Nutrient-Gene Interactions Methylenetetrahydrofolate Reductase C677T Polymorphism, Folic Acid and Riboflavin Are Important Determinants of Genome Stability in Cultured Human Lymphocytes

We tested the hypothesis that methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, folic acid deficiency and riboflavin deficiency, independently or interactively, are important determinants of genomic stability, cell death, cell proliferation and homocysteine (Hcy) concentration in 9-d human lymphocyte cultures. Lymphocytes of seven wild-type (CC) and seven mutant (TT) homozygotes were cultured under the four possible combinations of deficiency and sufficiency of riboflavin (0 and 500 nmol/L) and folic acid (20 and 100 nmol/L) at a constant L-methionine concentration of 50 mol/L. Viable cell growth was 25% greater in TT than in CC cells (P 0.05) and 32% greater at 100 nmol/L folic acid than at 20 nmol/L folic acid (P 0.002). The comprehensive cytokinesis-block micronucleus assay was used to measure micronuclei (MNi; a marker for chromosome breakage and loss), nucleoplasmic bridges (NPB; a marker of chromosome rearrangement) and nuclear buds (NBUD, a marker of gene amplification). The MNi levels were 21% higher in TT cells than in CC cells (P 0.05) and 42% lower in the high folic acid medium than in the low folic acid medium (P 0.0001). The NBUD levels were 27% lower in TT cells than in CC cells (P 0.05) and 45% lower in the high folic acid medium than in the low folic acid medium (P 0.0001). High riboflavin concentration (500 nmol/L) increased NBUD levels by 25% (compared with 0 nmol/L riboflavin) in folate-deficient conditions (20 nmol/L folic acid medium; P 0.05), and there was an interaction between folic acid and riboflavin that affected NBUD levels (P 0.042). This preliminary investigation suggests that MTHFR C677T polymorphism and riboflavin affect genome instability; however, the effect is relatively small compared with that of folic acid. J. Nutr. 134: 48–56, 2004. Folate plays an important role in the maintenance of genomic stability, mainly by providing methyl groups for the synthesis of deoxythymidine triphosphate (dTTP) from deoxyuridine monophosphate (dUMP), and methionine from homocysteine (Hcy) (1–4). Methionine is subsequently converted to S-adenosyl methionine (SAM), which provides methyl groups for the maintenance methylation of cytosinephosphate-guanosine dinucleotide (CpG) islands and other intervening sequences containing CpG. Figure 1 presents a simplified diagram of the relevant metabolic pathways and the key enzymes that control these pathways. Methylenetetrahydrofolate reductase (MTHFR) is a pivotal enzyme that controls the bioavailability of folate for dTTP synthesis and maintenance methylation of CpG. The activity of MTHFR can be reduced in three ways: 1) by polymorphisms in the gene sequence that alter its affinity for a substrate or cofactor; 2) by a high concentration of methionine or SAM, which inhibit MTHFR activity; and 3) by a low concentration of its cofactor flavin adenine dinucleotide (FAD) or of riboflavin, the precursor of FAD (5,6). Reducing MTHFR activity increases the 5,10-methylenetetrahydrofolate concentration, whereas it decreases the 5-methyltetrahydrofolate concentration. Such a situation is expected to 1) favor synthesis of dTTP over methylation of CpG, 2) minimize uracil incorporation into DNA and the chromosome breaks caused by uracil (7,8); and 3) increase the Hcy concentration. The C677T polymorphism reduces MTHFR activity by 50% and is associated with reduced risk for a variety of cancers, such as leukemia (9), lymphoma (10) and colorectal cancer (11), but also with increased risk for Down syndrome (12), neural tube defects (13) and cervical cancer (14). The polymorphism is also linked to reduced methylation of CpG DNA in lymphocytes (15). The contrasting effects of the C677T polymorphism may reflect the relative importance of CpG methylation and dUMP methylation in the etiology of the diseases listed above. Therefore, it is important to thor1 To whom correspondence should be addressed. E-mail: [email protected]. 2 Abbreviations used: APOP, apoptotic cell; BNC, binucleated cell; CBMN, cytokinesis-block micronucleus assay; CC, wild-type methylenetetrahydrofolate reductase C677T homozygote; CpG, cytosine-phosphate-guanosine dinucleotide; dTTP, deoxythymidine triphosphate; dUMP, deoxyuridine monophosphate; FAD, flavin adenine dinucleotide; Hcy, homocysteine; HF, high folic acid (100 nmol/L); HR, high riboflavin (500 nmol/L); LF, low folic acid (20 nmol/L); LR, low riboflavin (0 nmol/L); MNi, micronuclei; MTHFR, methylenetetrahydrofolate reductase; NEC, necrotic cell; NPB, nucleoplasmic bridge; NBUD, nuclear bud; SAM, S-adenosyl methionine; TT, mutant methylenetetrahydrofolate reductase C677T homozygote. 