Abstract Objective To investigate folic acid deficiency and MTHFR C677T genotype polymorphism genetic damage in lymphocytes of breast cancer patients and their differences with the control. Methods 9 days lymphocyte long-term culture supplemented cytoplasmic blocking the the cell group micronucleus (CBMN cyt), analysis of different concentrations of folic acid on three kinds of breast cancer MTHFR C677T genotype the crowd and health crowd binucleate cells micronucleus rate differences. The results of the of MTHFR C677T three genotypes (CC, CT, TT) of breast cancer individuals with control group lymphocytes in folate concentrations of 30 nmol / L when the dual-core cells micronucleus (MNBN) are significantly higher than the 60, 120 and 240 and 2 260 nmol / L test group (P <0.001 to 0.05), failed to find a significant difference in the 60 and 120 nmol / L between the two test groups, as well as 120, 240 and 2 260 nmol / L between the three test groups. In the the breast individual and lymphocytes in the control group, MTHFR677TT in any culture conditions, MNBN frequencies are significantly higher than the corresponding sample group of similar culture homozygous wild type (CC) (P <0.01 to 0.05); abatement sample their own genetic damage background after, the same genotype of the breast individuals and normal individuals lymphocytes MNBN frequency no significant difference. Conclusion In the case of in vitro, less than 120 nmol / L concentration of folic acid can increase human lymphocytes genetic damage, whether healthy people or patients with cancer of MTHFR 677TT homozygous individuals of folic acid deficiency are more sensitive.

Key words methylenetetrahydrofolate reductase; C677T polymorphism; folic acid; breast cancer

 Role of Folate Deficiency and MTHFR C677T Polymorphisms on Genetic Damage in Human Lymphocytes from Breast Cancer Case

    WU Xia  yu1, NI Juan1, ZOU Tian  ning2, LIANG Zi  Qing1, WANG Xu1

    1.School of Life Sciences, Yunnan Normal University, Kunming 650092, China; 2.Thirdary Affiliated Hospital of Kunming Medical College

    Corresponding Author: WANG Xu, E  mail: wangxu@fudan.edu.cnAbstract: Objective To investigate the role of folate deficiency and MTHFR C677T polymorphism on genetic damage in human lymphocytes from breast cancer cases and controls.Methods Lymphocytes with different MTHFR C677T genotypes from the donors were cultured for 9 days in media with different concentrations of folic acid (FA) and the frequencies of micronucleated binucleated cell (MNBN) was evaluated by cytokinesis  block micronucleus cytome assay (CBMN cyt). Results The frequencies of micronucleated binucleated cell (MNBN) in 30 nmol / L FA were significantly higher than those in 60, 120, 240, 2 260 nmol / L FA (P <0.001 ~ 0.05), but no significant differences were observed either between those in 60 and 120 nmol / L FA, or between those in 120, 240 and 2 260 nmol / L FA within either groups when the genotype was the same. Within either of cancer case and control groups, the frequencies of MNBN of mutant homozygotes (TT) was significantly higher than that of wild isozygotes (CC) (P <0.01 ~ 0.05) in any FA concentration group. After the genetic damage background of sample was reduced, there was no significantly difference in MNBN frequency between lymphocytes with the same genotype from case and control at the same FA concentration.Conclusion FA concentration below 120 nmol / L increased genetic damage in human lymphocytes in vitro. Individuals with MTHFR 677TT genotype may be more sensitive to folic acid deficiency regardless the cancer statues than those with MTHFR 677CC and 677 CT genotypes.

    Key words: MTHFR; C677T; Folic acid; Breast cancer
Folic acid is the body’s normal development of important nutrients. The one hand, it completed by providing methyl uracil deoxy nucleotide (dUMP) to thymidine nucleotide (dTMP) synthesis, on the other hand by homocysteine ​​(Hcy) synthetic methionine (Met) , the S  adenosylmethionine (SAM) biochemical processes that affect DNA methylation [1  4], folic acid plays an important role in maintaining genome stability. Methylenetetrahydrofolate reductase (MTHFR) control of folic acid to dTMP synthesis as well as a key enzyme of methylation of cytosine   guanine nucleotide phosphorylation (CpG), in the case of folic acid deficiency, MTHFR activity decreased may improve the of 5,10  Methylentetrahydrofolate concentration, reducing the concentration of of 5  methyl-tetrahydrofolate [5]. In this case, during the synthesis of dTMP may be stronger than the SAM synthetase over, thereby reducing dUMP mismatch to the DNA initiator chromosomal breakage, and may weaken the synthesis of methyl donor. Therefore MTHFR play a key role in DNA synthesis and methylation. MTHFR activity may be affected by the impact of MTHFR gene sequence variations, Met or SAM concentration, flavin adenine dinucleotide and riboflavin concentration factors [5  6].

