nutrients

Review
Methyl Donor Micronutrients that Modify DNA
Methylation and Cancer Outcome
Abeer M. Mahmoud 1,2, * and Mohamed M. Ali 1,2
1 Department of Physical Therapy, College of Applied Health Sciences, University of Illinois at Chicago,
1919 W. Taylor St. (AHSB), Room 433, Chicago, IL 60612, USA; [email protected]
2 Integrative Physiology Laboratory, College of Applied Health Sciences, University of Illinois at Chicago,
Chicago, IL 60612, USA
* Correspondence: [email protected]; Tel.: +1-312-753-9998




Received: 16 February 2019; Accepted: 7 March 2019; Published: 13 March 2019


Abstract: DNA methylation is an epigenetic mechanism that is essential for regulating gene
transcription. However, aberrant DNA methylation, which is a nearly universal finding in cancer,
can result in disturbed gene expression. DNA methylation is modified by environmental factors
such as diet that may modify cancer risk and tumor behavior. Abnormal DNA methylation has been
observed in several cancers such as colon, stomach, cervical, prostate, and breast cancers. These
alterations in DNA methylation may play a critical role in cancer development and progression.
Dietary nutrient intake and bioactive food components are essential environmental factors that may
influence DNA methylation either by directly inhibiting enzymes that catalyze DNA methylation
or by changing the availability of substrates required for those enzymatic reactions such as the
availability and utilization of methyl groups. In this review, we focused on nutrients that act as
methyl donors or methylation co-factors and presented intriguing evidence for the role of these
bioactive food components in altering DNA methylation patterns in cancer. Such a role is likely to
have a mechanistic impact on the process of carcinogenesis and offer possible therapeutic potentials.

Keywords: methyl donors; nutrients; DNA methylation; cancer; folate; Vitamin B; choline; betaine;
methyltransferase; dietary

1. Introduction
Cancer is an outcome of aberrant genetic and epigenetic events. Epigenetic mechanisms are
responsible for regulating gene expression without changing the DNA sequence. These mechanisms
mainly include chromatin remodeling, histone modification, and DNA methylation, the latter being
the most investigated mechanism and the focus of the current review [1]. The process of DNA
methylation includes the addition of methyl groups to the cytosine residues; a biological process
that depends on the availability of methyl groups and accordingly the function of methyl donors
and acceptors [2]. Micronutrients such as folate, choline, betaine, vitamin B12, and other B vitamins
contribute to DNA methylation as methyl donors and co-factors [3]. Therefore, the status of these
nutrients might correlate with DNA methylation and offer potential preventive and therapeutic targets
in pathological conditions such as cancer where aberrant DNA methylation is frequently observed.
It is widely accepted that DNA methylation profiles are dynamic and prone to modifications in
response to normal development and aging, environmental factors, and pathological conditions [4,5].
For example, DNA undergoes progressive global hypomethylation and gene-specific hypermethylation
as individuals age, leading to genomic instability or gene-specific suppression [4]. A similar pattern
has been observed in cancer where DNA is globally hypomethylated while tumor suppressor genes
are hypermethylated compared to normal tissues [6]. Whether these epigenetic patterns are a cause

Nutrients 2019, 11, 608; doi:10.3390/nu11030608 www.mdpi.com/journal/nutrients

Nutrients 2019, 11, 608 2 of 30

or an outcome of cancer is not entirely understood. Yet, cancer could be prevented through changes
in diet or lifestyle that might be attributed to the dynamic and adaptable nature of cancer-associated
epigenetic processes, particularly, DNA methylation.
In the present review, we summarized preclinical and clinical studies that provide intriguing
evidence on the interrelation between nutritional methyl donors and carbon one metabolism co-factors
and cancer through regulating DNA methylation patterns. Additionally, this review characterizes the
differential effects of micronutrient methyl donors on global versus gene-specific DNA methylation
and the effect of variables such as health status, source of biological samples that were analyzed, mode
(dietary versus supplemental) and quantity of nutrient exposure, and other confounders in an effort to
unravel potential sources of discrepancies in the literature

2. DNA Methylation
DNA methylation is an epigenetic mechanism that is essential for regulating gene transcription.
This mechanism is mediated via the addition of a methyl group to the cytosine residues in a
cytosine-guanine (CG) pair generating 5-methylcytosine. In the intergenic regions and repetitive
sequences of the human genome, CpG sites are sparse and mostly methylated. Hypomethylation
of CpG sites in these regions may lead to genomic instability and loss of gene imprinting, which
eventually result in the development of neoplastic cells [7]. On the other hand, gene promoters are
rich in CpG sites that are sometimes densely packed forming what is known as CpG islands. These
islands are mostly unmethylated in order to allow gene transcription. Aberrant hypermethylation of
these CpG sites may silence the expression of genes that are critical to cell homeostasis, DNA integrity,
or genome stability, resulting in cancer development and progression [8].
The process of DNA methylation is catalyzed by a group of enzymes called DNA methyltransferases
(DNMTs), namely, DNMT1, DNMT2, DNMT3a, DNMT3b, and DNMTL. DNMT1 maintains DNA
methylation during cell replication while the rest of the DNMT family, mainly DNMT3a and DNMT3b,
are responsible for de novo DNA methylation [9].
DNMTs that are responsible for de novo DNA methylation are highly expresses in developing
embryos than adult tissues; yet, there is growing evidence that they play a role in maintaining DNA
methylation patterns. For instance, combined genetic deletion or silencing of DNAMT1 and DNMT3b
reduced DNA methylation to a greater extent than deleting or silencing either genes alone, supporting
the critical role of de novo DNMTs in maintaining DNA methylation [10–12]. On the other hand,
some studies suggested that DNMT1 is required for de novo DNA methylation. For example, a study
by Liang et al. [13] showed that DNMT3a and DNMT3b did not induce de novo DNA methylation
efficiently in mouse embryonic stem cells in the absence of DNMT1 gene. Other studies have supported
this co-operativity between DNMTs in de novo DNA methylation [14–16]. The process of methyl
transfer starts by non-specific binding of DNMTs to DNA followed by recognition of specific DNA sites
and recruitment of the methyl group donor, S-adnosylmethionine. DNMTs incorporate the donated
methyl group into carbon 5 of the cytosine residue followed by a release of the DNMT enzyme and
s-adenosylhomocysteine [16].
When it takes place at gene promoters, DNA methylation results in transcriptional
repression either via interfering with the binding of transcription activating factors or by recruiting
transcriptional repressors such as methyl-binding proteins, histone deacetylases (HDACs), and
histone methyltransferases that reduces chromatin accessibility [17]. Absent or inactive DNMTs,
mainly DNMT1, will induce passive demethylation of the CpG sites and subsequently aberrant
gene expression [18]. DNA hypomethylation can also be induced via active demethylation by the
ten-eleven translocation methylcytosine dioxygenase family of enzymes (TETs) that helps create a
balanced methylation profile in the human genome [19]. TET enzymes oxidize 5-methylcytosine
(5-mC) to 5-hydroxy-mC (5-hmC), which is modified through several suggested mechanisms including
deamination and decarboxylation that ultimately lead to base excision repair and replacement with
an unmethylated cytosine [20]. TET1 is the most prominent member of the TET family, and previous
Nutrients 2019, 11, 608 3 of 30

studies showed that knockdown of TET1 results in increased global methylation in mice [21]. Other
suggested mechanisms for active DNA demethylation include: (1) base excision repair via DNA
glycosylase either by directly acting on 5-mC residue or following 5-mC deamination and conversion
into Thymine; (2) oxidative or hydrolytic removal of the methyl group; or (3) nucleotide excision repair
system that severs methylated CpG dinucleotides. Current efforts are underway to study the role of
DNA active demethylation in cancer and developmental diseases [22].