0022-3166/04 $8.00 © 2004 American Society for Nutritional Sciences. Manuscript received 24 June 2003. Initial review completed 31 July 2003. Revision accepted 6 October 2003. 48 by gest on A uust 4, 2017 jn.nition.org D ow nladed fom oughly understand the effect of the MTHFR C677T polymorphism and other metabolic factors that affect the activity of MTHFR on chromosomal instability, an important risk factor in cancer. To test these various factors we developed an in vitro system of culturing lymphocytes for 9 d with concentrations of micronutrients that are within or close to the physiological range (16–19). We used this system in combination with the cytokinesis-block micronucleus (CBMN) assay in its comprehensive mode (18,19) to measure various markers of genotoxicity and cytotoxicity that are important in assessing the effect of micronutrients on genomic stability and cell death. These markers include 1) micronuclei (MNi), a marker of chromosome breakage and/or loss; 2) nucleoplasmic bridges (NPB), a marker of chromosome rearrangement; 3) nuclear buds (NBUD), a marker of gene amplification; 4) necrosis (NEC); and 5) apoptosis (APOP) (Fig. 2). We previously used this system to show that MNi, NPB and NBUD levels increase markedly with a decline in folic acid concentration from 120 to 12 nmol/L, which coincides with the physiological range in the serum of individuals consuming an unsupplemented diet (8 to 35 nmol/L) (6,20,21). It is important to note that these markers of genome instability all positively correlate with each other, suggesting that folic acid deficiency affects the generation of breakage-fusion-bridge cycles, which is a hallmark of genomic instability in several types of cancer cells (22). However, we were unable to show that the MTHFR C677T polymorphism affected genomic stability under the conditions of in vitro culture used in that experiment. We reasoned that the supraphysiological concentrations of methionine (100 mol/L) and riboflavin (530 nmol/L) in the RPMI 1640 medium might have altered the activity of MTHFR T677T and MTHFR C677C in such a way that differing effects on genome instability between genotypes became indiscernible (17). The range of serum concentration of methionine in healthy subjects is 20 to 30 mol/L (23) and that of riboflavin varies from 5 to 50 nmol/L (6). An excess of methionine may increase SAM concentration, which inhibits MTHFR, whereas an excess of riboflavin may increase FAD concentration, which may be expected to promote MTHFR FIGURE 1 The main metabolic pathways by which folate, cobalamin, choline, methionine, pyridoxine and riboflavin affect DNA methylation, synthesis and repair. Abbreviations: B6, pyridoxine; B12, cobalamin; BHMT, betaine: homocysteine methyltransferase; DHF, dihydrofolate; DMG, dimethylglycine; FAD, flavin adenine dinucleotide; 5-MeTHF, 5-methyltetrahydrofolate; 5,10-MeTHF, 5,10-methylenetetrahydrofolate; MS, methionine synthase; MTHFR, methylenetetrahydrofolate reductase; SAM, S-adenosyl methionine; SHM, serine hydroxymethyltransferase; THF, tetrahydrofolate; TS, thymidylate synthase. FIGURE 2 Genome damage and cell death biomarkers scored in the comprehensive cytokinesis-block micronucleus (CBMN) assay. The CBMN assay allows the measurement of all possible outcomes following a genome damage event. In this assay a cell with genome damage either may undergo cell death via apoptosis or necrosis or may survive and undergo further nuclear division. In the latter case, dividing cells are recognized as binucleated cells (BNC) by blocking cytokinesis with cytochalasin-B. The BNC are then scored for the following genome damage events: 1) micronuclei (MNi), which originate from lagging whole chromosomes or broken chromosome fragments and are therefore a marker of chromosome breakage and chromosome loss events, the latter being due to defects in centromere or spindle structure; 2) nucleoplasmic bridges (NPB), which originate from dicentric chromosomes caused by misrepair of chromosome breaks and are therefore a marker of chromosome rearrangement; and 3) nuclear buds (NBUD), which are the mechanism by which the nucleus eliminates amplified DNA resulting from breakage-fusion-bridge cycles generated by NPB. The NBUD level therefore provides a measure of gene amplification. An increase in MNi, NPB and NBUD levels is indicative of an increase in genome instability commonly seen in cancer. For futher details of the CBMN assay, refer to Fenech (18,19). MTHFR, FOLIC ACID, RIBOFLAVIN AND GENOME STABILITY 49 by gest on A uust 4, 2017 jn.nition.org D ow nladed fom