Review
Folate (vitamin B9) and vitamin B12 and their function in the maintenance of nuclear and mitochondrial genome integrity

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Abstract

Folate plays a critical role in the prevention of uracil incorporation into DNA and hypomethylation of DNA. This activity is compromised when vitamin B12 concentration is low because methionine synthase activity is reduced, lowering the concentration of S-adenosyl methionine (SAM) which in turn may diminish DNA methylation and cause folate to become unavailable for the conversion of dUMP to dTMP. The most plausible explanation for the chromosome-breaking effect of low folate is excessive uracil misincorporation into DNA, a mutagenic lesion that leads to strand breaks in DNA during repair. Both in vitro and in vivo studies with human cells clearly show that folate deficiency causes expression of chromosomal fragile sites, chromosome breaks, excessive uracil in DNA, micronucleus formation, DNA hypomethylation and mitochondrial DNA deletions. In vivo studies show that folate and/or vitamin B12 deficiency and elevated plasma homocysteine (a metabolic indicator of folate deficiency) are significantly correlated with increased micronucleus formation and reduced telomere length respectively. In vitro experiments indicate that genomic instability in human cells is minimised when folic acid concentration in culture medium is greater than 100 nmol/L. Intervention studies in humans show (a) that DNA hypomethylation, chromosome breaks, uracil incorporation and micronucleus formation are minimised when red cell folate concentration is greater than 700 nmol/L and (b) micronucleus formation is minimised when plasma concentration of vitamin B12 is greater than 300 pmol/L and plasma homocysteine is less than 7.5 μmol/L. These concentrations are achievable at intake levels at or above current recommended dietary intakes of folate (i.e. >400 μg/day) and vitamin B12 (i.e. >2 μg/day) depending on an individual's capacity to absorb and metabolise these vitamins which may vary due to genetic and epigenetic differences.

Introduction

Folate and vitamin B12 play an important role in DNA metabolism [1] (Fig. 1). Folate is required for the synthesis of dTMP from dUMP. Under conditions of folate deficiency dUMP accumulates and as a result uracil is incorporated into DNA instead of thymine [2]. There is good evidence suggesting that excessive incorporation of uracil in DNA not only leads to point mutation but may also result in the generation of single and double stranded DNA breaks, chromosome breakage and micronucleus formation [3], [4]. The mutagenic effects of uracil are underscored by the observation that of eight known human glycosylases, four (UNG, TDG, hSMUG1, MBD4) are dedicated to the removal of uracil [5]. Folate and vitamin B12 are also required for the synthesis of methionine and S-adenosyl methionine (SAM), the common methyl donor required for the maintenance of methylation patterns in DNA that determine gene expression and chromosome conformation [6]. When the concentration of vitamin B12 and methionine is low, S-adenosyl methionine synthesis is reduced, methylation of DNA is reduced, inhibition by SAM of methylenetetrahydrofolatereductase (MTHFR) is minimised resulting in the irreversible conversion of 5,10,methylenetetrahydrofolate to 5,methyltetrahydrofolate, thus favouring an increase in the dUMP pool and uracil incorporation into DNA. Because vitamin B12 is an essential cofactor of methylmalonyl CoA mutase (MMCoA MUTASE), its deficiency also leads to accumulation of methylmalonic acid (MMA) which disrupts mitochondrial metabolism leading to generation of excess reactive oxygen species (ROS) [7], [8], [9]. ROS are also generated as a consequence of vitamin B12 deficiency-induced inflammatory cytokines such as tumour necrosis factor alpha (TNF-α) [10], [11], [12] and increased homocysteine [13] as a consequence of methionine synthase (MTR) inhibition caused by lack of vitamin B12 cofactor. Deficiencies in folate and vitamin B12 therefore can lead to (a) elevated DNA damage and altered methylation of DNA, both of which are important risk factors for cancer [3], [4], [5] and (b) an increased level in homocysteine status, an important risk factor for cardiovascular disease [14]. These same defects may also play an important role in developmental and neurological abnormalities [3], [4].

