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-methyl-THF), 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 chromatography͞MS 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 (T͞T) and 187 homozygous for the wild-type (C͞C) MTHFR genotype. The results show that genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels (P < 0.01). T͞T genotypes had a diminished level of DNA methylation compared with those with the C͞C wild-type (32.23 vs.62.24 ng 5-methylcytosine͞g DNA, P < 0.0001). When analyzed according to folate status, however, only the T͞T subjects with low levels of folate accounted for the diminished DNA methylation (P < 0.0001). Moreover, in T͞T 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.

Collectively, the evidence from epidemiologic, animal and human studies strongly suggests that folate status modulates the risk of developing cancers in selected tissues, the most notable of which is the colorectum. Folate depletion appears to enhance carcinogenesis whereas folate supplementation above what is presently considered to be the basal requirement appears to convey a protective effect. The means by which this modulation of cancer risk is mediated is not known with certainty, but there are several plausible mechanisms which have been described. Folate plays a major role in the formation of S-adenosylmethionine, the universal methyl donor, as well as in the formation of purine and thymidine synthesis for DNA and RNA. Therefore, most mechanistic studies performed to date have focused on alterations in DNA methylation, disruption of DNA integrity and disruption of DNA repair, all of which have been observed with folate depletion. These aberrations in DNA are believed to enhance carcinogenesis by altering the expression of critical tumor suppressor genes and proto-oncogenes. Recently, the role of a common polymorphism of the methylenetetrahydrofolate reductase gene has been highlighted as well. This review presents those mechanisms which are the most likely candidates to explain folate's effects and it proposes an integrated scheme to explain how these mechanisms might interact.

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.

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.

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 cell's unique gene expression patterns that are determined during cellular differentiation. Interestingly, these cell-specific gene expression patterns can be affected by an organism's 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 organism's 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.

Eukaryotic DNA is methylated at some cytosine residues, and this epigenetic feature performs critical functions. We developed a method for quantitative determination of 5-methyl-2'-deoxycytidine in human DNA using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The DNA was enzymatically hydrolyzed by sequential digestion with three enzymes. DNA hydrolyzates were subsequently separated by reversed-phase high-performance liquid chromatography in isocratic mode. The four major DNA bases and 5-methyl-2'-deoxycytidine were resolved and eluted in 13 min. Identification of 2'-deoxycytidine and 5-methyl-2'-deoxycytidine was obtained by combined diode array UV spectra analysis and mass spectra of chromatographic peaks. The isotopomers [15N3]-2'-deoxycytidine and (methyl-d3,ring-6-d1)-5-methyl-2'-deoxycytidine were used as internal standards. Ions of m/z 126 and 130 were used to detect 5-methyl-2'-deoxycytidine and its isotopomer, and ions of m/z 112 and 115 were used to detect 2'-deoxycytidine and its stable isotopomer, respectively. The DNA methylation status was calculated on the basis of the amount of 5-methyl-2'-deoxycytidine per microgram of DNA with percent relative standard deviations (%RSD) for a method precision of 7.1 (within-day) and 5.7 (day-to-day). This method also allows the measurement of 5-methyl-2'-deoxycytidine expressed as a percentage of total deoxycytidine residues in genomic DNA with %RSD for method precision of 1.9 (within-day) and 1.7 (day-to-day). This LC/MS method for quantitative determination of genomic DNA methylation status is rapid, sensitive, selective, and precise.

(Gut 1996; 39: 732-740)

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.