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© 2008 American Society for Nutrition J. Nutr. 138:703-709, April 2008

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Postweaning Dietary Folate Deficiency Provided through Childhood to Puberty Permanently Increases Genomic DNA Methylation in Adult Rat Liver1–3,

Joanne Kotsopoulos4, Kyoung-Jin Sohn5 and Young-In Kim4–6,*

4 Department of Nutritional Sciences and 5 Department of Medicine, University of Toronto, Toronto, Canada, M5S 1A8 and 6 Division of Gastroenterology, St. Michael's Hospital, Toronto, Canada, M5B 1W8

* To whom correspondence should be addressed. E-mail: youngin.kim{at}utoronto.ca.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Folate plays an important role in the pathogenesis of several chronic diseases by its potential ability to modulate DNA methylation. We hypothesized that the postweaning period might be a highly susceptible period to dietary folate intervention for DNA methylation patterning. We determined the effects of timing and duration of dietary folate intervention provided during the postweaning period on genomic DNA methylation in adult rat liver. In study 1, weanling rats were randomized to receive an amino acid-defined diet containing 0 (deficient), 2 (control), or 8 (supplemented) mg folic acid/kg until 8 wk of age, after which all the rats were fed the control diet until 30 wk of age. In study 2, weanling rats were fed the control diet until 8 wk of age and then randomized to receive the diet containing 0, 2, or 8 mg folic acid/kg until 30 wk of age. In study 3, weanling rats were randomized to receive these diets until 30 wk of age. Dietary folate deficiency, but not supplementation, provided during the postweaning period through childhood to puberty significantly increased genomic DNA methylation by 34–48% (P < 0.04) in rat liver that persisted into adulthood following a return to the control diet at puberty. In contrast, dietary folate deficiency or supplementation continually imposed at weaning or at puberty did not significantly affect genomic DNA methylation in adult rat liver. Our data suggest that early folate nutrition during postnatal development plays an important role in epigenetic programming that can have a permanent effect in adulthood.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
DNA methylation of cytosine in the cytosine-guanine (CpG)7 dinucleotide sequences is a heritable, tissue- and species-specific, postsynthetic epigenetic modification of mammalian DNA (1,2). DNA methylation is an important epigenetic determinant in gene expression (an inverse relation), maintenance of DNA integrity and stability, chromatin modifications, and development of mutations (1,2). As such, aberrant patterns and dysregulation of DNA methylation are mechanistically related to the development of several human diseases, including cancer (1,2). Seventy to 80% of all CpG sites in human DNA are normally methylated (1,2). However, this methylation occurs primarily in the bulk of the genome where CpG density is low, including exons, noncoding regions, and repeat DNA sites, and allows correct organization of chromatin in active and inactive states (3). By contrast, most CpG-rich areas clustered in small stretches of DNA termed CpG islands, which span the 5' end of approximately one-half of the human genes including the promoter, untranslated region, and exon 1, are unmethylated in normal cells, thereby allowing transcription (1,2). When methylated, CpG islands cause stable heritable transcriptional silencing (1,2). DNA methylation is a dynamic process between active methylation, mediated by CpG DNA methyltransferases (DNMT) using S-adenosylmethionine (SAM) as the methyl donor and removal of methyl groups from 5-methylcytosine residues by both passive and active mechanisms (4). After transfer of the methyl group, SAM is converted to S-adenosylhomocysteine (SAH), a potent inhibitor of most SAM-dependent methyltransferases (Fig. 1) (5).


Figure 1
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  		FIGURE 1  Simplified scheme of the role of folate in DNA methylation. B12, vitamin B-12; BADH, betaine aldehyde dehydrogenase; BHMT, betaine:homocysteine methyltransferase; CH3, methyl group; CHDH, choline dehydrogenase; DHF, dihydrofolate; DHFR, dihydrofolate reductase; MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; SHMT, serine hydroxymethyltransferase; THF, tetrahydrofolate.