The blood levels of folate and vitamin B12 required to prevent anaemia and hyperhomocysteinemia are properly defined, however, it is becoming increasingly evident that such accepted levels of sufficiency may be inadequate to minimise chromosome damage and optimise DNA methylation status. In this paper evidence is provided from in vitro studies with human cells, in vivo studies in rodents, and in vivo cross-sectional and intervention studies in humans to identify the concentration or intake level at which potential genotoxic effects of low folate and vitamin B12 status may be prevented. In addition the potential impact of genetic polymorphisms in key transport molecules and enzymes required for the metabolism of folate and vitamin B12 are discussed as factors that should be considered when determining dietary reference values (DRVs) of these vitamins based on DNA damage prevention. The concept of DRVs for DNA damage prevention was recently reviewed and identified the following genome integrity biomarkers as being demonstrably sensitive to the DNA damage impact of malnutrition: chromosome aberrations, micronuclei (biomarker of chromosome breakage or loss), DNA strand breaks, base damage (e.g. uracil incorporation, oxidized guanine), mitochondrial DNA deletions, telomere length, CpG methylation [15]. This review draws on all the evidence currently available that indicates the impact of folate and vitamin B12 on these biomarkers of nuclear and mitochondrial genome integrity.

Section snippets

Evidence from in vitro cultures with human cells

It has been shown that fragile sites in chromosomes are expressed when human lymphocytes are cultured in the absence of folic acid and thymidine in culture medium [16], [17]. Furthermore under these conditions chromosome breakage and micronucleus (MN) expression are increased simultaneously suggesting a similar mechanism underlying the expression of fragile sites and chromosome breakage [16], [17], [18]. Reidy's experiments showed that lymphocytes cultured in folic acid deficient medium exhibit

Evidence from in vivo studies in rodents

Results from studies in rodents suggest that extreme folate deficiency (i.e. on diets without folic acid that also include succinyl sulphathiazole, an antibiotic that eradicates folate producing bacteria in the gut) causes DNA strand breaks, hypomethylation of DNA, increased uracil and apurinic sites in DNA [35], [36], [37] and caffeine synergistically increased folate-deficiency-induced micronucleus frequencies in peripheral blood erythrocytes [38]. However, marginal folate deficiency (400 

Evidence from in vivo studies in humans

The early evidence of chromosome damage in human cells in vivo from folate and vitamin B12 deficiency was first obtained from studies linking the expression of Howell–Jolly bodies in erythrocytes with megaloblastic anaemias [50], [51], [52]. Howell–Jolly bodies are whole chromosomes or chromosome fragments that lag behind at anaphase during production and maturation of the red blood cell and, in fact, they are the same as micronuclei, the alternative and most commonly used term for this

Environmental and genetic factors that determine the bioavailability of folate and vitamin B12

Alcoholism is associated with significantly reduced levels of tissue folate, vitamin B12 and vitamin B6 in humans; at intakes greater than 3.0 g/kg/d there was a doubling in the level of DNA hypomethylation of lymphocytes [96]. The reduced folate level in alcoholics may be due to reduced absorption or sub-optimal dietary intake. However, if results in the rat model reflect the situation in humans, then there is a good probability that the microbial metabolism of alcohol can result in exceedingly

Recommended dietary intakes (RDIs) for folate and vitamin B12 based on genomic stability

There is now increasing interest to redefine recommended dietary intakes (RDIs) of minerals and vitamins not only to prevent diseases of extreme deficiency but also to prevent developmental abnormalities and degenerative diseases of old age as well as optimising cognition [128]. Prevention of DNA damage is an important parameter for the definition of new dietary reference values because increased loss of genome integrity (e.g. lymphocyte micronuclei, chromosome aberrations and telomere

Knowledge gaps

Some of the important knowledge gaps include the following:

  • (1)

    Is supra-physiological concentration of folate or vitamin B12 genotoxic and does it depend on the form of folate or vitamin B12?

  • (2)

    Which of the DNA damage biomarkers is most sensitive to folate deficiency or excess?

  • (3)

    Do folate and vitamin B12 requirements for DNA damage prevention vary greatly depending on common genetic polymorphisms of genes involved in their uptake, transport and metabolism? Which of these common genetic polymorphisms are

Conclusion

The accumulated evidence to date suggests that folate and vitamin B12 play an important role in genomic stability and that DNA damage biomarkers are remarkably sensitive to relatively small changes in the concentration of folate and vitamin B12 within the physiological range. There are various mechanisms by which folate and vitamin B12 deficiency, independently or interactively, increase DNA damage (summarized in Fig. 2, Fig. 3). Above RDI intakes of these vitamins may be required for optimal

Key to unit conversion

1 ng/mL folic acid = 2.26 nmol/L folic acid; 1 pg/mL vitamin B12 = 0.74 pmol/L vitamin B12.

Conflict of interest

There are no conflicts of interests.

Acknowledgements

This research was partially funded by the National Health and Medical Research Council of Australia, the Australian Academy of Sciences and the European Union IRSES MICROGENNET project.

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