3. DNA Methylation in Cancer

The two main salient features of aberrant DNA methylation in cancer are (1) global DNA
hypomethylation that takes place in the intergenic regions and repetitive DNA sequences and
(2) local DNA hypermethylation in CpG islands located in specific gene promoters [23]. The latter
phenomenon is induced by de novo DNA methylation that is mediated via DNMT3a and DNMT3b
and is accompanied by a suppressed transcription of corresponding genes [24]. Several studies
have shown that DNA hypermethylation in cancer cells targets tumor suppressor genes explicitly,
resulting in growth selection and uncontrolled cell proliferation [25–28]. Many tumor suppressor
genes are inactivated by this mechanism in cancer such as the adenomatous polyposis coli (APC) [29],
retinoblastoma (Rb) [30], Von Hippel-Lindau (VHL), BRCA1 [31], and several other genes that are
involved in DNA repair (MGMT; O-6-Methylguanine-DNA Methyltransferase), cell cycle progression
(p16INK4a , p15INK4b ), apoptosis (DAPK; death-associated protein kinase-1), or antioxidation (GSTP1;
Glutathione S-Transferase P-1) [32]. The extent to which DNA de novo methylation contributes to
cancer development and progression varies among different types of cancer; very high in colon cancer,
while rare in brain tumors [33].
There is also growing evidence indicating that global hypomethylation may lead to genome
instability and DNA breakage [4]. Furthermore, global hypomethylation may be accompanied by
loss of imprinting of some oncogenes leading to cancer development such as insulin-like growth
factor 2 (IGF-1) in colon cancer [34]. The oncogenes that are linked to cancer and activated via
hypomethylation are protease urokinase, mesothelin, cancer-testis genes, claudin4, S100A4, heparinase,
and the proopiomelanocortin gene [35]. Global DNA hypomethylation in cancer cells has been
suggested to be attributed to one of the following causes: (1) a discoordination between DNA
replication in cancer cells and DNMT-1 activity; (2) a natural selection of hypomethylated DNA
patterns accompanying overexpression of specific oncogenes or genomic instabilities that facilitate
cancer cell growth and expansion; or (3) a consequence of chromatin dysregulation and nuclear
disorganization that occur during cancer progression [36].
Under normal conditions, cells possess standard methylation profiles by maintaining a balance
between DNA methylation and demethylation processes. This balance is disturbed under pathological
conditions such as inflammation, oxidative stress and cancer resulting in diverse phenotypes [37].
Due to the dynamic and reversible nature of the DNA methylation process, it has been viewed as an
attractive target for cancer therapy. The de novo DNA methylation in cancer has been successfully
targeted via the DNMT potent inhibitor, 5-aza-20 -deoxycytidine (5-AZA) in experimental studies and
in the clinical settings for treating leukemia [38]. Treatment with 5-AZA reduced DNMT3b in breast
cancer cells and restored the expression of TSGs such as RASSF1A (Ras association domain family
member 1) in hepatocellular carcinoma, P53 in melanoma, and cyclin-dependent kinase-2B (CDKN2B)
in myelodysplastic syndrome [39–42].
There is a cumulative body of evidence indicating DNA methylation adaptability to environmental
factors including diet and nutritional elements [43,44]. Nutrients have been shown to modify DNA
methylation either globally or at specific CpG sites by inducing the formation of methyl donors, acting
as co-enzymes, or modifying DNMT enzymatic activity [45]. The interaction between nutrients and
epigenetics has been referred to as “Nutri-epigenomics”, which is an emerging and promising field that
offers opportunities for future applications of bioactive nutrients as epigenetic modifiers in diseases
where aberrant DNA methylation occurs [46,47].
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4. Effects of Nutrients and Bioactive Food Components on DNA Methylation

One of the proposed mechanisms by which nutrients may modify DNA methylation is by
participating in a cellular process known as “one-carbon metabolism” that provides methyl groups for
biological methylation of DNA, protein, or phospholipids [48]. One carbon metabolism is a process
that is involved in amino acid and nucleotide metabolism and comprises a group of biochemical
reactions that are catalyzed by a unique set of enzymes and coenzymes (Figure 1). This set of reactions
is referred to as one-carbon metabolism since they transfer one-carbon groups from donors to protein
or DNA. During this process, cyclical chemical reactions take place in mammalian cells where nutrients
such as folate, vitamin B12, vitamin B6, betaine, choline, and methionine play a significant role as
co-factors or methyl acceptors or donors [49]. This process starts with a one-carbon transfer from the
amino acid serine to tetrahydrofolate; the latter is a form of vitamin B-9 that is naturally present in
many food sources and is essential for the nucleic acid synthesis and other vital biological processes
in the body. This reaction is catalyzed by the enzyme serine hydroxymethyltransferase, a vitamin
B6-containing enzyme. The outcome of this reaction is 5,10 methylene tetrahydrofolate, which, in turn,
is transformed into 5-methyl tetrahydrofolate via the enzyme tetrahydrofolate reductase, a vitamin
B2-containing enzyme. The product 5-methyl tetrahydrofolate is the primary methyl donor for the
reaction that remethylates, homocysteine into methionine. The latter reaction is catalyzed by the
enzyme methionine synthase, which contains vitamin B12 as a co-factor. Methionine is then converted
to s-adenosylmethionine, the DNMT cofactor and the universal methyl donor for all the biological
methylation process inside mammalian cells including DNA methylation [2,50,51].
The negative feedback mechanism in this cycle is catalyzed by two enzymes, glycine
N-methyltransferase that converts s-adenosylmethionine to s-adenosylhomocysteine and
s-adenosylhomocysteine hydrolase that converts s-adenosylhomocysteine to homocysteine [52].
This reaction is considered as a negative regulator of the DNA methylation process since
s-adenosylhomocysteine binds to DNMTs with a higher affinity than s-adenosylmethionine resulting
in an inhibition of the DNMT activity. The conversion of s-adenosylhomocysteine to homocysteine is a
reversible chemical reaction, and accordingly high levels of homocysteine in the body are associated
with reduced DNMT activity. This negative regulation of the “one-carbon metabolism” cycle is
resolved by the re-methylation of homocysteine to methionine via the folate-dependent pathway or the
diet-derived betaine or choline in the liver and kidney [53]. The alternative pathway for homocysteine
metabolism is through transsulfuration to cystathionine, via cystathionine β-synthase. The latter is
further metabolized to cysteine via the enzyme, cystathionase. Both reactions utilize the cofactor
pyridoxal-50 -phosphate [54].
The “one-carbon metabolism” cycle is an example of how levels of nutrient exposure impact DNA
methylation in the human body (Figure 2). In this example, nutrients either maintain or change
the balance between s-adenosylmethionine and s-adenosylhomocysteine, which, in turn, regulate
the availability of methyl donors and the activity of DNMTs [2]. There is a growing body of
evidence indicating that nutrients, such as the green tea polyphenol, epigallocatechin-3-gallate, or
the soy isoflavone, genistein, are capable of inhibiting DNMT activity directly and competitively [49].
Furthermore, dietary supplements that have alpha-ketoglutarate may affect the activity of the active
demethylation enzymes, TETs [55]. In the current review, we will discuss reported studies and
observations for the mechanism and role of dietary methyl donors in modifying DNA methylation
and subsequently cancer risk or progression. These studies are summarized in Tables 1 and 2.
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Nutrients 2018, 10, x FOR PEER REVIEW 21 of 34

Figure
Figure1. 1.Micronutrient
Micronutrientmethyl methyldonors
donorsthatthat are involved in
are involved in the
theone
onecarbon
carbonmetabolism
metabolismand and
subsequently
subsequently in DNA
in DNA methylation.
methylation. Dietary
Dietary folate
folate is is
converted
converted toto
dihydrofolate
dihydrofolate (DHF)
(DHF) viavia
the
dihydrofolate synthase (DHFS)
the dihydrofolate synthaseenzyme
(DHFS)then enzyme
to tetrahydrofolate
then to (THF) by the dihydrofolate
tetrahydrofolate (THF) by reductase
the
(DHFR) enzyme; inreductase
dihydrofolate both steps,(DHFR)
vitaminenzyme;
B3 (B3 ) actsin as a co-factor.
both THF is then
steps, vitamin B3 (Bconverted
3) acts as to 5,10-methyl
a co-factor.
THF via the enzyme serine hydroxymethyltransferase (SHMT) that has
THF is then converted to 5,10-methyl THF via the enzyme serine hydroxymethyltransferase vitamin B6 (B 6 ) as a coenzyme.
This reaction is followed by a reduction of 5,10-methyl THF to 5-methyl
(SHMT) that has vitamin B6 (B6) as a coenzyme. This reaction is followed by a reduction THF via the enzyme
of
methylenetetrahydrofolate
5,10-methyl THF to 5-methyl reductaseTHF (MTHFR)
via the andenzyme
the co-enzyme, vitamin B2 (B2 ). At thereductase
methylenetetrahydrofolate end of this
(MTHFR)
cycle, 5-methylandTHFthe co-enzyme,back
is transformed vitamin
to THF B2by(Bthe
2). enzyme
At the end of this cycle, 5-methyl THF is
5-methyltetrahydrofolate-homocysteine
transformed back
methyltransferase (MTR)to that THF by the
utilizes vitaminenzymeB2 as 5-methyltetrahydrofolate-homocysteine
a co-enzyme. The same enzyme, MTR,
methyltransferase
converts homocysteine(MTR) (Hcy) that utilizes vitamin
to methionine. Betaine as aas
B2 acts co-enzyme.
an indirectThe same
methyl enzyme,
donor for the MTR,
latter
converts
reaction. homocysteine
Methionine, (Hcy)ittoismethionine.
whether endogenously Betaine acts as an
synthesized orindirect methyl
diet-derived is donor
criticalfor forthe
the
latter reaction. Methionine, whether it is endogenously synthesized
synthesis of S-adenosylmethionine (SAM), which acts as a DNA methyltransferase (DNMT) cofactor or diet-derived is critical
andfor the synthesis
a universal of S-adenosylmethionine
methyl-donor for DNA methylation. (SAM), which acts asthat
The enzyme a DNA methyltransferase
catalyzes this reaction is
(DNMT) cofactor and a universal methyl-donor for DNA
methionine adenosyltransferase (MAT). Glycine N-methyltransferase (Glycine N-MT) methylation. The enzyme
convertsthat SAM
catalyzes this reaction is methionine adenosyltransferase (MAT). Glycine
to s-adenosylhomocysteine (SAH), which could be reversibly converted to Hcy via the enzyme SAH N-methyltransferase
(GlycineFinally,
hydrolase. N-MT)the converts
activatedSAMDNMT to s-adenosylhomocysteine
enzyme will catalyze the(SAH), transferwhich could group
of a methyl be reversibly
to carbon
converted to Hcy via the enzyme SAH hydrolase. Finally, the activated DNMT enzyme will catalyze
5 of cytosines in the DNA to produce methylated DNA (mDNA).
the transfer of a methyl group to carbon 5 of cytosines in the DNA to produce methylated DNA
(mDNA).
Nutrients 2019, 11, 608 6 of 30

Table 1. Clinical studies of the impact of methyl donor micronutrients on DNA methylation.