 
Epigenetic changes are gradual in onset and are progressive and their effects are dose-dependent and potentially reversible (6). As such, they may be modulated by dietary and lifestyle factors and pharmacologic manipulations. For instance, diets deficient in different combinations of methyl group donors (choline, folate, methionine, and vitamin B-12), which are involved in mediating 1-carbon transfer for the provision of SAM, the primary methyl group donor for most biological methylation reactions including that of DNA (Fig. 1) (5), have been observed to induce genomic and site- and gene-specific DNA hypomethylation and upregulation of DNMT in rat liver in some, but not all, studies (7–17). Folate, a water-soluble B vitamin, has been the focus of intense interest because of its role in the pathogenesis of several chronic diseases, including cancer, and its potential ability to modulate DNA methylation (6,18). Folate, in the form of 5-methyltetrahydrofolate, is involved in remethylation of homocysteine to methionine, which is a precursor of SAM (Fig. 1) (5). However, dietary folate deficiency and supplementation have generally failed to induce significant changes in genomic DNA methylation in a predictable manner in animal models using rodents (6,18). These studies adopted a means of inducing folate depletion and repletion by continuously providing different amounts of folic acid (the fully oxidized monoglutamyl form of folate used in supplements and in fortified foods) in the diet at weaning for a variable length of intervention.

DNA methylation patterns are reprogrammed during embryogenesis by genome-wide demethylation early in embryogenesis, which erases large parts of the parental DNA methylation, followed by de novo methylation, which establishes a new DNA methylation pattern soon after implantation, with methylation mostly limited to non-CpG island areas, which is maintained postnatally (4,19). Therefore, during embryogenesis, epigenetics of developing fetus may be highly susceptible to environmental modifiers, including dietary factors (20). Indeed, studies using viable yellow agouti mice showed that maternal dietary methyl group supplementation containing folic acid permanently altered the phenotypic coat color of the offspring via increased CpG methylation in the promoter of the agouti gene (21,22). Similarly, a methyl group-rich diet has been shown to significantly reduce the proportion of progeny with a kinked tail in the AxinFused mice by one-half via increased CpG methylation in the promoter of the AxinFu gene (23). We posit that epigenetics in early infancy, childhood, and puberty might also be susceptible to effects of dietary folate intervention. In this study, we determined the effects of timing and duration of dietary folate intervention provided during the postweaning period on genomic DNA methylation in adult rat liver.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Rats and diets. This study was approved by the Animal Care Committee of the University of Toronto. Specific pathogen-free, weanling female Sprague-Dawley rats (~50 g; Charles River Laboratories) were singly housed and maintained at 24 ± 2°C and 50% humidity with a 12-h-light/-dark cycle. Amino acid-defined diets (Dyets) containing 0, 2, or 8 mg folic acid/kg diet were used. These diets constitute a standard method of inducing folate deficiency or providing supplemental dietary folate in rodents (24) and have been utilized extensively in previous studies of folate and DNA methylation (25–31). The diet containing 0 mg folic acid/kg produces progressive folate deficiency of a moderate degree through wk 3–5, after which systemic folate indicators stabilize (25–31). Although this diet is completely devoid of folate, severe folate deficiency is not induced because of de novo synthesis of folate by intestinal bacteria, some of which is incorporated into the tissue folate of the host (32) and animals survive over 30 wk without anemia, growth retardation, or premature death (28,29). The diet containing 2 mg folic acid/kg (control) is generally accepted as the basal dietary requirement for rodents (33). The diet containing 8 mg folic acid/kg represents folate supplementation 4 times the basal dietary requirement and may reflect the amount of the North American population taking supplemental folic acid in the postfortification era (34–37). The detailed composition of the diets has been published previously (24,30). These diets contained 50 g cellulose/kg, 60% of energy as carbohydrates, 23% as fat (or 10% by weight), and 17% as L-amino acids (24,30). The amount of methyl donors L-methionine, choline, and vitamin B-12 was 8.2 g/kg, 2.0 g/kg, and 50 µg/kg diet, respectively (24,30).

    Dietary intervention. In study 1, rats (n = 30) were randomized to receive the diet containing 0, 2, or 8 mg folic acid/kg diet (n = 10/group) from weaning at 3 wk of age for 5 wk, at which time (i.e. 8 wk of age) the initial diets were terminated and all the rats were placed on the control diet (2 mg folic acid/kg diet) for 22 wk until the time of killing at 30 w of age (Supplemental Fig. 1).

