fortification of food with folic acid. While genome-wide DNA methylation was assessed with several techniques that targeted genic and CpG rich regions, intergenic regions were less well interrogated. Wider implications of the findings: Overall, our findings provide evidence that short term exposure to low dose folic acid supplements of 400 μg/day, over a period of 3 months, a duration of time that might occur during infertility treatments, has no major impact on the sperm DNA methylome.

Dataset Information

The Effects of Vitamin B-6 Status on Homocysteine, Oxidative Stress, One-carbon Metabolism and Methylation: Cross-section, Case-control, Intervention and Follow-up Studies in Colorectal Cancer

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