Advances in Nutritional Epigenetics—A Fresh Perspective for an Old Idea. Lessons Learned,Limitations,and Future Directions

Old ideas and one carbon metabolism

Methylation has been a focus for nutritional epigenetic researchers since the mid-nineties when a group of leading researchers discovered associations between folate deficiency, 5,10 methylenetetrahydrofolate deficiency, methylenetetrahydrofolate reductase (MTHFR) inefficiency, neural tube defects, and cardiovascular disease.^34-41 Today, a PubMed search of the MTHFR enzyme at the National Library of Medicine generates over 7000 entries and has been associated with hundreds of conditions. ⁴² Single nucleotide variations (SNV) of MTHFR, the primary regulator of one-carbon metabolism and methyl group synthesis, was said to be the leading cause of active folate deficiencies leading to neural tube defects. ³⁵

B vitamins, including folate (B9) molecules derived from the diet, are considered cofactors for one-carbon metabolism, function as carriers of one-carbon methyl units ² and are well known for the epigenetic modulation of methylation. ⁴³ Analysis of metabolites and cofactors of one-carbon metabolism in erythrocytes, serum, and plasma, provide insight into vitamin deficiency, one-carbon metabolism activity and methylation. ²

Fortification of grains, including wheat flour and corn maze for the prevention of folate deficiency, had been discussed since 1974, ⁴⁴ but was not implemented in the United States until 1997 following repeated associations with folic acid depletion and the incidence of neural tube defects. ⁴⁴

DNA methylation

Methylation is a primary epigenetic mark essential for the regulation of gene expression and is generally associated with gene repression. ⁴⁵ Methyl groups for endogenous methylation of histones, nucleotides, and proteins are said to be derived from one-carbon metabolism. ² DNA methylation is a widely studied but not very understood epigenetic modification. DNA methyltransferase enzymes (DNMT) initiate DNA methylation through the binding of a methyl group to position 5′ of cytosine bases neighboring guanosine (CpG), generating 5-methyl-cytosine (5-mC). 5-mC is then oxidized to 5-hydroxymethylcytosine (5-hmC), which is said to promote de-methylation. ⁴⁵ The Ten-Eleven Translocation di-oxygenase (TET) family of enzymes are shown to contribute to the removal of the methyl group from cytosine, forming 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). ⁴⁵

CpG’s are found in clusters which are referred to as islands, spanning approximately 1000 bp at the promoter region of a gene and are generally found unmethylated. ⁴⁶ Methylated CpG’s at the promoter region of a gene are associated with gene repression. ⁴⁵ Differential methylation regions (DMR) are regions upstream of gene promoters that also influence a gene’s expression. ⁴⁷

5′ methylcytosines contribute to the rate of evolution as they are shown to deaminate at a much greater rate than unmethylated cytosines and are considered mutational hot spots within the genome.^48,49 Thymine DNA glycosylate (TDG) is essential for base excision repair throughout the DNA methylation and remethylating process. ⁴⁵

Promoter DNA methylation of any gene which results in a loss of gene function can have profound effects on phenotype and has the potential to cause disease. ⁵⁰ Inhibition and gene knock out studies which result in loss of function can often predict phenotypical outcomes of DMR or promoter region hypermethylation. ⁴⁷ For example; both promoter region CpG hypermethylation and DMR of the estrogen receptor1 (ESR1) which encodes the estrogen receptor alpha protein results in reduced ESR1 expression.^47,51 DMR of ESR1 has been implicated in a range of hormone-related diseases including ovarian endometriosis and resistance to hormone therapy.^47,51

Tryptophan hydroxylase 2 (TPH2) is essential for the first rate-limiting step in the synthesis of the 5-hydroxytryptophan (5-HTP), the immediate precursor to serotonin. ⁵² In animal models, reduced TPH2 activity is associated with depression-related behavior, aggression and altered sexual preference.^53,54

DNA methylation and reduced expression of TPH2 in humans has also been associated with suicide attempts in major depression. ⁵²

Histone methylation

The genome is comprised of hundreds of genes encoding writer histone methyltransferase enzymes capable of writing methylation to the histone. ¹⁹ Methylation of a repressive histone residue such as histone-3 lysine-9 results in a heterochromatin structural change to the epigenome and the exclusion of RNA polymerase from the genome, preventing gene expression. ¹⁹

Methyl groups derived from one-carbon metabolism are donated by a S-adenosylmethionine (SAM) dependant histone methyltransferase to a specific histone residue. ¹⁹ Dietary consumption of high folate foods, fortification or supplementation with cofactors of one-carbon metabolism such as folate and B12 contribute directly to histone methylation.^2,55

Histone demethylation

Histone demethylase enzymes erase the previously written methylation of the histone residue induced by the histone methyltransferase. ¹⁹

The enzymatic demethylase reaction by Jumonji C (JmjC)-domain-containing demethylases produce succinate and carbon dioxide, and is dependent on the presence of oxygen (O2), Fe(II) and the endogenous tricarboxylic acid (TCA) metabolite alpha ketoglutarate (aKG). ⁵⁶ Dietary derived ascorbate was also revealed to be an essential cofactor for JmjC-domain-containing histone demethylases ⁵⁷ which is supported in stem cell culture, with vitamin C treatment reducing global H3K9me2. ⁵⁸ Demethylase reactions are showing to be necessary for natural killer cell activation and expression of interferon-y (INF-y) in the anti-viral response. ⁵⁹ Inhibition of demethylase results in upregulation of glycolytic genes and downregulation of proinflammatory cytokines. ⁵⁹ Hypoxia and environmental substances such as nitric oxide (NO) have also demonstrated down-regulation of histone demethylase activity. ²⁹