In study 2, rats (n = 30) were placed on the control diet from weaning at 3 wk of age for 5 wk, at which time the rats were randomized to receive the diet containing 0, 2, or 8 mg folic acid/kg diet (n = 10/group) for 22 wk until the time of killing at 30 wk of age (Supplemental Fig. 1).

In study 3, rats (n = 30) were placed on the diet containing 0, 2, or 8 mg folic acid/kg diet (n = 10/group) from weaning at 3 wk of age for 27 wk until the time of killing at 30 wk of age (Supplemental Fig. 1).

Rats consumed diets and water ad libitum. The daily food intake of each group was measured on a predetermined day of each week. Body weights were recorded weekly.

    Sample collection. Blood was withdrawn from the lateral tail vein of each rat at 8 wk of age and from the heart at necropsy and centrifuged at 800 x g; 10 min at 4°C. Serum was stored at –70°C in 0.5% ascorbic acid for serum folate assay. The rats were killed by carbon dioxide inhalation followed by cervical dislocation at 30 wk of age. The liver from each rat was harvested, snap-frozen in liquid nitrogen, and stored at –70°C for determination of hepatic folate concentration and for DNA extraction.

    Determination of folate concentration. Serum folate concentrations were determined by a standard microbiological microtiter plate assay using Lactobacillus casei (38). Hepatic folate concentrations were measured by the same microbiologic assay (38), utilizing a previously described method for the determination of tissue folates (39). Intra-assay CV for serum and hepatic folate concentrations were 3 and 5%, respectively. Inter-assay CV for serum and hepatic folate concentrations were both 5%.

    DNA extraction. DNA from the liver was extracted by a standard technique using proteinase K followed by organic extraction (40). The resulting DNA was precipitated with 1 mol/L NaCl and 100% ethanol and was treated with RNase. The size of DNA estimated by agarose-gel electrophoresis was >20 kb in all instances. No RNA contamination was detected on agarose-gel electrophoresis. The final preparations had a ratio of A260:A280 between 1.8 and 2.0. The concentration of each DNA sample was determined as the mean of 3 independent spectrophotometric readings.

    Genomic DNA methylation determination. The methylation status of CpG sites in genomic DNA from the liver was determined by the in vitro methyl acceptance capacity of DNA using [3H-methyl]SAM (New England Nuclear) as a methyl donor and a prokaryotic CpG DNMT, Sss1 (New England Biolabs) as previously described (25,27–30,41,42). The manner in which this assay is performed produces a reciprocal relationship between the endogenous DNA methylation status and the exogenous 3H-methyl incorporation into DNA. All analyses were performed in duplicate. Both intra- and inter-assay CV of the DNA methylation assay were 5%.

    Statistical analysis. Between-group comparisons of continuous variables were assessed using the Kruskal-Wallis and Mann-Whitney nonparametric tests. Serum folate concentrations between 8- and 30-wk time points of dietary groups in which the same diet was provided continually starting at 8 wk of age until 30 wk of age in each study were also compared using repeated measures ANOVA. All significance tests were 2-sided and were considered significant at P < 0.05. Results are expressed as means ± SEM. Statistical analyses were performed using SPSS (version 10) for Windows.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Body weight and daily food consumption. Growth curves were not significantly different among the 3 dietary groups in all 3 studies (data not shown). This finding indicates that folate deficiency in the rats fed 0 mg folic acid/kg diet was moderate; otherwise, growth retardation or premature death would have occurred (41–43). The mean daily food consumption, which was determined on a preassigned day of each week, did not differ among the 3 groups in all 3 studies (data not shown).

    Serum and hepatic folate concentrations. In study 1 where dietary folate intervention was initiated at weaning, serum folate concentrations differed among the 3 dietary groups at 8 wk of age (i.e. 5 wk of dietary folate intervention) (P < 0.001; Table 1). At 8 wk of age, serum folate concentrations did not differ among the 3 dietary groups in study 2 where all the rats were fed the control diet from weaning for 5 wk (Table 2). In study 3 where dietary folate intervention was initiated at weaning, serum folate concentrations differed among the 3 dietary groups at 8 wk of age (P < 0.001; Table 3). Serum folate concentrations of the 3 dietary groups in all 3 studies at this time point were comparable with those observed in rodents that were fed the corresponding diets for 4–5 wk in previous studies (27,31).