    View of folic acid deficiency and MTHFR gene polymorphisms are related to the operational status of folate metabolism pathway and DNA of various metabolic activities affect the stability of the chromosome of the structure and function of DNA and tumor occurrence, development is closely related to the study by cyt CBMN [7] to explore the impact of different folate concentrations and the MTHFR C677T polymorphism on breast cancer patients and their controls lymphocyte genetic damage.

    1 Materials and methods

    1.1 Materials

    From the Third Affiliated Hospital of Kunming Medical College, 19 in the case of informed consent, pre-treatment of women with breast cancer (35 to 52 years old) sample collection of 20 healthy individuals of the same age, at the same time from Kunming Blood Center, each extracted peripheral blood 5 ml isolated lymphocytes were cultured in vitro.

    1.2 Reagents

    Folate, folic acid-free RPMI  1640 medium, L  glutamyl μ amine, cytochalasin B were purchased from Sigma Chemical Co.; penicillin and streptomycin were purchased from the General Pharmaceutical Factory of Harbin Pharmaceutical Group; conventional RPMI  1640 from Gibco ; newborn calf serum was purchased from Hangzhou Evergreen Biological Engineering Materials Co., Ltd.; dialyzed calf serum was purchased from Hyclone Company (SH30079.02); lymphocyte separation medium was purchased from the Shanghai Hua refined bio-tech Co., Ltd.; interleukin  Ⅱ purchased from Shenzhen the new Jill pharmaceutical, heparin (12 500 u) were purchased from the Shanghai Biochemistry preparation plant; Hinf Ⅰ, Mbo Ⅱ purchased from Huamei.

    1.3 PCR  RFLP analysis of MTHFR C677T genotype

    The 500μl peripheral blood genomic DNA was extracted, PCR amplification Get MTHFR gene fragments, PCR products were digested with Hinf Ⅰ, Mbo Ⅱ agarose gel electrophoresis to identify 677CC, 677CT, 677TT genotype, as described in [6].

    1.4 Cell culture

    According to human plasma folate physiological concentrations (20 to 40 nmol / L) [2  3], experimental folate concentrations Set 30,60,120,240,2 260 nmol / L (normal RPMI1640 medium), medium The composition and concentration consistent with RPMI1640 medium. Lymphocyte separation medium lymphocytes isolated from the peripheral blood of the tested individual cell concentration of 0.5 × 106 / ml were cultured in 1 ml containing 5% dialyzed calf serum, 100 units of double-antibody, 2 mmol / LL  glutamine 10 μl / ml PHA, pH 7.0, with different concentrations of folic acid medium. Cells at 37oC, 5% CO2 incubator culture, once the appropriate medium was changed every 3 days, 8 days of culture, adding cytochalasin B (final concentration of 4.5 μg / ml) and cultured for 28 h, the 9th day of 1000 r / min centrifugation supernatant was removed, adding about 20 μl of 6% DMSO containing fetal calf serum, and mix gently, allowed to stand at room temperature for 8 min, smears, air drying 1 h, methanol and acetic acid (3:1) for 10 min air drying, 5% Giemsa (pH 7.0) staining 5 min, air-dried, were mounted, CBMN analysis the the binucleate cells micronucleus rate (MNed BN / 1 000 BN cells MNBN) [7  8], each piece analysis 000 pairs of nucleated cells per concentration group analysis of more than 5000 dual-core cells.

    1.5 statistical methods

    SPSS12.0 one  way ANOVA analysis of different concentrations of folic acid, C677T gene polymorphism in lymphocytes of the breast individuals and normal individuals genotoxic effects, comparing breast cancer patients and normal individuals of the same genotype on folate deficiency caused by Independent Samples Test the genotoxic sensitivity differences, and to Diference of difference abatement background differences within each group of samples to further resolve the difference in sensitivity of breast cancer patients and normal individuals of folate deficiency [9], Sigma plot 10.0 plotted.