Laboratory techniques

Seventeen years following experimentation of MTHFR within cultured cells,^60,61 the same researchers were the first to develop a polymorphism detection technique using the restriction enzymes produced by Bird ⁶² and self-synthesized primers derived from the RNA of a 90 bp porcine liver MTHFR gene. ⁶³ Restriction enzymes are still used today for detection of mutation and commercial microarray genotyping. ⁶⁴ However, the use of restriction enzymes for SNV detection have their limitations as they are incapable of differentiating between a deletion, non-methylated base, or unrepaired deaminated nucleotide making next-generation sequencing a preferable option for accurate genotyping.^65,66

It is now understood that cell culture storage and preparation conditions such as the use of formaldehyde interfere directly with the epigenome and contribute to DNA damage and mutation, making many early studies of enzyme activity unreliable. ⁶⁷ Moreover, cell culture media is enriched with a variety of vitamins, minerals, and amino acids, including folic acid, ⁶⁸ which is known to interact directly with the dynamic epigenetic activities of a cell. ²

Epigenetic patterns and gene expression are unique to each cell. ⁶⁹ However, studies of MTHFR deficiency are frequently performed on non-specific, unsynchronised cultured dermal fibroblasts,^60,70 despite Rosenblatt’s early discovery which demonstrated MTHFR activity of fibroblasts performed differently to lymphoblasts when exposed to SAM. ⁶⁰

Cell specificity and feedback inhibition of one-carbon metabolism

Stokstad and Kutzbach first described feedback inhibition of MTHFR activity in cell culture with the addition of SAM, the primary methyl donor required for methylation reactions.^71,72 This finding was later supported by Rosenblatt who found the addition of SAM resulted in the inhibition of the one-carbon metabolic enzymatic reactions in some cells but not others, also demonstrating cell-specific regulation of methyl group production. ⁶⁰ Collectively; this makes diagnosis and treatment of neurological disease, based on the MTHFR enzymatic activity of the skin or oral mucosa, unreliable.

Rosenblatt also showed how the enzymatic activity was dependant on the mitotic stage of cell cycle replication, demonstrating epigenetic fluctuation of gene expression in cell culture. ⁷³ S-adenosyl-homocysteine has shown to be a potent inhibitor of SAM, establishing multiple mechanisms that regulate one-carbon metabolism and methyl group availability. ⁷⁴

In 1977, Rosenblatt showed inhibition of MTHFR enzymatic activity in cell culture with the addition of 5-methyltetrahydrofolate (5-MTHF) to the media, again demonstrating feedback inhibition of one-carbon metabolism. ⁶⁰

Spontaneous deamination

In DNA methylation identification via sequencing; sodium bisulfite is used to induce spontaneous mutation of 5′ methylcytosine. Methylated cytosines remain as C, where as unmethylated cytosines are deaminated to uracil and then thymine. ⁷⁵ Compared to normal aging cells, immortalized cultured human fibroblasts are well known to deaminate resulting in a loss of their methylation marks due to epigenetic changes induced by constituents within the media. ⁷⁶ A consequence of the cyclic process of DNA methylation, demethylation and remethylation is the deamination of cytosine and the underrepresentation of CpG’s during the transition and prior to base excision repair by TDG. ⁴⁹ A loss of CpG’s in the genome is thought to be due to unrepaired deaminated bases, ⁷⁷ and therefore a deficiency in TDG results in a super-mutator phenotype and the progressive loss of CpG in the genome. ⁷⁸ These unrepaired transition mutations are said to account for half of the pathogenic mutations that occur within the human body, ⁷⁷ and are a significant contributor to evolution in viruses, ⁷⁹ eukaryotes and prokaryotes, ⁸⁰ and may also play a role in antibiotic resistance ⁸¹ increased viral pathogenicity or cross-species infection. ⁷⁹

Rosenblatt also noticed that MTHFR was thermolabile, and its enzymatic activity was strongly inhibited following a 30-minute incubation at 55°. ⁶¹ Heat and alkaline conditions have also demonstrated to increase the rate of deamination of 5 mC dramatically ⁸² suggesting that heat-induced deamination may have contributed to the inhibition of this enzyme, and folates in cell-culture media and DNA preparation techniques, including heat-inducing polymerase chain reaction (PCR), may influence the rate of deamination resulting in a change in gene expression and the under-representation of cytosine in sequencing. Overall this makes accurate detection of MTHFR single nucleotide variants with microarray genotyping difficult.

Evolution and the MTHFR polymorphism

Populations that have swayed radically from traditional diets do the worst when it comes to health and longevity. ⁸³ Using a model of hypertension, Laing explains how epigenetic modification as a result of a rapidly changing diet and environment explains this phenomenon. ⁸⁴ Studies of positive selection and great ape diversification found the most significant acceleration of evolution in processes essential for environmental adaptation. ⁸⁵ For example, the loss of human L-gulono-γ-lactone oxidase (GLO), the gene responsible for the final step in vitamin C synthesis, is said to be the result of changing oxidative conditions and increasing dietary consumption of fruit and vegetables. ⁸⁶ Similarly; the well documented LCT gene encoding the enzyme lactase-phlorizin hydrolase has shown strong positive selection and is associated with lactase persistence after weaning. ⁸⁷ However, shifts in allele frequencies alone do not explain this phenomenon, and DNA methylation of the LCT gene has also shown to contribute to lactose metabolism and its phenotype, indicating the epigenetic influence on alleles and evolution. ⁸⁸