Authors Population/Tissue Study Design Methylation Assay Conclusion/Outcome

Folate
Higher folate levels were associated with
Adults with history of colorectal Randomized, double-blind Gene-specific quantitative bisulfite
higher levels of ERα (estrogen receptor
Wallace et al. [56] adenoma controlled trial pyrosequencing
alpha) and SFRP1 (Secreted Frizzled
Colorectal tissues 1 mg/day for 3 years ERα and SFRP1 genes
Related Protein 1) methylation
High folate status was associated with
decreased plasma homocysteine and
Colorectal adenoma and cancer Case-control study
Global DNA methylation via [(3)H] increased colonic DNA methylation.
Pufulete et al. [57] patients and heathy controls Estimates of dietary intake and serum
methyl incorporation Low folate intake and colonic DNA
Colonic tissues and erythrocyte folate
hypomethylation were associated with
increased risk for adenoma and cancer
Patients with cervical Cross-sectional study Global DNA methylation via Folic acid fortification did not change
Piyathilake et al. [58] intraepithelial neoplasia Dietary intake pre and post folic acid Immunohistochemical staining for global DNA methylation in cells
Cervical tissues fortification 5-methyl cytosine involved in cervical carcinogenesis
Global DNA methylation was
Patients with bladder cancer Case-control study
Global DNA methylation via 5-methyl significantly lower in cases than control.
Moore et al. [59] and controls Dietary intake via food frequency
cytosine antibody No significant differences in folate intake
Blood questionnaire (FFQ)
between cases and control
Patients with cervical Blood cell (but not cervical cell) DNA
Global DNA methylation via bisulfite
intraepithelial neoplasia and was hypomethylated in cases compared
Case-control study pyrosequencing LINE-1 (Long
Piyathilake et al. [60] controls to controls and hypermethylated in the
Serum levels measured Interspersed Nucleotide Element 1)
Blood and exfoliated cervical highest folate compared to the lowest
analysis
cells folate tertile.
Observed weak inverted associations
Healthy adults Cross-sectional study Global DNA methylation via [(3)H]
Pufulete et al. [61] between serum and erythrocyte folate
Colonic tissues Serum and erythrocyte levels measured methyl incorporation
and colonic DNA hypomethylation
Patients with colorectal Randomized, double-blind Folate treatment significantly reversed
Global DNA methylation via
O’Reilly et al. [62] adenoma controlled trial global DNA hypomethylation in
methylation-sensitive restriction enzymes
Colonic tissues 600 µg folic acid/day for 6 months colonic tissues
Folate supplementation reversed DNA
Patients with colorectal Randomized, controlled, cross-over study
Global DNA methylation via [(3)H] Hypomethylation, which returned to
Cravo et al. [63] adenoma 5 mg/day for 3 months then switched to
methyl incorporation baseline values after switching to
Colonic tissues placebo for additional 3 months
placebo treatment
Patients with colorectal Randomized, double-blind Folate supplementation increased
Kim et al. [64] adenoma controlled trial Global DNA methylation genomic DNA methylation at 6 months
Colonic tissues 5 mg/day for 1 year and 1 year
Nutrients 2019, 11, 608 7 of 30

Table 1. Cont.

Authors Population/Tissue Study Design Methylation Assay Conclusion/Outcome

Gene-specific quantitative bisulfite
pyrosequencing
Patients with colorectal cancer APC (adenomatous polyposis coli),
Cross-sectional analysis Low folate levels were associated with
Coppedè et al. [65] Colonic tissues (cancer and MGMT (Methylguanine-DNA
Serum levels were measured hMLH1 hypermethylation
adjacent healthy) Methyltransferase), hMLH1 (MutL
homolog 1), RASSF1A and CDKN2A
(cyclin-dependent kinase 2A) genes
The Pathways Study: a prospective Genome-wide methylation analysis via Higher folate intake was associated with
Breast cancer patients
Christensen et al. [66] cohort study Illumina GoldenGate methylation a trend toward increased CpG
Breast cancer tissues
Estimates of dietary intake bead-array platform methylation in several genes
Folate was associated with increased
Nested case-control study in The
Patients with lung cancer and methylation levels of RASSF1A (Ras
European Prospective Investigation into Genome-wide quantitative bisulfite
Vineis et al. [67] healthy controls association domain family member 1)
Cancer and Nutrition (EPIC) pyrosequencing
Blood and MTHFR
Serum levels were measured
(methylenetetrahydrofolate reductase)
Methylation-specific PCR (polymerase
chain reaction) for APC-1A
(adenomatous polyposis coli-1A), Gene promoters were hypermethylated
p14(ARF) (alternate reading frame in patients with low folate intake
van Engeland Patients with colorectal cancer Netherland Cohort Study (NLCS)
protein of cyclin-dependent kinase 2A), compared with high folate intake;
et al. [68] Colorectal biopsies Estimated dietary intake via FFQ
p16(INK4A) (cyclin-dependent kinase differences were not statistically
inhibitor 4A), hMLH1, O(6)-MGMT significant
(O-6-methylguanine-DNA
methyltransferase), and RASSF1A genes
IGF2 promoter methylation was not
Pregnant women Cross-sectional study
Ba et al. [69] Methylation-specific PCR for IGF2 gene associated with serum folate levels in
Maternal and cord blood Serum levels were measured
either cord or maternal blood
Pregnant women Cross-sectional study Gene-specific (IGF-2) quantitative IGF-2 methylation decreased with
Hoyo et al. [70]
Cord blood Estimated dietary intake via FFQ bisulfite pyrosequencing increasing folate intake
Folate depletion-repletion clinical trial Observed global DNA hypomethylation
Healthy non-pregnant women Global DNA methylation via [(3)H]
Shelnutt et al. [71] 115 µg/day for 7 weeks followed by 400 during depletion and increases in DNA
Blood methyl incorporation
µg/day for additional 7 weeks methylation during repletion
Nutrients 2019, 11, 608 8 of 30

Table 1. Cont.

Authors Population/Tissue Study Design Methylation Assay Conclusion/Outcome

Vitamin B
Patients with the highest quartile of
Gene-specific methylation analysis via vitamin B12 intake showed significantly
Patients with head and neck Cross-sectional study
Colacino et al. [72] Illumina Goldengate Methylation Cancer less tumor suppressor gene methylation
cancer Estimated dietary intake via FFQ
Panel compared with those in the lowest
quartile
A direct association was reported
Patients with lung cancer
Cross-sectional study Global DNA methylation via [(3)H] between vitamin B-12 and global DNA
Piyathilake et al. [73] Cancer tissue and adjacent
Tissue levels were measured methyl incorporation methylation in cancer tissues but not in
normal bronchial tissue
normal tissues
School-age children Cross-sectional study Global DNA methylation via bisulfite No association between vitamin B12 and
Perng et al. [74]
Blood Plasma levels were measured pyrosequencing LINE-1 analysis global DNA methylation
Clinical trial
Old adults Global DNA methylation via bisulfite Vitamin B supplementation had no effect
Hubner, et al. [75] 500 µg folic acid, 500 µg vitamin B12 and
Blood pyrosequencing LINE-1 analysis on global DNA methylation in blood cells
50 mg vitamin B6 for 1 year
Folate and vitamin B12, maintain a high
Women positive for human Gene-specific (HPV(human papilloma degree of methylation at specific CpG
Cross-sectional study
Piyathilake et al. [76] papilloma virus virus)-16) quantitative bisulfite sites in the HPV E6 gene and
Plasma levels were measured
Exfoliated cervical cells pyrosequencing subsequently reduce the risk of cervical
intraepithelial neoplasia
Choline and betaine
Global DNA (hydroxy)methylation was Choline and betaine intake in the first
MANOE (MAternal Nutrition and
Pregnant women measured in blood using LC-MS/MS weeks was negatively associated with
Pauwels et al. [77] Offspring’s Epigenome) cohort study
Blood (liquid chromatography-mass DNA hydroxymethylation (a step that
Estimated dietary intake via FFQ
spectrometry/mass spectrometry) precedes demethylation)
Total choline + betaine intake was
Cross-sectional study from The Nurses’ Plasma total homocysteine measurement inversely associated with homocysteine
Healthy women
Chiuve et al. [78] Health Study (NHS) via HPLC (high performance liquid (measured as a surrogate biomarker for
Blood
Estimated dietary intake via FFQ chromatography) effective methyl donation and
DNMT activity)
Randomized, double-blind
Obese adults controlled trial Plasma total homocysteine measurement Betaine supplementation decreased the
Schwab et al. [79]
Blood Betaine supplements (6 gm/day) for via HPLC plasma homocysteine concentration
12 weeks
Randomized, double-blind controlled
Healthy men trial Plasma total homocysteine measurement Choline supplementation decreased the
Olthof et al. [80]
Blood Choline supplements (2.6 gm/day) for via HPLC plasma homocysteine concentration
2 weeks
Nutrients 2019, 11, 608 9 of 30