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  		TABLE 1 Effects of postweaning dietary folic acid intervention for 5 wk followed by the control diet on serum and hepatic folate concentrations and on hepatic genomic DNA methylation in rats (study 1)1

 

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  		TABLE 2 Effects of dietary folic acid intervention starting at puberty and continually provided into adulthood on serum and hepatic folate concentrations and on hepatic genomic DNA methylation in rats (study 2)1

 

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  		TABLE 3 Effects of postweaning dietary folic acid intervention provided continually into adulthood on serum and hepatic folate concentrations and on hepatic genomic DNA methylation in rats1

 
At necropsy (i.e. 30 wk of age), serum and hepatic folate concentrations did not significantly differ among the 3 dietary groups in study 1 where all the rats were fed the control diet at 8 wk of age for 22 wk (Table 1). At necropsy, serum and hepatic folate concentrations differed among the 3 dietary groups in studies 2 and 3 where dietary folate intervention started at 8 wk of age and at weaning, respectively (P < 0.001; Tables 2 and 3). Serum and hepatic folate concentrations of the 3 dietary groups in all 3 studies at this time point were comparable with those observed in rodents that were fed the corresponding diets for 20–24 wk in previous studies (25,27,31,44).

In all 3 studies, serum folate concentrations were lower at 30 wk of age than at 8 wk of age in dietary groups in which the same diet was provided continually starting at weaning at 3 wk of age through puberty (i.e. 8 wk of age) until the time of killing at 30 wk of age (P < 0.01; Tables 1–3).

    Effect of dietary folate intervention on genomic DNA methylation in the liver. In study 1, where dietary folate intervention was started at weaning for 5 wk, until 8 wk of age, after which all of the rats were fed the control diet until 30 wk of age. The rats that were fed the folate-deficient diet initially for 5 wk at weaning had a 34–48% less 3H-methyl incorporation into hepatic DNA than the rats that were fed the control diet (P < 0.035) or folate-supplemented diet (P < 0.023) initially (Table 1), suggesting an increase in genomic DNA methylation. However, the extent of hepatic genomic DNA methylation did not differ between rats that were fed the control diet and the rats that were fed the folate-supplemented diet initially for 5 wk followed by the control diet.

In study 2 (Table 2), weanling rats were fed the control diet for 5 wk until 8 wk of age and then consumed 0, 2, or 8 mg folic acid/kg diets until 30 wk of age and in study 3 (Table 3), weanling rats consumed diets containing 3 different levels of folic acid until 30 wk of age. In both of these studies, the extent of hepatic genomic DNA methylation did not differ among the 3 dietary groups.


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Our data indicate that a moderate degree of dietary folate deficiency started at weaning and continued through early infancy, childhood, and puberty until 30 wk of age did not significantly modulate genomic DNA methylation in adult rat liver. Furthermore, this degree of dietary folate deficiency, which started at puberty and continued until 30 wk of age, did not significantly alter genomic DNA methylation in adult rat liver. However, an unexpected, interesting finding is that the same degree of dietary folate deficiency started at weaning and continued through early infancy and childhood for 5 wk until puberty followed by the control diet for 22 wk until 30 wk of age induced a significant 34–48% increase in genomic DNA methylation in adult rat liver compared with the control and folate-supplemented diets provided in the same manner. We previously showed a 56%, albeit nonsignificant, increase in genomic DNA methylation associated with a more severe degree of dietary folate deficiency provided at weaning for 6 wk in rat liver (41). Also, the same moderate folate-deficient diet provided at weaning for 5 wk in mice was shown to induce a significant 56% increase in genomic DNA methylation in the liver (30). We posit that a compensatory upregulation of DNMT and of the choline and betaine-dependent transmethylation pathway in response to folate deficiency during the postweaning period resulted in genomic DNA hypermethylation in the liver and this newly established DNA hypermethylation pattern was maintained in the presence of adequate folate and other methyl group supply when the animals were fed the control diet at puberty into adulthood. In support of this, methyl group donor deficiency has previously been shown to upregulate DNMT in rat liver (10–14,16,17,45–47). Methionine biosynthesis from homocysteine is dependent on 2 separate but closely related transmethylation pathways utilizing folate and choline/betaine, catalyzed by methionine synthase and betaine:homocysteine methyltransferase, respectively (Fig. 1) (48). Folate and choline/betaine transmethylation pathways are intimately interrelated, with perturbations in one pathway affecting the other (49,50). Previous studies have shown that folate deficiency and folate antagonists upregulate betaine:homocystiene methyltransferase and increase the utilization of betaine and choline in rat liver to maintain critical hepatic methionine concentrations (50,51).