DNA methylation patterns differentiate between ethnicity in population studies where a more significant percentage of methylation is seen in African-American, and Han-Chinese Americans compared to Caucasian-Americans. ⁸⁹ Researchers have confirmed methylation regulatory genes, MTHFR and betaine homocysteine s-methyltransferase (BHMT), are also subject to positive selection as a result of increased DNA methylation from environmental pressures including fortification and ultraviolet radiation exposure.^90-94 A genetic drift model of positive selection of the reduced function MTHFR single nucleotide variant, rs1801133 was demonstrated at high altitude in Tibet, which was suspected to be associated with increased exposure to ultraviolet radiation. ⁹⁵

It was previously thought that methane was not utilised by humans and was therefore passed unmetabolized through faeces or breath, however it has been recently documented that methane-rich saline, can alter immune function via changes in gene expression. ⁹⁶ Therefore, increasing levels of greenhouse gases, such as methane and carbon could drive the increased incidence of MTHFR single nucleotide variations through positive selection, resulting in the downregulation of endogenous methyl group production in some populations. It is therefore also likely that cells exposed directly to methane may require less endogenous methyl group synthesis and prolonged fortification may result in greater persistence of the reduced function MTHFR alleles such as the C677T genotype. In support of this hypothesis, environmental methyl radicals have shown to directly induce the formation of 5-methylcytosine of DNA and are therefore capable of influencing both epigenetics and evolution. ⁹⁷ This is proposing that activation of one-carbon metabolism may only be required when methyl groups are reduced, making dietary and supplemental enhancement of systemic methylation unnecessary. Additionally, the consequent increase in atmospheric temperature due to methane induced climate change, may also contribute to increased rates of evolution through increasing the rate of spontaneous deamination.

Neural tube defects

The mechanism by which folic acid supplementation prevents neural tube defects is still unclear, however, knock out and hypermethylation studies of various genes, including Paired Box 3 (PAX3) and repressive H3K27 histone methyltransferase Enhancer of Zeste Homolog (EZH2) impair neural tube closure.^98,99 Folic acid supplementation acts by reactivating these genes, which would be possible through either histone methylation of an activating residue, such as H3K4 or the inhibition of one-carbon metabolism. ¹⁰⁰ Mandatory fortification has shown to be effective as the incidence of neural tube defects has declined by 47%. ⁴⁴ However, the biological effects of fortification upon other cell types throughout embryonic differentiation have been less explored. Research suggests that prenatal folic acid may have increased the incidence of ankyloglossia, ¹⁰¹ which is also associated with infantile developmental delay. ¹⁰² As a result of fortification and supplementation, circulating levels of unmetabolized folates has largely increased over the past two decades. ¹⁰³ Elevated maternal serum folate in the 3rd trimester has been associated with reduced foetal growth ¹⁰⁴ and a murine model of high dietary folate supplementation exhibited impaired gestational development and protein utilization, proposing exogenous folates to be xenobiotic as opposed to nutritional. ¹⁰⁵

Methylation and autism spectrum disorder (ASD)

Studies demonstrating the pathogenic effects of folate supplementation during neural development and their association with neurological disorders such as ASD are growing in number. ¹⁰⁶ Researchers have proposed that the offspring of Mothers who have supplemented with folic acid have an increased risk of ASD. ¹⁰³

Polymorphic variations in the previously mentioned methyltransferase and chromatin remodeling gene EZH2 are also associated with the incidence of ASD, suggesting environmental and epigenetic adaptation of this gene may have played a role in the etiology. ¹⁰⁷

Methylation and development

It is well understood that histone modifications fluctuate during development. For example; fluctuation in the degree of expression of EZH2 is required for appropriate neural stem cell differentiation. ¹⁰⁸ High expression of EZH2 and consequent upregulation of H3K27me3 results in oligodendrocyte differentiation. ¹⁰⁸ In contrast, reduced expression results in the reduction H3K27me3 allowing gene expression for astrocyte or neuron differentiation.^109,110 It is understood that DNA methylation of CpG of promoter regions during development remain stable. ¹¹⁰ However, it has been demonstrated that deposition of the demethylation metabolite 5-hydroxymethylcytosine (5 hmC) in gene bodies during neurogenesis is associated with upregulation of EZH2 expression and the persistence of the repressive H3K27me3 mark, ¹¹¹ collectively representing the dynamic nature of DNA methylation and epigenetic regulation of gene expression very different to that of embryonic stem cells during differentiation. ¹¹¹ This implies that the use of folic acid during pregnancy may increase the expression of EZH2 during neurulation and prevent neural tube defects, but may also increase EZH2 expression persistently during neural cell development, with unknown neurological developmental consequences.

Folate and excitotoxicity

Natural sources of folate are conjugated to a mono, di or poly L-glutamate tail. ¹¹² Early studies of folic acid activity demonstrated folic acid administration to have excitatory activity in neurons which is likely due to L-glutamate being the major excitatory neurotransmitter in the brain.^113,114 High dose folate supplementation during pregnancy has shown to alter synaptic transmission, lowering the threshold and increasing the susceptibility for seizure in offspring. ¹¹⁵ Glutamic acid decarboxylase (GAD) enzyme is responsible for the conversion of excitatory neurotransmitter L-glutamate to the inhibitory neurotransmitter gamma-aminobutryric acid (GABA). ¹¹⁶ Due to reduced GABA synthesis, anti-GAD antibodies in the brain are associated with a range of neurological disorders, ¹¹⁷ including stiff person syndrome, cerebellar ataxia, Miller Fisher syndrome, eye movement disorders and epilepsy. ¹¹⁸ In pancreatic islet beta cells, GAD is the target for autoantibodies and autoreactive T cells in insulin and non-insulin-dependent diabetes mellitus.^116,118 In a laboratory; monoclonal antibodies against GAD are produced by injecting mice with 70 to 210 mcg dose of GAD. ¹¹⁹ Similarly, L-glutamate induces overexpression of GAD, potentially contributing to the production of GAD antibodies. ¹¹⁶ Moreover; folate polyglutamate tails have also demonstrated antagonism of glutamate receptor binding sites, potentially interfering directly with neural development or GAD antibody production. ¹²⁰