Table 1. Cont.

Authors Population/Tissue Study Design Methylation Assay Conclusion/Outcome

Methionine
Methionine was associated with
Vineis et al. [67] Details are in the folate section of the table
decreased methylation of RASSF1A gene
A high intake of methionine showed
Pauwels et al. [77] Details are in the choline and betaine section of the table lower DNA hydroxymethylation (a step
that precedes demethylation)
Multi-Ethnic Study of Atherosclerosis
Healthy adults Global DNA methylation via bisulfite Dietary methionine was not associated
Perng et al. [81] (MESA) Stress Study
Blood pyrosequencing LINE-1 analysis with global DNA methylation
Estimated dietary intake via FFQ
Cross-sectional study from the Western
Breast cancer patients and Methylation-specific PCR of E-cadherin, Dietary intake of methionine was not
New York Exposures and Breast Cancer
Tao et al. [82] control p16, and RAR-β(2) (retinoic acid receptor associated with promoter methylation of
Study (WEB Study)
Breast cancer tissue beta 2) genes E-cadherin, p16, and RAR-β(2) genes
Estimated dietary intake via FFQ

Table 2. Clinical studies of the impact of methyl donor micronutrients on cancer.

Authors Population/Tissue Study Design Conclusion/Outcome

Folate
The Nurses’ Health Study, and the Health Professionals Follow-up
High dietary folate was inversely associated with risk of
Giovannucci et al. [83] Male and female adults Study
colorectal adenoma in women and men
Estimated dietary intake via FFQ
Significant association between folate intake and lower risk of
The NHANES I Epidemiologic Follow-up Study (NHEFS)
Su et al. [84] Male and female adults colon cancer among men and non-alcohol drinkers, but not
Estimated dietary intake via FFQ
women or alcohol drinkers
The Nurses’ Health Study Higher folate intake reduces the risk of colon cancer associated
Fuchs et al. [85] Female adult
Estimated dietary intake via FFQ with a family history of the disease.
The American Cancer Society Cancer Prevention Study II Nutrition
Higher intake of folate was associated with a nonsignificant
Stevens et al. [86] Male adult Cohort
decrease in the risk of advanced prostate cancer
Estimated dietary intake via FFQ
Patients with colorectal Low plasma levels of folate were associated with a reduced risk
Gylling et al. [87] The Nurses’ Health Study
cancer and matched controls of colorectal cancer
A nested case-control study in the population-based Northern
Giovannucci et al. [88] Female adult Sweden Health and Disease Study Folate intake was associated with a lower risk for colon cancer
Estimated dietary intake via FFQ
Nutrients 2019, 11, 608 10 of 30

Table 2. Cont.

Authors Population/Tissue Study Design Conclusion/Outcome

The Netherlands Cohort Study The study reported an inverse association between colon
Konings et al. [89] Male and female adults
Estimated dietary intake via FFQ cancer risk and total dietary folate intake.
A nested case-control study in the Canadian National Breast
Patients with colorectal Folate intake was inversely associated with the risk of
Terry et al. [90] Screening Study
cancer and matched controls colorectal cancer
Estimated dietary intake via FFQ
A nested case-control study in the Nurses’ Health Study (NHS) and Folate intake was associated with lower risk of colon cancer;
Patients with colorectal
Wei et al. [91] the Health Professionals Follow Up Study (HPFS) however, rectal cancer cases tended to have slightly
cancer and matched controls
Estimated dietary intake via FFQ higher folate
There were no independent associations of folate with
Population-based Iowa Women’s Health Study cohort incidence of colon cancer; however, relative risk was lower
Harnack et al. [92] Female adults
Estimated dietary intake via FFQ among those who had a combined high folate and high vitamin
B-12 or high folate and vitamin B6.
Colorectal cancer and A case-control study Folate intake was associated with reduced risk of
Benito et al. [93]
matched controls Estimated dietary intake via FFQ colorectal cancer
Colorectal cancer and A case-control study There was a trend of a protective effect of high folate intake
Ferraroni et al. [94]
matched controls Estimated dietary intake via FFQ against colorectal cancer development
Colorectal cancer and A case-control study Folate intake was associated with a reduced risk of rectal
Freudenheim et al. [95]
matched controls Estimated dietary intake via FFQ cancer but not colon cancer
A nested case-control study within the Alpha-Tocopherol No association between serum folate and colorectal cancer.
Patients with colorectal
Glynn et al. [96] Beta-Carotene Study cohort of male smokers High dietary folate intake was protective against colorectal
cancer and matched control
Estimated dietary intake via FFQ and serum levels were measured cancer.
Patients with colorectal Case-control study No association between dietary folate and risk of
La Vecchia et al. [97]
cancer and matched control Estimated dietary intake via FFQ colorectal cancer
Patients with colorectal Case-control study Decreased risk of colorectal cancer in subjects who consume
Le Marchand et al. [98]
cancer and matched control Estimated dietary intake via FFQ high levels of folate and vitamin B6
Patients with colorectal Case-control study No significant association between folate intake and
Levi et al. [99]
cancer and matched control Estimated dietary intake via FFQ colorectal cancer
Boutron-Ruault Patients with colorectal Case-control study Folate intake prevents adenoma formation and protective
et al. [100] cancer and matched control Estimated dietary intake via FFQ against adenoma growth associated with alcohol
A nested case-control study in the New York University Women’s The risk of colorectal cancer in the subjects in the highest
Patients with colorectal
Kato et al. [101] Health Study cohort quartile of serum folate concentrations was half that of those in
cancer and matched control
Serum levels were measured the lowest quartile
Patients with colorectal Randomized, double-blind controlled trial Folic acid at 1 mg/day does not reduce the risk of colorectal
Cole et al. [102]
adenoma 1 mg/day of folic acid for 3 years adenomas or their advancement to neoplastic lesions
Norwegian Vitamin Trial and Western Norway B Vitamin Folic acid plus vitamin B12 supplementations were associated
Male and female adults with
Ebbing et al. [103] Intervention Trial with increased cancer outcomes and all-cause mortality in
ischemic heart disease
folic acid (0.8 mg/day) plus vitamin B12 (0.4 mg/day) for 6–7 years patients with ischemic heart disease
Nutrients 2019, 11, 608 11 of 30

Table 2. Cont.