In contrast, although the new DNA hypermethylation pattern might have been established in the rats that were fed the moderate folate deficiency diet at weaning period and continued through early infancy, childhood and puberty until 30 wk of age via the same compensatory mechanisms, it was not maintained because of the lack of folate in the diet provided from puberty to adulthood. Furthermore, it has previously been shown that upregulation of the betaine and choline-dependent remethylation of homocysteine in response to dietary folate deficiency is not sufficient to maintain hepatic SAM in the long run (51). Consistent with our observation, the same moderate folate-deficient diet for 5 wk in mice induced a significant 56% increase in genomic DNA methylation in the liver; however, the level of genomic DNA methylation returned to that of the baseline by 8 wk (30). These data suggest that compensatory genomic DNA hypermethylation in rat liver induced by dietary folate deficiency is established during the postweaning period and that this pattern is maintained only if adequate levels of dietary folate are provided at puberty and continued into adulthood. However, if continual dietary folate deficiency is imposed, this compensatory hypermethylation pattern is not maintained. This finding illustrates the importance of timing of folate deficiency and subsequent supply of folate in establishing and maintaining the DNA methylation pattern in rat liver.

Our data indicating no significant change in genomic DNA methylation in adult rat liver resulting from continual dietary folate deficiency for 22–27 wk starting at weaning or at puberty are consistent with prior observations. Although isolated folate deficiency has been shown to reduce SAM concentrations and SAM:SAH ratios and increase SAH concentrations in rat liver (25,41,51–53), conflicting data exist for the effect of isolated dietary folate deficiency provided in a similar manner as in the present study on genomic DNA methylation in rat liver (6,18). One study reported a significant 20% decrease in genomic DNA methylation associated with a severe degree of dietary folate deficiency for 4 wk in rat liver (52), whereas we showed a 56% (P = 0.1), increase in genomic DNA methylation associated with the same severe folate deficient diet for 6 wk in rat liver (41). The same moderate degree of dietary folate deficiency, as in our study, provided for 15–24 wk did not significantly alter genomic or c-myc protooncogene-specific DNA methylation in rat liver (25). The same moderately folate-deficient diet fed to mice for 5 wk induced a significant 56% increase in genomic DNA methylation in the liver followed by the return of genomic DNA methylation value to that of the baseline by 8 wk (30). Taken together, these observations suggest that moderate dietary folate deficiency imposed continually starting at weaning or at puberty does not significantly affect genomic DNA methylation in adult rat liver. In contrast, dietary deficiency of methyl group donors provided continually starting at weaning for a variable duration of intervention has generally been shown to induce genomic DNA hypomethylation in rat liver despite significantly upregulated DNMT (7–17). This difference is likely related to the fact that most diets used in these studies were severely devoid of methyl group donors and thus, optimal genomic DNA methylation could not be maintained despite upregulated DNMT because of the lack of methyl group donors, thereby resulting in persistent genomic DNA hypomethylation.

In all 3 studies, dietary folate supplementation at 4 times the basal dietary requirement did not significantly increase genomic DNA methylation in adult rat liver regardless of timing or duration of supplementation. These observations are also consistent with prior studies. Dietary folate supplementation (4–20 times the basal dietary requirement) continually provided starting at weaning for 4–20 wk did not significantly increase genomic DNA methylation in adult rat liver (27,54). However, a recent study has indicated that dietary folate supplementation at 4 times the basal dietary requirement continually provided for 20 wk starting at 1 y of age can increase genomic DNA methylation in the liver of elder rats (55).