Methylation and affective disorders

In a murine model; increasing maternal or post-weaning folic acid alters gene expression resulting in anxiety-like behavior and hyperactivity. ¹²¹ Folic acid toxicity showed both down and upregulation of genes, including the upregulation of X-inactive specific transcript (XIST). ¹²¹ Overexpression of XIST is often present in affective disorders including bipolar and psychosis. ¹²²

Macrocytic anemia

Since 1931, folate and B12 deficiencies have been associated with macrocytic anemia, a condition of anemia due to a defect in erythrocyte differentiation resulting in abnormally large red blood cells.^44,123 During erythropoiesis, dynamic fluctuations in the expression of epigenetic modifiers and transcription factors are essential for effective differentiation of the erythroblast into a reticulocyte. ¹²⁴ The mature erythrocyte is anucleate and therefore no longer requires nuclear methylation, implying folate depletion throughout differentiation may be a natural consequence, ¹²⁵ and reduced erythrocyte folate may be the outcome for the appropriate use of substrate as opposed to a deficiency or a requirement for folate repletion.

Therefore, over-supplementation of folate may lead to epigenomic inflexibility and inadequate differentiation. Moreover, a PubMed search of macrocytic anemia shows significant evidence of mitochondrial dysfunction and reduced expression of cell cycle transcription factors playing a significant role in the development of macrocytic erythropoiesis.^126-128 This suggests that folate supplementation may contribute to disease in genetically susceptible individuals through enhanced methylation and its effectiveness for those who are treated successfully may have been due to feedback inhibition of one-carbon metabolism.

Homocysteine

Early studies have demonstrated elevated plasma or urinary homocysteine to be associated with inadequate methylation and are hallmark risk factors for cardiovascular disease.^129,130 Homocysteine is said to be converted to methionine by the vitamin B12 dependant methionine synthase (MS) in a reaction that also transforms 5-methyltetrahydrofolate into tetrahydrofolate, and by the zinc dependant BHMT enzyme.^131,132 From here, the ATP dependant methionine adenosyltransferase (MAT) converts methionine to SAM for epigenetic histone and DNA methylation reactions. ¹³³ Excess homocysteine is catalyzed to cystathionine by the enzyme cystathionine beta-synthase (CBS). ¹³⁴

Folic acid is shown to reduce plasma homocysteine levels; however, its efficacy is irregular and dependent on the cell-specific DNA methylation status of either BHMT or CBS ¹³⁴ further supporting self-regulated feedback mechanisms of one-carbon metabolism and demonstrates the phenotypical effects of biological manipulation to be highly individualized.

It is understood that s-adenosylhomocysteine is a primary by-product of DNA methylation and a potent inhibitor of DNMT enzymes. However, Ponnaluri and colleagues demonstrated overexpression of s-adenosylhomocysteine hydrolase (AHCY), the key enzyme responsible for the last step in the synthesis of homocysteine to bind DNMT1 increasing its expression and participating in global DNA methylation inheritance and global hypermethylation. ¹³⁵ Together, this suggests that elevated homocysteine can also increase DNA methylation as opposed to being a hallmark feature of reduced methylation as demonstrated by earlier studies.

Histone acetylation

Acetylation is a primary writer of epigenetic modification and is essential for both gene accessibility and activation. ¹⁹ In contrast to methylation, acetylation of the same histone residue can result in open euchromatin allowing genes to be accessible to RNA polymerase for transcription (Figure 1). ¹⁹

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Figure 1.

Heterochromatin and euchromatin. The methyl group (a) bound to the histone tail maintains a positive charge resulting in tight hydrogen bonds and compact genomic structure; heterochromatin (b) Heterochromatin excludes ribonucleic acid polymerase (RNA) (c) from binding the gene and initiating gene transcription. The acetyl group (d) neutralises the methylating positive charge maintaining heterochromatin, loosening the hydrogen bonds and enabling open structural conformation; euchromatin (e) and RNA polymerase to access the genome and initiate gene transcription.

Despite the early discovery, ¹⁸ the importance of acetylation has not been recognized until recently. Histone acetyltransferases (HAT) or histone lysine acetyltransferases (KAT) are the primary enzymes responsible for acetylation. ¹⁹ Acetyl groups are generated through oxidation of pyruvate, fatty acid beta-oxidation, and direct synthesis using acetate as a substrate.^138,139 Like one-carbon metabolism, acetyl-CoA synthesis is epigenetically self-regulated, ¹⁴⁰ and a cellular reduction in acetyl-CoA production or a loss of mitochondrial DNA copy numbers results in genome-wide hypoacetylation.^141,142

When a HAT binds an acetyl group to a histone residue, it neutralizes the positive charge maintained by methylation (Figure 1), weakening the chromatins tightly wound hydrogen bonds, making histone acetylation directly antagonistic to histone methylation. ¹⁴³

Evidence indicates that HATs are essential for the expression of the vast majority of genes, including the epigenetic modulating demethylases, ¹⁴⁴ and one-carbon metabolic genes, including MTHFR, ¹⁴⁵ making acetylation essential for all biological processes.

