The Role And Mechanisms Of Folate And Some Metabolic Factors On Human Genomic Stability

Folate(FL) is an important micronutrient which plays a critical role in the DNA metabolism. Human being is not able to synthesize this vitamin and have to obtain from food or supplements. Folic acid(FA) is the synthetic form of FL which is found in supplements or added to fortified foods. Folate provides methyl group for DNA methylation and conversion of dUMP to dTMP. It is therefore involved in DNA replication, DNA repair, gene expression and cell proliferation. Some key enzymes and relative co-factors are extramly important for the metabolism of folate such as methylene tetrahydrofolate reductase (MTHFR) which is a pivotal enzyme that controls the balance for dTMP synthesis and maintaining methylation of DNA. Riboflavin(RF), as a precursor of FAD( MTHFR cofactor),may affect on the activity of MTHFR and as a result, it could cause some effects on folate metabolism.This research aimed to explore the comprehensive effects of folate, riboflavin and MTHFR polymorphism on human genomic stability, define optimal Recommended Dietary Allowances(RDA) of folate and riboflavin for genomic stability. Radioimmunoassay, cytokinesis-block micronucleus assay (CBMN) were employed to compare the differences of plasma/red cell folate concentrations and of the genomic instability under folic acid deficiency between control and breast cancer patients. The mechanism of the genetic response differences between 2 groups was analyzed as well. The efficiency of FA and 5-methyltetrahydrofolate (MeTHF) in their capacity to prevent genetic damage and mal-disjunction of human chromosome 17 and 21 were determined by CBMN and fluorescence in situ hybridization, respectively. The distribution of MTHFR C677T, A1298C genotypes was analyzed by multiplex PCR-RFLP. The role and possible mechanisms of different concentration combinations of folic acid - riboflavin on genomic stability of lymphocytes with different MTHFR polymorphisms were evaluated by CBMN.The results showed that (1) The concentrations of foalte in plasma/red cell were significantly lower in the breast cancer group compared with control(P<0.001) and it implied that folate may be associated with risk of breast cancer. (2) 30nM FA was associated with increased frequencies of micronucleated binucleated cell (Misled BNC), nucleoplasmic bridges (NPB), nuclear buds (BUD), relative to 120nM and 240nM FA (P < 0.001) in lymphocytes of case and control groups in vitro, however there were no significant differences between 120nM and 240nM FA within each sampling group. It suggests that 120nM FA is the optimal concentration for maintaining lymphocyte genomic stability in vitro.(3) The genetic damage and necrosis were significantly increased in breast cancer group than that of control at all concentrations of FA (P≤0. 005). However, Difference of Difference Analysis(DDA) showed that breast cancer cases were not abnormally sensitive to the genome-damaging and necrosic effects of folate deficiency, it indicated that both higher genetic damage baseline and genetic instability induced by folic acid deficiency were the risk factors of breast cancer. FA concentration explained 60%, 39%, 39%, 52% and 71% of the variance of MNed BNC, NPB, BUD , APO and NEC respectively compared to breast cancer status which only explained 6% and 7% of the variance of MNed BNC and NEC, FA concentration is much more important than breast cancer status in determining genomic instability and cell death. (4) The frequency of MNed BNCs was significantly lower at 120nM FA and 120nM MeTHF compared with 12nM concentration at both forms of folate (P < 0.05).The induction of aneuploidy under folate deficiency showed similar tendency. 120nM of MeTHF and FA caused a 26%-35% reduction in frequency of aneuploidy of chromosome 17 and of chromosome 21 relative to 12 nM MeTHF and FA. The result suggests that folate possesses important role on maintaining genomic stability. However, MeTHF was less efficient than FA in preventing genetic damage in this in vitro system. (5)No significant differences were found on the distribution of MTHFR C677T and A1298C genotypes.(6)The genetic damage achieved maximum at low FA-low RF combination medium in both case and control groups and was minimized at high FA-low RF medium. The frequencies of MNed BNC, BUD, NPB were 43%, 48% and 44% lower in the high FA medium than in the low FA medium among the control group with C677T polymorphism , and 35%,45%, 34% among case group with C677T polymorphism. However, high RF only reduced those genetic damage by 3~10%. The frequencies of MNed BNC,BUD,NPB were 42%, 55% and 36% lower in the high FA medium than in the low FA among the control group with A1298C polymorphism , and 37%,45%, 36% among case group with A1298C polymorphism. However, high RF reduced BUD and NPB by 11% and 18% in control and NPB by 11% in breast cancer group. The result suggests that the efficiency of genomic stability maintaining of FA is much stronger than that of RF. (7) The genomic stability of 677CC, 1298AA is higher than the mutation homozygotic 677TT and 1298CC.(8) The genomic stability of breast cancer patients with 677CC or 1298AA was significantly lower than the control group with same genotype. However, DDA analysis revealed that those 2 groups didn't show different sensitivity to FA and RF deficiency. We thereby conclude that the genomic stability is not only affected by FA ,RF and MTHFR polymorphism but also interfered by other endogenous factors.The research explored the role and the mechanisms of folate, riboflavin and MTHFR polymorphisms on human genomic stability from the vision of Nutrigenomics. We conclude that 120nM FA is the optimal concentration for maintaining lymphocyte genomic stability in vitro. In vitro data may not predict precisely in vivo folic acid requirement, however, they provide a useful guide of optimal concentration range for genome health. MTHFR C677T, A1298C polymorphisms and RF affected genome stability but the effects were relatively weak compared with that of FA. The research showed that there was no significant sensitivity differences toward FA and RF deficiency between case and control, i.e., their role on genomic stability maintaining in lymphocytes of both breast cancer patients and controls was on a similar extent. However low intake of folate for long term could increase genetic damage and further aggravate the genomic instability of breast cancer-prone subjects. Adequate FA intake may be of benefit to breast cancer prevention.