    2 Results

    The breast patients or controls MNBN of frequency at 30 nmol / L test group was significantly higher than 60,120,240 2 260 nmol / L four test groups (P <0.001 to 0.05), and 60 nmol / L and 120 nmol / no statistically significant difference between the concentration of L two 120,240,2 260 nmol / L three test groups, no statistically significant difference, on this basis, we have statistics folate concentrations were 30,120 and 240 nmol / L, different genotypes (wild-type homozygous CC genotype, heterozygous CT type, mutant homozygous TT genotype) human lymphocyte micronucleus rates are shown in Table 2, Figure 1 and 2. The results show that both the cases or control lymphocytes TT genotype in three concentration group, MNBN are significantly higher than similar training wild homozygous CC genotype (P <0.01 to 0.05); removal of the breast cancer individuals with the control group background values, when folate concentrations between the same genotype of the breast individuals with normal individual MNBN frequency differences were not statistically significant. Table 1 folate concentrations blocking lymphocyte micronucleus frequency of breast cancer patients and the control group of individuals cytoplasmic

    3 Discussion

    MTHFR folate metabolism in a key enzyme, the main catalytic 5,10  Methylentetrahydrofolate to the 5  methyltetrahydrofolate’s conversion, is a very important enzyme for of balanced DNA synthesis and DNA methylation. The MTHFR enzyme activities affect the the the plasma 5,10  methylene tetrahydrofolate concentration of 5,10  Methylentetrahydrofolate concentration of dUMP to dTMP into play a crucial role in affecting DNA synthesis and repair. 5,10  Methylentetrahydrofolate beneficial high intracellular concentration of the synthesis of dTMP, thereby enhancing the stability of DNA [10  11]. MTHFR gene 677 points in C → T replacement [12] 677TT genotype and risk of the incidence of many cancers associated: the TT homozygote colon cancer risk may be reduced, and gastric cancer, cervical cancer risk increased tendency [13]. 677TT mutant homozygote individual plasma homocysteine ​​was significantly higher than the 677CC wild-type homozygote individual red blood cell folate concentrations were significantly lower than 677CC wild-type individuals [10], the reports also display the 677TT individual with respect 677CT and the 677CC individual, there may be a higher breast cancer (or other disease) risk of [10,12]. When insufficient intake of folic acid, the same will increase dUMP mismatch in the DNA of the opportunity. This study found that the the same folate concentrations 677TT cases and controls lymphocyte micronucleus rates were significantly higher than those in the sample group 677CC genotype, suggesting that the genotype folate deficiency more sensitive, however, how it affects MTHFR gene polymorphism DNA stability and malignant transformation has not accurately reported. MTHFR is irreversible catalytic 5,10 the  methylene tetrahydrofolate to 5  conversion of methyltetrahydrofolate, 5  methyltetrahydrofolic folic acid eventually to provide methyl complete the synthesis of SAM, this biochemical reactions on DNA methylation and gene expression is essential [14], suggesting that the decline of MTHFR activity may reduce the concentration of 5  methyltetrahydrofolate, the degree of DNA methylation. The results of this trial do not get the expected assumption homozygous MTHFR 677 locus gene mutation increased breast cancer genetic instability. Genome genetic damage in breast cancer patients with folic acid concentration increased significantly reduced, and that the intake of folic acid may reduce the risk of breast cancer. In this study, we compared through five concentration group found that folic acid concentration of 30, 60 and 120 nmol / L, the micronucleus rate with increasing folate concentrations decreased folate concentrations of 120, 240 and 2 260 nmol / L, the micronucleus rate no longer with folate concentrations increased significantly change, therefore we believe that the case vitro, less than 120 nmol / L folate concentration increased human lymphocytes genetic damage, which in the past we ‘s findings coincide [15  18]. View of normal human plasma folate physiological concentration of 20 to 40 nmol / L, below we maintain genetic stability the optimal folate concentrations, suggesting that additional supplemental folic acid may help reduce the body’s genetic risk of injury and disease.