Histone deacetylase

Like histone demethylases reverse methylation reactions, histone deacetylases reverse the action of acetylation with the removal of the acetyl group from the histone residue. ¹⁴⁶

Nicotinamide adenosine dinucleotide (NAD) and Zn2+ are the primary cofactors for class 1 and 2 and sirtuin (class 3) histone deacetylase enzymes. ¹⁴⁶

Classically, the sirtuin reaction removes the acetyl group specifically from a lysine residue. ¹⁴⁷ The first step is the release of nicotinamide (B3) from NAD+, followed by the release of a peptidyl ADP-ribose intermediate attached the to the acetyl group. ¹⁴⁷

B3 is an essential component of the NAD molecule and has demonstrated to increase NAD levels in the blood, increasing the activity of SIRT3 and improving mitochondrial function. ¹⁴⁸ However; in cell culture, B3 has been widely studied for its non-selective inhibition of sirtuin histone deacetylase activity. ¹⁴⁸ Again, this demonstrates time-specific substrate-level feedback regulation of epigenetic mechanisms. Moreover; fluctuation of SIRT3 acetylation is showing to regulate additional epigenetic modifiers through controlling the available source of succinate-CoA ¹⁴⁹ and acetyl CoA ¹⁵⁰ for histone acetylation and succinylation.

Histone deacetylase inhibition

In 1978, Davie, Candido, and Reeves revealed epigenetic inhibitory modulation of histone deacetylase with the bacterially produced fatty-acid butyrate ¹⁵¹ and supplemental butyrate prevented insulin resistance and obesity in mice fed a high-fat diet. ¹⁵² Histone deacetylases are pH sensitive, and a variety of acids, including endogenous lactate, have shown histone deacetylase inhibitory (HDACi) activity. ¹⁵³ The valerian derived valeric acid and analog Valproic acid was developed as a solvent in 1881 and used medically as an anticonvulsant by the late sixties. ¹⁵⁴ Its action as an HDACi was not discovered until 2001, ¹⁵⁵ and its ability to alter chromatin structure was not identified until 2005. ¹³⁷ Today; the mood-stabilizing medicine is used for the treatment of bipolar disorder, migraine, depression, epilepsy and some cancers.^154,155

Ketones and ketogenic diets

In 2014, an endogenous ketone beta-hydroxybutyrate was found to have HDACi activity. ¹⁵⁶ Ketogenic diets are highly beneficial for the management of epilepsy, depression, reducing mortality and improving memory.^157-159 Together this demonstrates the importance for open chromatin and active gene expression in the management of neurological disease.

Energy metabolism

Energy metabolism can also be used to demonstrate epigenetic cross-talk (Figure 3). For example; respiratory quotients (VCO2/VO2) have been used to estimate the use of energy substrate during rest and exercise. ¹⁷¹ RQ > 0.7 is said to represent mix substrate utilization, 0.8-0.9 protein utilization, 1 glycolysis only and >1.2 anaerobic metabolism. ¹⁷² Making RQ below 1 desired for high energy output and sustainability.

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Figure 3.

Epigenetic cross talk in energy conservation. Pyruvate dehydrogenase (PDH) (a) synthesized acetyl-CoA enters the tricarboxylic acid (TCA) (b) cycle for mixed energy substrate and sustainable adenosine triphosphate (ATP) (c) synthesis. ATP is used for epigenetic methionine synthase, (MAT) (d) ubiquitination, phosphorylation, and chromatin remodeling complexes. Acetyl-CoA catabolized acetyl groups participate in protein and histone acetylation. Oxygen (O₂) (e) intake increases activity jmjC demethylation, reducing methylation and enabling active gene expression and high energy output. Hypoxic reduction of O₂ dependant demethylase and consequent upregulation of methylation marks including activating histone-3-lysine-4 methylation (H3K4me) (f), repressive histone-3-lysine-9 trimethylation (H3K9me) (g) and protein arginine methyltransferase activity (PRMT) (h) and histone-3-arginine-2 dimethylation (H3R2me) (i). Upregulation of PRMT5 and consequent proteolysis of methylated arginine increases asymmetric dimethylarginine (ADMA) (j) resulting in downregulation of endothelial nitric oxide (eNOS) (k). Upregulation of H3K4me results in the induction of hypoxia-inducible factor (HIF) (l) and subsequent upregulation of eNOS, increasing nitric oxide (NO) (m). Upregulated HIF, upregulates pyruvate kinase (PDK) (n), inhibiting PDH, reducing acetyl CoA production and acetylation. Increased HIF induced NO inhibits demethylation, causing persistent methylation, conserving energy and oxygen through switching to glycolysis only metabolism and advancing to respiratory quotient (RQ) (o) = >1. Dihydrofolate reductase (DHFR) (p) promotes eNOS coupling and NO synthesis. Nitrosylation of DHFR stabilises the protein and prevents uncoupling. eNOS is self-regulated by eNOS protein nitrosylation, inhibiting its expression. Hypoxic upregulation of inhibiting H3K9me3 reduces expression of DHFR, resulting in eNOS uncoupling, reduced NO synthesis, superoxide generation, and upregulation of demethylase activity signifying precise epigenetic regulation of energy and O2 conservation in hypoxic conditions.