Authors Population/Tissue Study Design Conclusion/Outcome

Vitamin B
Patients with colorectal Case-control study Neither vitamin B2, vitamin B6, nor vitamin B12 were
Otani et al. [104]
cancer and matched control Estimated dietary intake via FFQ significantly associated with colorectal cancer
Patients with prostate cancer Case-control study Serum concentrations of vitamin B12 were associated with an
Hultdin et al. [105]
and matched control Serum levels were measured up to three-fold increase in prostate cancer risk
Plasma levels of vitamin B12 were inversely associated with
Gylling et al. [87] Details are in the folate section of the table
rectal cancer risk
Choline and Betaine
Serum betaine but not choline was inversely associated with
Patients with breast cancer A hospital-based case-control study
Du et al. [106] risk of breast cancer development in subjects with
and matched control Serum levels were measured
below-median dietary folate intake
Total choline intake was inversely associated with colorectal
Patients with colorectal Case-control study
Lu et al. [107] cancer risk however no significant associations were observed
cancer and matched control Estimated dietary intake via FFQ
for betaine or total choline plus betaine intakes
Patients with nasopharyngeal Case-control study Intakes of total choline, betaine, and combined choline and
Zeng et al. [108]
cancer and matched control Estimated dietary intake via FFQ betaine were inversely associated with nasopharyngeal cancer
Patients with liver cancer and Case-control study Higher intake of choline and betaine was associated with a
Zhou et al. [109]
matched control Estimated dietary intake via FFQ lower risk of liver cancer
A nested case-control study within the European Prospective Higher betaine and choline concentrations were associated
Patients with colorectal
Nitter et al. [110] Investigation into Cancer and Nutrition (EPIC) with lower risk of colorectal cancer especially in subjects with
cancer and matched control
Plasma concentrations were measured lower folate concentrations
Methionine
A direct association between higher methionine intake and
Patients with prostate cancer Case-control study
Feigelson et al. [111] prostate cancer risk was observed only in men who have at
and matched control Estimated dietary intake via FFQ
least one MTHFR A1298C allele
Methionine intake was inversely associated with risk of having
Giovannucci et al. [83] Details are in the folate section of the table
larger adenomas (1 cm or larger)
Significantly increased risk of colon cancer in men who
Su et al. [84] Details are in the folate section of the table consume low-methionine diet compared to those who consume
high methionine diet
Higher intake of methionine reduces the risk of colon cancer
Fuchs et al. [85] Details are in the folate section of the table
associated with a family history of the disease
Methionine concentrations were inversely associated with
Nitter et al. [110] Details are in the betaine and choline section of the table
colorectal cancer risk with borderline significance
Nutrients 2018, 11,
Nutrients 2019, 10, 608
x FOR PEER REVIEW 12 of
22 of 30
34

Figure 2. Schematic representation of DNA methylation and its effect on gene transcription. DNA
methyltransferase
Figure 2. Schematic(DNMT) convertsof
representation cytosine to 50 methyl-cytosine.
DNA methylation The process
and its effect on geneinvolves the transfer
transcription. DNA
of a methyl group(DNMT)
methyltransferase from S-adenosylmethionine (SAM) to the cytosine
converts cytosine to 5′methyl-cytosine. resulting
The process in the the
involves conversion
transfer
of
of DNA to methylated
a methyl group from DNA and methylated (SAM)
S-adenosylmethionine SAM to to non-methylated S-adenosylhomocysteine
the cytosine resulting in the conversion of
(SAH).
DNA toDNA methylation
methylated DNArecruits histone deacetylase
and methylated (HDAC), methyl
SAM to non-methylated binding proteins (MBPs),
S-adenosylhomocysteine and
(SAH).
other
DNA transcription repressing
methylation recruits factors.
histone These modifications
deacetylase resultbinding
(HDAC), methyl in closed chromatin
proteins conformation,
(MBPs), and other
inaccessibility
transcription to transcriptional
repressing machinery,
factors. and eventually
These modifications gene in
result silencing.
closed chromatin conformation,
inaccessibility to transcriptional machinery, and eventually gene silencing.
5. Micronutrients and DNA Methylation and Their Impact on Cancer
Author Contributions: A.M.M.: Conceptualization, editing, and reviewing final draft. M.M.A.:
5.1. Folate
Conceptualization, editing, and reviewing final draft.
Folate naturally occurs in a wide variety of foods. Rich sources include leafy green vegetables,
Funding: This research and the APC were funded by the National Institute of Health grant number
beans, peas, lentils, fruits like lemons, bananas, and melons, and fortified and enriched products,
1K99HL140049-01 (AMM).
such as breads and cereals [112]. Folate is essential for several biological cell processes such as DNA
Acknowledgments:
synthesis and methylation,This production
review article and was supported
maintenance of newby cells,
the following
and aminofunding source: [50].
acid metabolism NIH
1K99HL140049-01 (AMM).
In several studies, folate supplementation has been shown to increase DNA methylation. For
example,
Conflicts in of aInterest:
clinical No
trialpotential
conducted by Wallace
conflicts of interestetwere
al. [56], folate supplementation increased DNA
disclosed.
methylation of two proto-oncogenes in colorectal mucosa, estrogen receptor alpha (ER-α) and secreted
References protein-1 (SFRP-1). Similarly, in an in vitro model of neuroblastoma cells, Li et al. [113]
frizzled-related
have
1. shown S.;
Biswas, that treatments
Rao, with folic
C.M. Epigenetics acid (20
in cancer: and 40 mmol/L)
Fundamentals increased
and beyond. s-adenosylmethionine
Pharmacol. to
Ther. 2017, 173, 118–134.
s-adenosylhomocysteine
2. Ducker, G.S.; Rabinowitz, ratioJ.D.
and subsequently
One-carbon downregulated
metabolism in health and thedisease.
abnormally phosphorylated
Cell Metab. tau
2017, 25, 27–42.
protein
3. by inhibiting the demethylation of its regulator, PP2A (protein
Zeisel, S. Choline, other methyl-donors and epigenetics. Nutrients 2017, 9, 445. phosphatase 2A). Additionally,
animal
4. studies
Jung, suggest
M.; Pfeifer, G.P.that folate
Aging anddeficiency or supplementation
DNA methylation. BMC Biol. 2015,modifies
13, 7. the methylation status of
DNA
5. promoterK.A.;
Lillycrop, in genes
Burdge,thatG.C.
areEpigenetic
critical in mechanisms
carcinogenesis.linkingFolate-rich diet increased
early nutrition DNA
to long term methylation
health. Best Pract.
of genes suchEndocrinol.
Res. Clin. as p16 [114] in 2012,
Metab. mouse 26, colon
667–676. and protooncogenes such as PDGF-B (platelet-derived
growth
6. factor-B),
Klutstein, M.; Ras (rat D.;
Nejman, sarcoma), andR.;
Greenfield, survivin
Cedar, H. inDNA
a mouse model in
methylation of cancer
gliomagenesis
and aging.[115].
Cancer Res. 2016,
Conflicting
76, 3446–3450. results regarding the effect of folate status on DNA methylation have been reported.
For
7. example, global DNA
Kulis, M.; Queiros, hypomethylation
A.C.; Beekman, was reported
R.; Martin-Subero, in three
J.I. Intragenic DNA different murine
methylation studies in
in transcriptional
regulation, normal differentiation and cancer. Biochim. Biophys. Acta 2013, 1829, 1161–1174.
response to either a folate-deficient or a folate-rich diet provided during gestation, lactation, or after
8. Bakshi,
weaning A.; Bretz, C.L.;
[116–118]. Cain, T.L.;
Another study Kim, J. Intergenic
showed that and intronic DNA hypomethylated
a folate-deficient diet reduced regions
tumor as putative
size in a
regulators
colorectal cancer of imprinted
mouse model domains.
withEpigenomics
no effect on 2018, 10, 445–461.
global or gene-specific DNA methylation. However,
9. reduction
this Lyko, F. The inDNA
tumormethyltransferase
size was onlyfamily: observed A versatile
when toolkit for epigenetic regulation.
the folate-deficient diet was Nat. Rev. Genet.
administered
2018, 19, 81–92.
after tumor development, and no effect was observed when folate deficiency was induced before
Nutrients 2019, 11, 608 13 of 30