Our data demonstrating the maintenance of the hypermethylation pattern of genomic DNA resulting from postweaning dietary folate deficiency for 5 wk followed by dietary folate sufficiency starting at puberty into adulthood is quite surprising. Prior studies have shown that genomic DNA hypomethylation induced by dietary methyl group donor deficiency for 4–9 wk is reversed to normal extent after refeeding of a methyl donor group-sufficient diet for 1–45 wk in rat liver (7,17) in conjunction with restoration of elevated DNMT activities to normal values (7). However, hypomethylation of certain sites of specific genes persisted even after refeeding of the methyl group donor-sufficient diet (7). In rats exposed to a longer duration of postweaning methyl group donor deprivation (18–36 wk), refeeding of a methyl group donor-sufficient diet for 18–36 wk did not reverse the genomic DNA hypomethylation in the liver (17). We posit that a compensatory upregulation of DNMT and of the choline- and betaine-dependent transmethylation pathway in response to folate deficiency during the postweaning period results in genomic DNA hypermethylation in the liver and this newly established DNA hypermethylation pattern is maintained by the maintenance methylation machinery, including DNMT1 (4), only in the presence of adequate folate and other methyl group supply in adolescence and adulthood. Our findings and conjecture are further supported by a recent observation that demonstrated that dietary deficiency of methyl donors, including folic acid, during the postweaning period for 60 d followed by a control diet for 100 d caused dramatic loss of imprinting at the insulin-like growth factor 2 that persisted into adulthood in mouse kidney (56). Furthermore, it has recently been shown that diet-induced epigenetic changes are permanent and can be transmitted to future generations (57).

Given the fact that the effects of folate deficiency on DNA methylation appear to depend on cell type, target organ, and stage of transformation (6,18,58), whether or not the observed folate deficiency-induced DNA hypermethylation pattern established during postweaning period and maintained in adulthood in rat liver is also operative in other organs needs to be investigated. Furthermore, given the fact that the effects of folate deficiency on DNA methylation are gene- and site-specific and that the changes in genomic and site-specific DNA methylation in response to folate deficiency may not be in the same direction (6,18,41,59), a comprehensive survey of the effect of dietary folate intervention provided during the postweaning period, childhood, and puberty on gene- and site-specific DNA methylation in adulthood is of great interest. Because aberrant patterns and dysregulation of DNA methylation are mechanistically related to the development of several chronic diseases, including cancer (1,2), how folate deficiency-induced DNA hypermethylation established during the postweaning period and maintained in adulthood may be mechanistically related to malignant transformation needs to be elucidated. The findings from this study clearly beg for future studies that investigate mechanistic explanations for the establishment and maintenance of new DNA hypermethylation associated with postweaning dietary folate deficiency. It will be important to measure intracellular SAM and SAH concentration, to determine expression and activity of de novo and maintenance DNMT, and to determine other epigenetic regulatory and transcriptional machinery responsible for the changes in genomic DNA methylation pattern observed in this study. Given the small sample size in the current study, type II error cannot be entirely ruled out in the analysis of genomic DNA methylation and, hence, studies are warranted to confirm our findings.

In summary, we showed that dietary folate deficiency of a moderate degree, but not supplementation, provided during the postweaning period through early infancy and childhood to puberty significantly increased genomic DNA methylation in rat liver that persisted into adulthood following a return to the control diet at puberty. In contrast, dietary folate deficiency or supplementation continually imposed at weaning or at puberty did not significantly affect genomic DNA methylation in adult rat liver. In addition to the well-established permanent and heritable epigenetic effect of a methyl group-rich maternal diet on certain phenotype of the offspring (21–23,57), our data suggest that early folate nutrition during postnatal development and preadulthood may play an important role in epigenetic programming that can have a permanent effect in adulthood.


   ACKNOWLEDGMENTS
 
We thank Rochelle Martin for assistance with statistical analyses.


   FOOTNOTES
 
1 Supported by a grant from the Canadian Institutes of Health Research (grant no. MOP-14126 to Y-I.K.). Back

2 Author disclosures: J. Kotsopoulos, K.-J. Sohn, and Y.-I. Kim, no conflicts of interest. Back

3 Supplemental Figure 1 is available with the online posting of this paper at jn.nutrition.org. Back

7 Abbreviations used: CpG, cytosine-guanine dinucleotide; DNMT, DNA methyltransferase; SAH, S-adenosylhomocysteine; and SAM, S-adenosylmethionine. Back

Manuscript received 5 September 2007. Initial review completed 6 November 2007. Revision accepted 17 January 2008.


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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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