JmjC histone demethylase enzymes have demonstrated direct cellular O2 sensing. ¹⁷³ A reduction in histone de-methylase substrates akG or O2 ⁵⁶ results in potent inhibition of histone demethylation and upregulation of repressive H3K9me2, ¹⁷⁴ and activating H3K4me ¹⁷⁴ resulting in reduced expression of DHFR ¹⁷⁴ and upregulation of hypoxia-inducible factor a (HIF1a) respectively. ¹⁷⁴ Upregulation of HIF1a is well known for its oxygen conservation via feedback inhibition of pyruvate dehydrogenase (PDH) activity, through upregulation of pyruvate dehydrogenase kinase (PDK) resulting in increased lactate synthesis and a reduction in the available acetyl CoA to enter the TCA and mitochondria for sustainable ATP synthesis, ¹⁷⁵ subsequently increasing RQ to 1 or >1.2, resulting in excessive muscle lactate. Moreover; upregulation of HIF1a has been associated with the upregulation of eNOS,^176,177 resulting in increased NO and greater downregulation of demethylase activity ²⁹ and O2 conservation.

B vitamins have long been associated with energy production; however, the mechanisms have remained undefined. The above describes how a reduction in sustainable ATP synthesis, through hypoxia, or hypermethylation at the level of acetyl CoA synthesis could result in downregulation of all primary ATP dependant epigenetic modifiers, relying on environmental or supplemental direct methyl group donation to drive methylation and glycolysis only metabolism (RQ > 1) which is unlikely to be sustainable for exercise such as endurance. Moreover; TCA cycle or mitochondrial ATP synthesis defects have been repeatedly implicated in diseases of cancer and dysfunctional immunity. ¹⁷⁸ As a result the excessive lactate production and consequent HDACi ¹⁵³ may result in acetylation of undesired genes as seen in the metabolic switch known as the Warburg effect, which is commonly detected in cancer cells. ¹⁷⁹

Chronic fatigue syndrome (CFS)/Myalgic encephalomyelitis (ME)

A reduced ability to produce ATP, which is often characterized as deregulation of glycolysis at the pyruvate dehydrogenase complex is seen in patients with post-exertional malaise, or CFS. ¹⁶ McGregor and colleagues revealed changes in glycolysis and concentrations of metabolic pyruvate, acetate and lactate influencing both acetylation and deacetylation which were indicative of hypoacetylation in patients with CFS/ME. ¹⁶

Epigenetic fluctuation

Immune cells fluctuate levels of epigenetic modifiers. ¹⁸⁰ Specifically, T cells require coordinated suppression of methylation for memory T cell differentiation and appropriate response to antigen. ¹⁸⁰ Similarly, the menstrual cycle ¹⁸¹ and circadian rhythms ¹⁸² require coordinated and a timely fluctuation of epigenetic modifications for regularity and synchronicity. Therefore, uncoordinated supplementation of methyl donors or modern epigenetic modifiers may result in dysregulated menstrual cycles, abnormal sleep patterns or immune dysfunction.

Endothelial damage

Two primary mechanisms that have been implicated in the development of endothelial dysfunction are elevated levels of asymmetric dimethylarginine (ADMA) and a lack of dimethylarginine dimethylaminohydrolase (DDAH) enzyme activity. ¹⁹³ ADMA is an endogenous eNOS inhibitor derived from the proteolysis of methylated arginine residues following arginine methylation by a group of epigenetic modifying enzymes referred to as protein-arginine methyltransferases (PRMT). ¹⁹³ PRMTs are dependent on the one-carbon metabolite SAM for methyl group donation. ¹⁹⁴ Increased ADMA has demonstrated superoxide release, uncoupling and inhibition of eNOS activity by up to 40%, and its presence predicts cardiovascular mortality, endothelial dysfunction in hypertension, hyperlipidemia, diabetes, and coronary artery disease. ¹⁹³

In contrast to MTX, in vivo vascular infusion of 5-MTHF has demonstrated improved endothelial function, eNOS coupling and decreased superoxide production. ¹⁹⁵ However; in other cells, overexpression of eNOS has exhibited both male and female infertility.^196,197 Validating the unpredictable nature of orally administered 5-MTHF.

Myeloproliferative neoplasms

An acquired JAK2 V617f mutation is present in the majority of patients with myeloproliferative neoplasms. ²⁰⁹ Several phenotypical manifestations result from the same mutation, and the presence of this mutation does not always present with disease. ²¹⁰ The phenotypical presentation is dependent on the JAK2 burden, which is the accumulative JAK2 RNA from JAK2 copy numbers and the ratio of JAK2 RNA between wild type and its mutants. ²¹¹ However, the neoplastic cell line UKE-1 shows the accumulation of JAK2 copy numbers in culture, suggesting epigenetic adaptation to the media. ²⁰⁹

As previously described, human fibroblast cell lines frequently deaminate, losing methylation marks resulting in longevity or immortalization of cells. ⁷⁶ Negative regulation of JAK2 is demonstrated by the upregulation of G9a methyltransferase at H3K9me2, and comparably the inhibition of G9a H3K9 methyltransferase increases JAK2 expression and ultimately H3Y41 phosphorylation, demonstrating direct epigenetic modulation by methylation. ²⁰¹ Similarly, in cell culture, folic acid and the folate analog methotrexate also inhibit JAK2 phosphorylation.^212,213

Like other cell lines, nutritional media for the UKE-1 cell line is also abundant in epigenetic modulators such as folic acid capable of supporting epigenetic inhibition of JAK2 and potentially a feedback loss of JAK2 methylation.^210,214 This suggests that chronic epigenetic modification may influence genomic adaptation, resulting in increased gene copy number or lead to constitutive activation of oncogenes such as JAK2 (Figure 4).

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Figure 4.