tumor development [119]. In a study by Kotsopoulos et al. [120], folate-deficient diet increased DNA
methylation in rat liver when administered post weaning and this effect continued through adulthood;
no changes in DNA methylation were observed when either a folate-deficient or a folate-rich diet was
administered at puberty. These findings may suggest that the dietary level of folate is not the only
determinant for DNA methylation status and that other confounding factors might modulate the role
of folate as a methyl donor.
Similarly, data from human studies that assessed the contribution of folate deficiency or folate
supplementation on cancer risk or progression is inconsistent and varies significantly based on
factors such as folate dose, mode of intake (dietary versus supplementary), stage of development
during exposure (prenatal versus postnatal), and the pathological status (normal versus neoplastic) of
tissues [121–124]. Other factors such as age, genetic and epigenetic background, alcohol intake, and
comorbidities might influence the outcome in folate consumers [125,126].
Global DNA hypomethylation and gene-specific hypermethylation are significant characteristics
of some cancers such as colorectal and prostate cancer and reversing these epigenetic changes has
been of great interest for cancer researchers [127,128]. Folate intake has been reported to correct
global DNA hypomethylation and imprint proto-oncogenes such as H-Ras in patients with colorectal
cancer [129]. In fact, several studies that investigated the effect of folate on cancer focused on colorectal
cancer. For example, an observational study by Giovannucci et al. [83] reported an inverse association
between high folate intake and the risk of colorectal adenoma in men and women from the Health
Professional Follow-up Study and the Nurses’ Health Study, respectively. Furthermore, findings from
the NHANES (National Health and Nutrition Examination Survey) Epidemiologic Follow-up Study
(NHEFS) support the association between folate intake and reduced risk of colon cancer [84]. In this
study, men who consumed more than 249 µg/day had a lower risk of colon cancer (Relative Risk
(RR): 0.40). Similarly, a prospective study of 88,758 women reported reductions in colon cancer risk
in women who consumed more than 400 µg/day (RR: 0.81), especially those with a family history of
colon cancer (RR: 0.48) [85]. These findings were reproduced by Stevens et al. in 43,512 men and 56,011
women from the Cancer Prevention Study II Nutrition Cohort. Folate intake was also shown to reduce
prostate cancer risk in the American Cancer Society Cancer Prevention Study II Nutrition Cohort [86].
However, conflicting evidence has emerged, indicating that the mechanisms associated with
folate effect on DNA methylation are more complex than previously thought and confounded by other
dietary, genetic, or tissue-related factors. For example, a prospective nested case-control study of
331 cases and 662 matched controls in the population-based Northern Sweden Health and Disease
Study demonstrated an association between low plasma levels of folate and reduced risk of colon
cancer [87]. This study concluded that low circulating levels of folate are protective against colon cancer.
Findings from other epidemiological studies were inconsistent; only five out of seven prospective
studies [84,88–92,130] and seven out of 11 case-control studies [93–101,131,132] have reported a
protective effect of folate intake against colon cancer. This discrepancy motivated researchers to
conduct meta-analyses of published observational studies to provide an overall estimate of the
association between folate intake and colorectal cancer risk. A meta-analysis by Sanjoaquin et al. [133]
indicated that dietary folate has a more protective effect on colorectal cancer than supplemental
folate. However, confounding factors such as gender, other dietary factors, and alcohol consumption
modify the association. A relatively recent meta-analysis reported a lack of association between folate
supplementation and total cancer incidence including colorectal cancer, lung cancer, prostate cancer,
or breast cancer [134]. Observational studies that assessed the association between folate status and
global DNA hypomethylation in cancer patients have also yielded mixed findings. An association
between global DNA hypomethylation and increased risk of colon, cervical, and bladder cancer was
established in these studies [57–59]. However, a role of low folate status (intake or blood levels) in this
association could not be consistently found. A significant association between folate status and DNA
hypomethylation was reported by Piyathilake et al. [60] in their study of 376 women with cervical
intraepithelial neoplasia. Other studies reported either a weak [61] or null association [58,59].
Nutrients 2019, 11, 608 14 of 30

This inconsistency extended to clinical trials. For example, in a randomized clinical trial,
folate supplements (600 µg/day) for two years significantly increased tissue folate and global DNA
methylation in twenty post-polypectomy Patients [62]. Similar results were obtained in other clinical
trials where folate was administered by patients with colorectal adenoma or carcinoma for shorter
periods (3–6 months) [61,63,64,135]. On the other hand, a randomized clinical trial by Cole et al. [102]
demonstrated that daily folic acid supplementation (1mg/day) for three to five years did not reduce the
risk of adenoma recurrence in 1021 men and women diagnosed with colorectal adenoma. Furthermore,
a combined analysis of participants from two randomized clinical trials (Norwegian Vitamin Trial
and Western Norway B Vitamin Intervention Trial) demonstrated significant increases in lung cancer
risk in participants who administered folic acid (800 µg/day) and vitamin B12 (400 µg/day) [103].
It is worth mentioning that folate doses in these two studies were twice the recommended daily
folate intake (400 µg/day) provided in the Dietary Reference Intakes (DRIs) [136] suggesting a
biphasic dose-dependent response to folate intake. This assumption is supported by a dose-response
meta-analysis by Zhang et al. [137] where a J-shaped correlation between folate intake and breast
cancer risk was found. In this meta-analysis, women who consumed 200–320 µg/day were at a lower
risk of developing breast cancer; however, women who consumed more than 400 µg/day had a
significantly higher breast cancer risk.
Thus far, studies that measured the association between folate intake and global DNA methylation
in cancer have yielded inconsistent results as shown above. This inconsistency could be related to
the use of varying doses over varying periods; however, a critical confounding factor that should be
considered is the type of methylation assays that were used and what they actually measure [138].
Some of these assays measure DNA methylation status in repetitive elements such as LINE (Long
Interspersed Nucleotide Element 1), SINE (Short interspersed nuclear elements), and Alu ( Arthrobacter
luteus restriction endonuclease) repeats and others examine differentially methylated regions via
methylation-sensitive restriction enzymes [139]. Furthermore, some of the assays cover the whole
genome while others cover only a certain percentage of the genome [138]. These assays, in general, have
different sensitivity and specificity and are prone to over- or under-estimation errors. Details about
global DNA methylation analysis methods are beyond the scope of the current review; the reader is
directed to a recent article by Kurdyukov et al. [140] that summarizes and compare these methods
in terms of outcomes and appropriateness for specific research questions. However, it is worth
mentioning that the correlation among these assays is poor, which could mainly be attributed to the
fact that they measure different subsets of DNA sequences in diverse regions of the genome [141].
Distinct methylation profiles of various repetitive DNA elements have been found among different
tissues and pathological conditions. Furthermore, loss of DNA integrity in cancer may compromise
the accuracy of global DNA methylation assay outcome [142]. Thus, using different global DNA
methylation assays to evaluate the effect of folate status on DNA methylation could be the reason
behind the reported inconsistent and irreproducible findings.
Compared with global DNA methylation, gene-specific methylation analyses in response to
folate status have demonstrated more consistent findings. A study by Wallace et al. [56] assessed
DNA methylation at specific CPG sites in estrogen receptor alpha (ER-α) and secreted frizzled-related
protein-1 (SFRP1) in patients with previous colorectal adenomas who administered folate supplements
for three years. In this study, higher levels of folate were associated with higher methylation in the
CpG-rich promoters of ER-α and SFRP1; the expression of which has been correlated with an increased
risk of colorectal cancer [143,144]. Similarly, promoter methylation of RASSF1A, a proto-oncogene,
correlated positively with serum folate concentration in nested cases with malignancies and controls
from a prospective study with reported dietary habits and lifestyles [67].
Together, these data suggest that folate might be protective against some cancers via inducing
promoter methylation of proto-oncogenes; however, different genes and CpG sites are not equally
susceptible to DNA methylation changes in response to folate intake. Accordingly, studies that utilize
a candidate gene-specific analysis to investigate the effects of folate on DNA methylation are expected
Nutrients 2019, 11, 608 15 of 30

to produce more accurate results than global methylation studies. Furthermore, studies that examine
thousands of CpG loci simultaneously will help elucidate the interaction between DNA methylation
and folate intake. These studies should take into consideration modifying factors such as age, gender,
genetic background, time of exposure (pre-cancer versus post-cancer development), mode of exposure
(dietary versus supplemental), and other dietary habits and lifestyles.

5.2. Riboflavin, Pyridoxine, and Cobalamin

B vitamins are water-soluble vitamins That are found in a variety of foods such as meat, wholegrains,
eggs, dairy products, legumes, nuts, dark leafy vegetables, and fruits (citrus fruits, avocados, and
bananas) [136]. There are several types of vitamin B including thiamin (vitamin B1), riboflavin (vitamin
B2), niacin (vitamin B3), pantothenic acid, vitamin B6, biotin (vitamin B7), and vitamin B12. While all
types of vitamins B are essential in regulating metabolism, hemoglobin synthesis, and maintaining
skin and nervous system integrity, vitamins B2, B3, B6 and B12 are specifically critical for one carbon
metabolism [145]. Accordingly, the intake of these vitamins may induce DNA methylation and gene
expression changes and modify the risk of diseases where DNA methylation play an important role
such as cancer. In support of this notion, increased intake of vitamins B2, B6, and B12 by humans have
been shown to inversely correlate with cancers such as esophageal cancer [146], cervical intraepithelial
neoplasia [76], colorectal cancer [104], and prostate cancer [105]. However, the association between
vitamin B levels or administration and DNA methylation was only reported in a few studies that
measured the joint effect of vitamin B and folic acid status or included vitamin B in a combined model
of other vitamins and micronutrients. Animal studies showed that vitamin B12 deprivation induced
global hypomethylation even when combined with a folate-rich diet, which emphasizes the crucial
role of vitamin B12 in catalyzing one-carbon metabolism and DNA methylation [147]. This and other
animal studies referred to the importance of maintaining a balance between folate and Vitamin B12 in
order to preserve physiological DNA methylation patterns [148].
In examining the relationship between vitamin B12 levels and homocysteine levels in the blood,
several human studies have reported an inverse association [149,150]. Furthermore, Vitamin B6 and
B12 intake was effective in reducing circulating levels of homocysteine [151,152]. These findings along
with the one-carbon metabolism pathway we discussed above suggest that B vitamins contribute
toward DNA hypermethylation. Nonetheless, studies that reported an association between vitamin B
intake and the reduced risk of cancer are inconsistent to whether this protective effect is associated with
an induced hypo- or hypermethylation either globally or at the candidate gene level. For example, in a
study by Colacino et al. [72], a methylation score for several tumor suppressor genes was calculated and
correlated with dietary intake of micronutrients in patients with head and neck cancer. In this study,
individuals with the highest quartile of vitamin B12 intake showed significantly less tumor suppressor
gene methylation compared with those in the lowest quartile. Additionally, two observational studies
have shown that levels of vitamin B12 correlated with global DNA hypomethylation in lung and breast
cancer tissues [73,153]. Furthermore, findings that provide minimal to no support of the assumed
effect of vitamin B on DNA methylation have been reported. For example, in two separate studies,
a lack of any significant changes or correlations between LINE-1 repetitive element methylation and
vitamin B levels or supplementations in school-age children or elderly was reported [74,75].

