Sang-Woon Choi

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.

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.

Epigenetics: A New Bridge between Nutrition and Health.

Nutrients can reverse or change epigenetic phenomena such as DNA methylation and histone modifications, thereby modifying the expression of critical genes associated with physiologic and pathologic processes, including embryonic development, aging, and carcinogenesis. It appears that nutrients and bioactive food components can influence epigenetic phenomena either by directly inhibiting enzymes that catalyze DNA methylation or histone modifications, or by altering the availability of substrates necessary for those enzymatic reactions. In this regard, nutritional epigenetics has been viewed as an attractive tool to prevent pediatric developmental diseases and cancer as well as to delay aging-associated processes. In recent years, epigenetics has become an emerging issue in a broad range of diseases such as type 2 diabetes mellitus, obesity, inflammation, and neurocognitive disorders. Although the possibility of developing a treatment or discovering preventative measures of these diseases is exciting, current knowledge in nutritional epigenetics is limited, and further studies are needed to expand the available resources and better understand the use of nutrients or bioactive food components for maintaining our health and preventing diseases through modifiable epigenetic mechanisms.

Gene-Nutrient Interactions and DNA Methylation

Many micronutrients and vitamins are critical for DNA synthesis/repair and maintenance of DNA methylation patterns. Folate has been most extensively investigated in this regard because of its unique function as methyl donor for nucleotide synthesis and biological methylation. Cell culture and animal and human studies showed that deficiency of folate induces disruption of DNA as well as alterations in DNA methylation status. Animal models of methyl deficiency demonstrated an even stronger cause-and-effect relationship than did studies using a folate-deficient diet alone. Such observations imply that the adverse effects of inadequate folate status on DNA metabolism are mostly due to the impairment of methyl supply. Recently, an interaction was observed between folate status and a common mutation in the gene encoding for methylenetetrahydrofolate reductase, an essential enzyme in one-carbon metabolism, in determining genomic DNA methylation. This finding suggests that the interaction between a nutritional status with a genetic polymorphism can modulate gene expression through DNA methylation, especially when such polymorphism limits the methyl supply. DNA methylation, both genome-wide and gene-specific, is of particular interest for the study of cancer, aging and other conditions related to cell-cycle regulation and tissue-specific differentiation, because it affects gene expression without permanent alterations in DNA sequence such as mutations or allele deletions. Understanding the patterns of DNA methylation through the interaction with nutrients is fundamental, not only to provide pathophysiological explanations for the development of certain diseases, but also to improve the knowledge of possible prevention strategies by modifying a nutritional status in at-risk populations.

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.

Epigenetics: The link between nature and nurture

While the eukaryotic genome is the same throughout all somatic cells in an organism, there are specific structures and functions that discern one type of cell from another. These differences are due to the cells unique gene expression patterns that are determined during cellular differentiation. Interestingly, these cell-specific gene expression patterns can be affected by an organisms environment throughout its lifetime leading to phenotypical changes that have the potential of altering risk of some diseases. Both cell-specific gene expression signatures and environment mediated changes in expression patterns can be explained by a complex network of modifications to the DNA, histone proteins and degree of DNA packaging called epigenetic marks. Several areas of research have formed to study these epigenetic modifications, including DNA methylation, histone modifications, chromatin remodeling and microRNA (miRNA). The original definition of epigenetics incorporates inheritable but reversible phenomena that affect gene expression without altering base pairs. Even though not all of the above listed epigenetic traits have demonstrated heritability, they can all alter gene transcription without modification to the underlying genetic sequence. Because these epigenetic patterns can also be affected by an organisms environment, they serve as an important bridge between life experiences and phenotypes. Epigenetic patterns may change throughout ones lifespan, by an early life experience, environmental exposure or nutritional status. Epigenetic signatures influenced by the environment may determine our appearance, behavior, stress response, disease susceptibility, and even longevity. The interaction between types of epigenetic modifications in response to environmental factors and how environmental cues affect epigenetic patterns will further elucidate how gene transcription can be affectively altered.

Epigenetic control of 11 beta-hydroxysteroid dehydrogenase 2 gene promoter is related to human hypertension.