Hypothetical cell culture epigenetic manipulation of Janus kinase-2. Folic acid is added to the UKE1 cell line culture media, influencing epigenetic methylation. Upregulation of G9a methyltransferase inhibits Janus kinase-2 (JAK2) (a). Upregulation of histone-lysine N-methyltransferase (SUV39H1) (b) induces trimethylation of histone-3-lysine-9 (H3K9me3 (c) stimulating the binding of heterochromatin protein 1 (HP1) (d) and initiating heterochromatin. Reduced expression of JAK2 is incapable of excluding HP1 and maintaining euchromatin. Excess methylation inhibits one-carbon metabolism (1 Cm) (e) and induces spontaneous deamination of methylated cytosine-phosphate-guanosine (CpG) (f) and loss of CpG resulting in JAK2 constitutive activation or copy number accumulation.

Estrogen

Animal organ and muscle estrogen can be stable up to 180 degrees and absorbed by the gastrointestinal tract of the consumer. ²²² Estrogen is a potent epigenetic modulator influencing gene expression and has been associated with cognition, memory, mood, immunity, and bones. ²²³ Estrogen has shown to interact directly with the previously mentioned epigenetic transcription factor JAK2,^224,225 signifying that a sudden reduction in consumption of biologically active molecules like estrogen may result in epigenetic adaptation resulting in hundreds of molecular changes to gene expression required to ensure adequate endogenous synthesis of estrogen. Therefore, concomitant hypermethylation, SNV or deletion in the genome due to advanced evolution within the hormonal synthesis pathway, may result in pathological symptomology and/or disease.

Prolactin

Prolactin is a pituitary hormone essential for lactation and is found abundantly in breast milk. ²²⁶ Prolactin is commonly associated with reproduction; however, it is also produced by many immune cells and can be considered a cytokine for it has demonstrated production of a variety of cytokines. ²²⁷ Activation of the prolactin receptor also stimulates activation of JAK2 which may result in histone post-translational phosphorylation.^228,229

In the gastrointestinal tract, prolactin has displayed stimulation of intestinal calcium absorption, increasing bone turnover and reducing renal calcium excretion. ²³⁰

Taken together, this suggests that a sudden withdrawal of dairy products from the diet may require epigenetic modification of the immune system, cytokine production and calcium absorption.

Serotonylation

Serotonin (5-hydroxytryptamine—5-HT) is a biogenic amine derived from the amino acid tryptophan and has demonstrated multiple functions within multiple organs. ²²⁷ Serotonin and its precursor 5-hydroxytryptophan (5-HTP) are found abundantly in animal tissues ²³¹ including breast milk,^232,233 is orally bioavailable and has a half-life of over 17 hours. ²³⁴

In the brain, both serotonin and its precursor are neurotransmitters, with serotonin having 3000 fold greater biological activity at some receptors but less affinity at others. ²³⁵ 5-HTP has also shown to be directly antagonistic to serotonin at the receptor. ²³⁶ It was once thought that serotonin was incapable of crossing the BBB, however more recent research demonstrates otherwise.^237-239 The results of earlier studies may have been due to a lack of active gene expression in cell culture.

Histone seronylation of the 5th glutamine residue of histone 3 (H3Q5) results in significant changes to gastrointestinal and brain gene expression profiles. ²⁴⁰ This demonstrates the potential for peripheral serotonylation to influence gene expression beyond the BBB. The Coeliac disease implicated transglutaminase 2 (TGM2) ²⁴¹ is said to initiate the serotonylation adjacent to the activating H3K4me3, enhancing the binding of chromatin transcription factors for gene expression. ²⁴¹

Dopaminylation

Dopamine is a neurotransmitter derived from the amino acid tyrosine. ²⁴² It plays a pivotal role in reward and movement regulation. ²⁴² Unmodified H3Q5 can also be dopaminylated. ²⁴³ The lack of marked serotonylation at this residue allows for dopaminlylation to occur, which likely explains the antagonistic relationship frequently reported between dopamine and serotonin.^244,245 In a murine model of cocaine addiction, a fall in H3Q5 dopaminylation demonstrated decreases in the rate of spontaneous action potentials and an increase in cocaine-seeking behavior ²⁴³ demonstrating direct epigenetic influence by stimulants and their withdrawal.

Dopamine has a relatively short half-life and poor oral bioavailability, and therefore the epigenetic effects of dietary consumed dopamine is likely to be limited. ²⁴⁶

Gamma-ammino butyric acid (GABA)

GABA is an inhibitory neurotransmitter found abundantly in almost all organisms and therefore can be obtained directly from many foods. ²⁴⁷ It is found in over 20 peripheral tissues ²⁴⁸ , and in all regions of the central nervous system. ²⁴⁹ GABA is renowned for its high potency. ²⁴⁹ As previously discussed, defective GABA function has been associated with a variety of neurological and non-neurological conditions including mood, cognition, motor function, flight response, sexual and reproductive behavior, anxiety, pain, violence, and aggression. ²⁴⁸

Like serotonin, GABA was once thought to be incapable of crossing the BBB; however, a handful of studies have demonstrated otherwise yet the mechanisms remain unclear. ²⁵⁰ Unlike serotonin, GABA has a short half-life of approximately 17 minutes, and therefore direct modulation of the brain following dietary consumption seems unlikely.^249,250 However; Yamastu and colleagues displayed rapid gastrointestinal absorption and a peak in blood concentrations of GABA 30 minutes after consumption. ²⁵¹ Moreover; GABA was revealed to successfully inhibit class I, II and III histone deacetylases, upregulate H3K9 acetylation and H4K12 acetylation in SH-SY5Y cells; therefore, we cannot rule out epigenetic modulation that may rapidly influence gene expression beyond the BBB. ²⁴⁷ In addition, neurosteroids are highly lipophilic and therefore, readily cross the BBB. ²⁵² Plasma and brain synthesised neurosteroids have demonstrated rapid effects on neuro-excitability via modulation of the GABA-a receptor. ²⁵³ Therapeutic neurosteroids have established varied oral bioavailability, ^254,255 the primary steroid cholesterol of which all neurosteroids are derived has an individualised oral bioavailability between 29 and 81%. ²⁵⁵ This suggests that a sudden reduction in dietary neurosteroids may negatively impact mood or cognition in a patient who has a genetic synthesis pathway defect.