5.3. Choline and Betaine

Choline is an essential nutrient that acts as an indirect methyl donor and is involved in several
physiological processes such as methylation reactions, cellular membrane integrity, neurotransmitter
synthesis, and metabolism [154]. The human body is capable of synthesizing choline in a minimal
amount that is not enough to meet the body needs [155]. Thus, the primary source of choline is dietary
including fish, poultry, eggs, cruciferous vegetables, and dairy products. The daily recommended
intake of choline is 425 and 550 mg for women and men, respectively [136]. Betaine can be obtained
from dietary sources or formed inside the body through irreversible oxidation of choline [156]. Betaine
Nutrients 2019, 11, 608 16 of 30

is a direct methyl donor; it donates a methyl group to homocysteine resulting in its conversion to
methionine [157]. The role of betaine in the methylation process becomes more crucial under conditions
of folate deficiency such as excessive alcohol consumption that impairs folate metabolism and interferes
with folate-dependent methylation [158,159].
Several studies reported inverse correlations between dietary choline or betaine intake or their
plasma levels and homocysteine bioavailability in humans. Melse-Boonstra et al. [160], reported that
plasma levels of betaine are significant determinants of fasting homocysteine levels in healthy humans.
Cross-Sectional analysis in 1477 women by Chiuve et al. [78], reported lower homocysteine levels
in the highest quintile of total betaine and choline intake compared to the lowest quintile. These
inverse associations were more pronounced in women who had low folate or high alcohol intake.
Similarly, in a study by Lee et al. [161], the association between choline and betaine intake and fasting
homocysteine concentrations were evaluated in 1325 males and 1407 females who participated in
the Framingham Offspring Study. In this study, higher choline and betaine intakes were associated
with lower homocysteine, especially in participants with low plasma levels of folate and vitamin B12.
This association was no longer detected in the post-fortification period where most of the products in
the United States were fortified with folic acid (140 g folic acid/100 g flour per grain).
In addition to these observational studies, supplementation with either betaine or choline has
been shown to reduce homocysteine concentrations in clinical trials. For example, a study by
Brouwer et al. [162], reported significant reductions in plasma total homocysteine in 15 healthy
participants who administered six grams of anhydrous betaine daily for three weeks. Similar findings
were observed in obese subjects who administered betaine (6 g/day) for 12 weeks [79] and healthy
men who ingested phosphatidylcholine (2.6 g/day) for two weeks [80]. Doses of betaine and choline
that were used in these short-term interventions are much higher than the recommended daily
intake we referred to above. Accordingly, further trials that utilize comparable doses to regular daily
consumptions are recommended.
Since choline and betaine can act as methyl donors and modify the bioavailability of homocysteine,
several studies, mostly animal-based, investigated the association between choline and betaine status
and global and candidate gene methylation [3]. Analyses of global and gene-specific DNA methylation
in rodent offspring exposed to maternal choline-deficient diet demonstrated global hypomethylation
and prompter hypomethylation of CDKN3 (cyclin-dependent kinase 3) [163] calbindin1 [164], VEGF-C
(vascular endothelial growth factor-C) and ANGPT2 (angiopoietin 2) genes [165]. Interestingly, in
a study by Kovacheva et al. [166], global hypomethylation and gene-specific hypermethylation
(insulin-like growth factor-2 (IGF-2)), were observed in rodent offspring exposed to maternal
choline-deficient diet. In this study, a concomitant hypomethylation was observed in DNMT1
promoter. These data suggest an early effect of choline deficiency where the lack of methyl groups and
impaired one-carbon metabolism result in global hypomethylation that encompasses DNMT1 promoter.
This is followed by a phase where the augmented DNMT1 expression subsequently increases DNA
methylation at specific gene loci such as IGF-2. This assumption necessitates further investigations;
however, if proven right, it may explain the encountered discrepancy among studies that investigated
the association between methyl-donating nutrients and DNA methylation status. In this case, factors
such as the duration of nutrient intake should be taken into consideration.
Secondary to their role in modifying DNA methylation, choline and betaine status is associated
with carcinogenesis [167]. Low choline diet increased levels of S-adenosylhomocysteine in rodents’
livers, reduced DNMT activity, and DNA methylation, and increased the incidence of developing
hepatocellular carcinoma [168,169]. While some oncogenes such as c-myc were hypomethylated
in this low choline-diet rodent model, the promoter of some tumor suppressor genes such as p53,
p16INK4a, PtprO, Cdh1, and Cx26 was hypermethylated [170–172]. In support of the role of betaine
and choline in protecting against hepatocellular carcinoma, Lupu et al., [173] reported that, in a mouse
model, the deletion of betaine-homocysteine methyltransferase gene, which is implicated in choline
Nutrients 2019, 11, 608 17 of 30

metabolism resulted in the spontaneous elevation of S-adenosylhomocysteine levels and development

of preneoplastic foci in the liver.
In human studies, four case-control studies demonstrated an inverse relationship between choline
and betaine intake and the risk of developing breast cancer [106], colon cancer [107], nasopharyngeal
cancer [108], and liver cancer [109]. Interestingly, in a nested case-control study within the European
Prospective Investigation into Cancer and Nutrition (EPIC) cohort, a significant inverse association
between plasma levels of betaine and colorectal cancer risk was observed only in participants with
low folate concentrations [110] suggesting that choline and betaine may serve as alternative methyl
group donors in states of folate deficiency. A meta-analysis of 11 epidemiological studies supported
the protective role of dietary betaine and choline against several types of cancer with the most
considerable effect reported for breast cancer followed by nasopharyngeal and lung cancers [174].
In this meta-analysis, it was found that an increment in dietary betaine and choline of 100 mg/day
reduced cancer incidence by 11%. In summary, cumulative evidence suggests a significant contribution
of choline and betaine to methyl metabolism and DNA methylation and accordingly to gene regulation
in carcinogenesis.

5.4. Methionine
Methionine is an essential sulfur-containing amino acid that plays a significant role in regulating
metabolism and synthesizing other amino acids, such as cysteine and taurine, and proteins, such as
carnitine and melatonin [175,176]. Methionine is not synthesized in the body and should be obtained
from foods. While nearly all protein-containing foods have some methionine, eggs, fish, soy, dairy
products, and some meats contain high amounts of this amino acid [177]. Methionine is an integral part
of the one-carbon metabolism since it serves as a precursor for s-adenosylmethionine, the universal
methyl donor for DNA methylation [178]. Thus, dietary intake of methionine is expected to correlate
with DNA methylation. However, due to the cyclical nature of the one-carbon metabolism process,
methionine excess may have a negative impact on DNA methylation via inhibiting homocysteine
methylation to maintain a balance between s-adenosylhomocysteine and s-adenosylmethionine [179,180].
Indeed, there is limited evidence suggesting that dietary methionine induces gene-specific DNA
hypermethylation [181]. Furthermore, the effect of methionine is tissue-specific, and studies that
measured the correlation between methionine intake and plasma s-adenosyl methionine to s-adenosyl
homocysteine ratio have yielded inconsistent findings. For example, three animal studies that have
examined the effects of dietary methionine intakes on hepatic levels of methionine, s-adenosyl
methionine and s-adenosyl homocysteine demonstrated a significant discrepancy. Finkelstein and
Martin [179] reported a lack of induction of hepatic methionine and reduction in the s-adenosyl
methionine to s-adenosyl homocysteine ratio in response to methionine-rich diet intake for seven days.
In their study, Regina et al. [180] demonstrated a 20-fold increase in hepatic and plasma methionine and
80% increase in the s-adenosyl methionine to s-adenosyl homocysteine ratio in rats fed methionine-rich
diet for 14 days. In this study, a lack of increase in the s-adenosyl methionine was observed in the
kidney, small intestine, and skeletal muscle. Lastly, Rowling et al. [182] observed an eight-fold increase
in hepatic s-adenosyl methionine to s-adenosyl homocysteine ratio in rats fed methionine diet for
10 days. The observed inconsistencies among these studies indicate the complex nature of the effect of
methionine that might vary among different tissues and might depend on several factors related to
age or duration of exposure.
These contradictory effects might explain the lack of robust correlations between methionine
intake and DNA hypermethylation. For example, rats fed a methionine-rich diet showed no changes
in p53 [183] or cystathionine beta-synthase promoter methylation [184]. Similarly, in human studies,
methionine was associated with decreased methylation levels of RASSF1A, a gene that is implicated
in several malignancies [67]. Despite this lack of association between dietary methionine and DNA
hypermethylation at the gene level, a positive association at the global level has been observed in
mouse gut [185]. In general, there is an evident lack in human studies that measured the effect of
Nutrients 2019, 11, 608 18 of 30