BACKGROUND Lower activity of 11 beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2) classically induces hypertension by leading to an altered tetrahydrocortisol- versus tetrahydrocortisone-metabolites (THFs/THE) shuttle. Recent cell culture and animal studies suggest a role for promoter methylation, a major epigenetic feature of DNA, in regulation of HSD11B2 expression. Little is known, however, of human HSD11B2 epigenetic control and its relationship with the onset of hypertension. OBJECTIVE To explore the possible relevance of HSD11B2 promoter methylation, by examining human peripheral blood mononuclear cell (PBMC) DNA and urinary THFs/THE ratio as a biochemical indicator of 11beta-HSD2 activity, in blood pressure control. METHODS Twenty-five essential hypertensives and 32 subjects on prednisone therapy were analyzed, the latter to investigate 11beta-HSD2 function in the development of hypertension. RESULTS Elevated HSD11B2 promoter methylation was associated with hypertension developing in glucocorticoid-treated patients in parallel with a higher urinary THFs/THE ratio. Essential hypertensives with elevated urinary THFs/THE ratio also showed higher HSD11B2 promoter methylation. CONCLUSIONS These results show a clear link between the epigenetic regulation through repression of HSD11B2 in PBMC DNA and hypertension.

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.

DNA methylation, an epigenetic mechanism connecting folate to healthy embryonic development and aging

Experimental studies demonstrated that maternal exposure to certain environmental and dietary factors during early embryonic development can influence the phenotype of offspring as well as the risk of disease development at the later life. DNA methylation, an epigenetic phenomenon, has been suggested as a mechanism by which maternal nutrients affect the phenotype of their offspring in both honeybee and agouti mouse models. Phenotypic changes through DNA methylation can be linked to folate metabolism by the knowledge that folate, a coenzyme of one-carbon metabolism, is directly involved in methyl group transfer for DNA methylation. During the fetal period, organ-specific DNA methylation patterns are established through epigenetic reprogramming. However, established DNA methylation patterns are not immutable and can be modified during our lifetime by the environment. Aberrant changes in DNA methylation with diet may lead to the development of age-associated diseases including cancer. It is also known that the aging process by itself is accompanied by alterations in DNA methylation. Diminished activity of DNA methyltransferases (Dnmts) can be a potential mechanism for the decreased genomic DNA methylation during aging, along with reduced folate intake and altered folate metabolism. Progressive hypermethylation in promoter regions of certain genes is observed throughout aging, and repression of tumor suppressors induced by this epigenetic mechanism appears to be associated with cancer development. In this review, we address the effect of folate on early development and aging through an epigenetic mechanism, DNA methylation.

Gene-Nutrient Interactions in One-Carbon Metabolism

Advances in molecular biology greatly contributed, in the past decades, to a deeper understanding of the role of gene function in disease development. Environmental as well as nutritional factors are now well acknowledged to interact with the individual genetic background for the development of several diseases, including cancer, cardiovascular disease, and neurodegenerative diseases. The precise mechanisms of such gene-nutrient interactions, however, are not fully elucidated yet. Many micronutrients and vitamins are crucial in regulating mechanisms of DNA metabolism. Indeed, folate has been most extensively investigated for its unique function as mediator for the transfer of one-carbon moieties for nucleotide synthesis/repair and biological methylation. Cell culture, animal, and human studies, clearly demonstrated that folate deficiency induces disruption of DNA synthesis/repair pathways as well as DNA methylation anomalies. Remarkably, a gene-nutrient interaction between folate status and a polymorphism in methylenetetrahydrofolate reductase gene has been reported to modulate genomic DNA methylation. This observation suggests that the interaction between a nutritional status and a mutant genotype may modulate gene expression through DNA methylation, especially when such polymorphism affects a key enzyme in one-carbon metabolism and limits the methyl supply. DNA methylation, both genome-wide and gene-specific, is of particular interest for the study of aging, cancer, and other pathologic conditions, because it affects gene expression without permanent alterations in the DNA sequence such as mutations or allele deletions. Understanding the patterns of DNA methylation through the interaction with nutrients is a critical issue, not only to provide pathophysiological explanations of a disease state, but also to identify individuals at-risk to conduct targeted diet-based interventions.