Heavy metals

It has been established that environmental heavy metals interact with the epigenome. ²⁵⁸ However, their epigenetic modulation is often contrasting and difficult to define due to their effects being cell and dose-specific. Metabolic transformation of inorganic arsenic produces metabolites monomethylarsinic, dimethylarsinic acid, and trimethylarsinic acid, and the one-carbon metabolic product SAM donates the methyl group in the reaction. ²⁵⁹ It was once thought that the methyl and glutathione conjugation of carcinogenic metals was essential for detoxification and elimination, however more recent research is demonstrating the conjugated metabolites to have greater toxicities.^259-261 Arsenic induced malignant cellular transformation is said to be due to global DNA hypomethylation and aberrant gene expression. ²⁶² The highly toxic lead and methyl-mercury have both demonstrated non-consistent differential epigenetic modifications which are dependent on the specific tissue.^263,264

Endocrine disrupting chemicals

Endocrine-disrupting chemicals found in pesticides and plastics such as bisphenol A (BPA) have also demonstrated interaction at the epigenetic level, potentially contributing to rapid evolution and disease.^34,265

Fluoride

In the 1930s researchers found an excess of the natural mineral fluoride in drinking water supplies was associated with mottled teeth enamel yet interestingly, also prevented dental caries. ²⁶⁶ The concentrations of natural sources of fluoride in water wells across the globe and regionally are highly variable. In the early 1900s natural sources fluoride In the United States were measured to be as low as 0.1 parts per million (ppm). ²⁶⁶

On January 5th 1945; Michigan state, United States were the first to introduce fluoride fortification of their town water, other states and Countries followed shortly after. ²⁶⁶ Today water concentrations of fluoride in water supplies can range anywhere between 0.7 ppm and 16 ppm. However; around the same time as fluoride fortification was implemented around the World, researchers in India were discovering that natural sources of fluoride in the water supply as low as 1ppm were associated with fluoride toxicity and skeletal fluorosis. ²⁶⁶ Fluoride toxicity and subsequent fluorosis have been assessed for its epigenetic influence upon genes associated with skeletal development. ²⁶⁷ Promoter region DNA hypermethylation and sequential downregulation of expression were demonstrated for genes associated with extracellular matrix deposition and cartilage formation. ²⁶⁷

Together, this demonstrates classic epigenetics and evolutionary differences in populations, where one man’s medicine may be the cause of another’s ailment and exemplifies the damaging effects of nutritional conformity and a need for personalised nutritional management.

Nutritional deficiency

Reduced erythrocyte, serum or plasma nutrient levels comparable to the average cohort have long been used to identify a nutritional deficiency. However, it is rarely considered that a reduction in an epigenetic modifier such as folate may be due to excessive use of substrate and enzymatic activity—for example; folate depletion due to excessive DNA methylation. The methylation requirement for each cell is different, making an evaluation of serum folate difficult to predict the level of folate within and required for glial cells compared to cardiomyocytes. Therefore; nutritional epigenetic research must look at nutrition from both angles keeping bio-individuality in mind, and practitioners should use caution in interpretation.

Rhythmicity and cell specificity

Outlining the cell-specific epigenetic regulation of rhythmic and coordinated biological processes will add to precision and patient management accuracy.

Laboratory techniques

Nutritional modulation of the epigenome in the laboratory is becoming increasingly popular and has profound disease modulatory potential. The cell, tissue and dose-specific epigenetic mechanisms for many environmental substances including bioactive peptides are yet to be determined; however, when it comes to experimentation, researchers should pay special attention to detail with the use of excipients, extraction, storage, reagents, media, and temperature as to avoid unintentional epigenetic modification and human error.

Accurate genotyping with the use of next-generation sequencing technologies is still an expensive operation, and the use of first-generation micro-array genotyping technologies is affordable and provides valuable insight into a patient’s ancestry and origin for precise nutritional health management. However; the use of restriction enzymes in this technique makes it impossible to differentiate a deletion from a methylated base or unrepaired deaminated base. Moreover; this technology does not provide information pertaining to haplotype selective expression, cell-specificity or gene epigenetic regulation, all of which largely affect the pathogenic effect of an SNV. This makes nutritional epigenetic modulation of specific genes based on these results currently unreliable, and therefore standardization of accurate genotyping is of great importance. It is now understood that regardless of genotype, epigenetics is the foundation of a gene’s regulation and therefore, RNA based sequencing techniques may provide a more reliable insight into cell-specific gene expression.

Novel epigenetic post translational modifications

A deeper understanding of the novel epigenetic post-translational modifications such as serotonylation and dopaminylation will aid suicide prevention and the natural management of mental health conditions and addiction.

Evolution

Understanding the impact of environments on gene evolution and epigenetics within populations or cultural subgroups should take priority as this opens up bio-individuality moving away from nutritional conformity and greater health for all populations.