dietary methionine on DNA methylation and whether high dietary methionine intake induces DNA
hyper- or hypomethylation is still to be determined. Moreover, epidemiologic findings as to whether
dietary methionine intake protects against cancer in humans are inconsistent [186]. Higher dietary
methionine intake was associated with increased prostate cancer risk (OR = 2.1; 95% CI 1.1–3.9) in
men with low Gleason score [187] and failed to demonstrate any association with breast cancer risk
in the American Cancer Society Cancer Prevention Study II Nutrition Cohort [111]. Additionally,
several ongoing studies are exploiting dietary methionine restriction as a potentiator for the effect of
cancer chemotherapy regimen in metastatic cancer [188–190], melanoma, and glioma [191]. On the
other hand, a meta-analysis of eight prospective studies that measured the association between dietary
methionine intake and risk of colon cancer in 431,029 participants reported a summary relative risks
(RRs) of 0.77 (95% CI = 0.64–0.92) for the highest versus lowest methionine intake [186]. Additionally,
in a dose-response meta-analysis, a linear dose-response relationship was found between methionine
intake and the risk of breast cancer; the risk was reduced by 4% for every 1 gram per day increment in
dietary methionine intake [192]. In summary, the available findings to date support the need for further
investigations to elucidate the direction of the association between dietary methionine and cancer risk
and highlight the underlying molecular and epigenetic mechanisms for this potential association.

5.5. The Impact of Alcohol and Smoking on Nutrient-Mediated DNA Methylation

Chronic alcohol consumption was a strong predictor of the association between methyl
donor nutrient deficiency and the disturbed one-carbon metabolism in several human
studies [78,133,158,159,161]. Alcohol has been shown to inhibit methionine synthase activity in the
liver resulting in a significant reduction in s-adenosyl methionine levels. Alcohol administration in
rodents resulted in a significant decrease in liver concentrations of s-adenosyl methionine and DNA
methylation [193]. Additionally, cancer related genes such as c-myc showed increased expression
that was accompanied by increased risk of alcoholic liver disease and hepatocellular carcinoma in
mice [194,195]. Similarly, tobacco smoke interferes with the one-carbon metabolism and subsequently
with the availability of methyl groups required for DNA methylation [196–198]. Smoking was also
a strong predictor for the association between vitamin B6 and B12 and methylation for several
multi-disease-related gene promoters in humans [67]. Additionally, previous cross sectional studies
demonstrated that hydrocarbons from tobacco smoke are capable of interacting with vitamin B12 and
folic acid resulting in their biological inactivity [199].

5.6. The Impact of Early Nutrition in Modulating DNA Methylation

There is growing evidence that maternal nutrition during pregnancy and early postnatal period
affects epigenetic profiles of their offspring [200]. Several animal studies reported changes in
global and gene-specific DNA methylation in the progeny of animals fed low-protein diet [201,202],
caloric-restriction diet [203], or high-fat diet [204,205]. These studies suggest that prenatal or early
postnatal periods are critical for the establishment of the epigenome and vulnerable to environmental
factors such as nutrition. In support of this assumption, choline supplementation in a maternal murine
model modified methionine metabolism genes and global DNA methylation in the offspring [206].
Human studies also supported this assumption and a role of maternal diet in modifying fetal
DNA methylation of growth and metabolic genes has been shown in individuals exposed prenatally
to famine, and conceivably extreme folate deficiency, during the Dutch Hunger Winter at the end of
World War II [207,208]. This association was also supported by Steegers-Theunissen et al. [209], where
maternal use of folic acid periconceptually resulted in 4.5% increase in IGF2 methylation and reduced
birth weight in children compared to those born to women who had not taken folic acid. Additionally,
a study by Ba et al. [69], showed that IGF2 methylation in fetal blood was positively associated with
vitamin B12 levels in maternal blood.
Positive associations were observed in the Maternal Nutrition and Offspring’s Epigenome
(MANOE) study for maternal intake of betaine and folate and gene-specific DNA methylation in infants,
Nutrients 2019, 11, 608 19 of 30

mainly RXRA (retinoid X receptor alpha) gene [210]. Furthermore, the MANOE study demonstrated
a time trend for the relationship between methyl donor intake and DNA methylation in pregnant
women where women with higher methyl-group intake exhibited higher methylation in the third
trimester, and not in the first two semesters [77]. This time-sensitive relationship between methyl
donors and DNA methylation in pregnant women might be reflected on their infants’ epigenetic
patterns. However, further studies are warranted to investigate this assumption.
In summary, these studies showed the possibility that methyl donor nutrients in the maternal
diet could induce DNA methylation levels in the offspring. However, null, even inverse, associations
between maternal methyl donor nutrient intake and fetal global or gene-specific DNA methylation
have been reported in other clinical studies [70,211,212]. Therefore, recent studies have reported
concerns regarding maternal supplementations of folic acid basing their argument on the demonstrated
unexpected alterations in normal fetal DNA methylation that could induce detrimental effects [213–216].
Indeed, the relationship between methyl donors and DNA methylation is complex and may involve
other factors such as other dietary components or health status. Accordingly, definitive linear
associations of maternal methyl donor intake with fetal DNA methylation could not be confirmed.
Furthermore, dietary versus supplemental intake of methyl donors could be a source of inconsistent
outcomes. For example, while folic acid deficiency has been clearly shown to be associated with
adverse health outcomes, high folate concentrations due to supplementation correlated to mixed
outcomes as we summarized above. Accordingly, the currently available evidence is insufficient to
determine whether methyl donor supplementation during pregnancy or early postnatal would induce
favorable or unfavorable epigenetic effects on the offspring.

6. Perspectives of Nutritional Modification of DNA Methylation

Nutrients can regulate several biological processes in our body via modifying epigenetic mechanisms
that are critical for gene expression such as DNA methylation. These epigenetic modifications may
contribute to the status of our health or the health of our offspring. This dynamic interaction between
nutrients we consume throughout our lifetime and our epigenetic signature has now been identified
as Nutri-epigenomics. In the current review, we summarized the current literature that reported
findings and concepts related to the influence of direct and indirect methyl donor nutrients on DNA
methylation and cancer risk. Despite the promising insight Nutri-epigenomics could provide for
how to target health from a nutritional standpoint, our knowledge in this topic is still limited and
mostly animal-based. Data related to human consumption of methyl donor nutrients were mostly
collected from observational studies that are inherently prone to inaccuracy and recall bias. DNA
methylation status was assessed by either global methylation approach that lacks a robust clinical
relevance or candidate gene-driven approach with limited genome coverage and sensitivity. Further
studies in human subjects utilizing sensitive, high-throughput quantitative technologies with a broader
range of coverage to systemically analyze the role of methyl donors in modifying DNA methylation
and subsequently cancer risk or progression are required. Future work is needed to understand
better the interactions among methyl donors involved in one-carbon metabolism and strengthen our
understanding of their biological role in health and disease states. The fact that epigenetic marks
are potentially reversible and sensitive for nutritional supplementation or pharmaceutical therapies
makes the subject of Nutri-epigenomics very attractive, promising, and worthy of study. Yet, we must
acknowledge the complexity of the interaction between nutrients and DNA methylation, which is
not indicative of a single nutrient. Instead, DNA methylation is a highly regulated process that is
gene-sensitive and tissue-dependent and reflects the outcome of several pathobiological processes
and environmental exposures. Accordingly, future research should be designed in a way that dissects
the effect of single versus combined intake of methyl donors, dietary versus supplementary mode
of intake, and dose response relationship. Furthermore, researchers should consider the differential
response to methyl donors between healthy tissues and cancerous lesions where aberrant methylation
profiles might exist and modify the outcome. Finally, large scale clinical trials in both healthy people
Nutrients 2019, 11, 608 20 of 30

and cancer patients are needed in order to provide specific recommendations for methyl donor intake
that maintains normal methylation profiles in each group.

Author Contributions: A.M.M.: Conceptualization, editing, and reviewing final draft. M.M.A.: Conceptualization,
editing, and reviewing final draft.
Funding: This research and the APC were funded by the National Institute of Health grant number
1K99HL140049-01 (AMM).
Acknowledgments: This review article was supported by the following funding source: NIH 1K99HL140049-01
(AMM).
Conflicts of Interest: No potential conflicts of interest were disclosed.

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