Folic Acid And Some Of Its Related Metabolic Molecules With Human Genetic Stability

A lot of study confirmed that folate is an important micronutrient in human body. It is required for the synthesis of dTMP from dUMP. Under condition of folate deficiency dUMP accumulates, and as a result, uracil is incorporated into DNA instead of thymine. The excessive incorporationof uracil in DNA not only leads to point mutation but also results in single and double strand DNA break, chromosme breakage and micromucleus formation. S-adenosylmethionine is the common methyl donor required for the maintenance of methylation patterns in DNA that determine gene expression and DNA conformation. The experiment aimed to evaluate the genotoxic effects of Met and B₁₂ deficiency in human lymphoblast cell line carrying BRCA1 mutation.30 health and 24 breast cancer women aged 35-65 years and not receiving anti-folate therapy or cancer treatment were recruited for the study. Patients donated a fasted blood sample (5ml in lithium-hepairminized vacuettes). Lymphocytes were isolated using Ficoll-Paque. Lyphocyte cultures were prepared at a concentration of 0.5×10⁶cells/ml in 1ml RPMI 1640 medium containing 30nM, 120nM, 240nM FA, 8% dialyzed fetal bovine serum, 10U/ml interlukin-2, 2mM L-Glutamine, 100U/ml penicillin 100ug/ml streptomycin. The concertration of folate chosen were intended to incestigate effects within the deficiency and sufficiency concentration range for which we had already shown clear diffierence in genomic stability. Mitogenesis was stimulated by the addition of phytohaemagglutinin (45ug/ml, PHA) and cultures were incubated at 37℃ and 5% CO₂ in a humidified incubator. After 3 days, The cultures were continued in 0.8ml fesh medium and 0.2ml "conditioned" medium from the previous 3 day culture. The components of medium were same as above but without PHA. This process of counting and re-culturing cells was repeated 6 days post-PHA treatment.At 8 days post-PHA treatment, two 750ul aliquots of each culture were transferred to 6ml culture tubes for the CBMN assay to measure chromosome damage. Cytochalasin B (4.5ug/ml) was added to each tube and approximately 28h later cells were harvested onto microscope slides using a cyto-centrifuge. Slides were then air dried, fixed and stained with DMSO. Coded slides were scored for the frequency of MNed BNCs, MNed monos, NBUDs, NPBs. 1000-1500 BNCs were scored per culture to demine frequency of MNed BNCs, NPBs and NBUDs. MNed monos cell frequency was determined by scoring 500 MONO cells. The scoring criteria usedwere those described by Fench.However the lymphoblast cells (GM13705, BRCAl mutation, GM12593) were cultured under 12 combinations of deficiency and sufficiency of vitamin B12 (75-1200 pM) and methionine (15, 50 uM) medium for 9 days, Common RPMI 1640 culture was set as the control. The method of culture and scoring method as the previous.The result showed that:1 , folic acid concentration correlated significantly (/K0.0001) and negatively (r=-0.248—0.832) with all markers of chromosome damage;The markers of chromosome damage in 30nM folic acid groups were significantly increased comparing with 120nM, 240nM of folic acid (/><0.0001); No significant differences were found among those two groups, which were minimized at 120nM folic acid much greater that concentrations assumed "normal", but necessarily optimal in plasma. Conclusion: 120nM of folic acid was able to minimize genetic, damage of the lymphoblast Cell under the current experimental condition.2 % Met-Bn concentration correlated significantly (pO.OOOl) and negatively (r=-0.519—0.856) with all markers of chromosome damage of two tested cell lines; All markers of chromosome damage in both cell lines were significantly higher in 15uM of Met combined with all concentrations of B12 than that of 50 uM of Met with all concentrations of B12 (p<0.05) . The frequencies of MNBN of normal and breast cancer cell line were remarkably reduced when 50uM of met and 600pM of B12 were used; In same concentration of Met-Bi2the genome with BRCAl mutation cell line may be more sensitive to the stress of B12 deficiency than normal cell line. Conclusion: We conclude that 50 uM of Met and 600 pM of B12 are optimal combination for stabilizing the genome of the test cell line.This preliminary investigation suggests Met and B12 deficiency causes genomic instability. It is considerable to supply some adequate amount of Met and B12U) keep genome stability.