Effects of folic acid deficiency and MTHFR C677T polymorphism on spontaneous and radiation-induced micronuclei in human lymphocytes.

Folic acid plays a key role in the maintenance of genomic stability, providing methyl groups for the conversion of uracil to thymine and for DNA methylation. Besides dietary habits, folic acid metabolism is influenced by genetic polymorphism. The C677T polymorphism of the methylene-tetrahydrofolate reductase (MTHFR) gene is associated with a reduction of catalytic activity and is suggested to modify cancer risk differently depending on folate status. In this work the effect of folic acid deficiency on genome stability and radiosensitivity has been investigated in cultured lymphocytes of 12 subjects with different MTHFR genotype (four for each genotype). Cells were grown for 9 days with 12, 24 and 120 nM folic acid and analyzed in a comprehensive micronucleus test coupled with centromere characterization by CREST immunostaining. In other experiments, cells were grown with various folic acid concentrations, irradiated with 0.5 Gy of gamma rays and analyzed in the micronucleus test. The results obtained indicate that folic acid deficiency induces to a comparable extent chromosome loss and breakage, irrespective of the MTHFR genotype. The effect of folic acid was highly significant (P < 0.001) and explained >50% of variance of both types of micronuclei. Also nucleoplasmic bridges and buds were significantly increased under low folate supply; the increase in bridges was mainly observed in TT cells, highlighting a significant effect of the MTHFR genotype (P = 0.006) on this biomarker. Folic acid concentration significantly affected radiation-induced micronuclei (P < 0.001): the increased incidence of radiation-induced micronuclei with low folic acid was mainly accounted for by carriers of the variant MTHFR allele (both homozygotes and heterozygotes), but the overall effect of genotype did not attain statistical significance. Treatment with ionizing radiations also increased the frequency of nucleoplasmic bridges. The effect of folic acid level on this end-point was modulated by the MTHFR genotype (P for interaction = 0.02), with TT cells grown at low folic acid concentration apparently resistant to the induction of radiation-induced bridges. Finally, the effect of in vitro folate deprivation on global DNA methylation was evaluated in lymphocytes of six homozygous subjects (three CC and three TT). The results obtained suggest that, under the conditions of this work, folic acid deprivation is associated with global DNA hypermethylation.

[1]  M. Fraga,et al.  DNA methyltransferases control telomere length and telomere recombination in mammalian cells , 2006, Nature Cell Biology.

[2]  V. Torri,et al.  Methylenetetrahydrofolate reductase 677C/T gene polymorphism, gastric cancer susceptibility and genomic DNA hypomethylation in an at‐risk Italian population , 2006, International journal of cancer.

[3]  H. Powers.  Interaction among folate, riboflavin, genotype, and cancer, with reference to colorectal and cervical cancer. , 2005, The Journal of nutrition.

[4]  M. Fenech,et al.  Folic acid deficiency increases chromosomal instability, chromosome 21 aneuploidy and sensitivity to radiation-induced micronuclei. , 2005, Mutation research.

[5]  T. Lange,et al.  Shelterin: the protein complex that shapes and safeguards human telomeres , 2005 .

[6]  T. Milewicz,et al.  Uracil misincorporation into DNA of leukocytes of young women with positive folate balance depends on plasma vitamin B12 concentrations and methylenetetrahydrofolate reductase polymorphisms. A pilot study. , 2005, The Journal of nutritional biochemistry.

[7]  M. Fenech.  The Genome Health Clinic and Genome Health Nutrigenomics concepts: diagnosis and nutritional treatment of genome and epigenome damage on an individual basis. , 2005, Mutagenesis.

[8]  M. Wolters,et al.  Folic acid and colorectal cancer prevention: molecular mechanisms and epidemiological evidence (Review). , 2005, International journal of oncology.

[9]  J. Stempak,et al.  Cell and stage of transformation-specific effects of folate deficiency on methionine cycle intermediates and DNA methylation in an in vitro model. , 2005, Carcinogenesis.

[10]  B. Henderson,et al.  The MTHFR C677T Polymorphism and Colorectal Cancer: The Multiethnic Cohort Study , 2005, Cancer Epidemiology Biomarkers & Prevention.

[11]  M. Lucock,et al.  Folic acid |[mdash]| vitamin and panacea or genetic time bomb? , 2005 .

[12]  A. Neugut,et al.  One-carbon metabolism, MTHFR polymorphisms, and risk of breast cancer. , 2005, Cancer research.

[13]  A. V. D. Van Der Zee,et al.  Methylenetetrahydrofolate reductase (MTHFR) and susceptibility for (pre)neoplastic cervical disease , 2005, Human Genetics.

[14]  Tom J. Petty,et al.  Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks , 2004, Nature.

[15]  Tony Kouzarides,et al.  Methylation of H3 Lysine 4 at Euchromatin Promotes Sir3p Association with Heterochromatin* , 2004, Journal of Biological Chemistry.

[16]  J. Schlesselman,et al.  Polymorphisms in the methylenetetrahydrofolate reductase gene and prostate cancer risk. , 2004, International journal of oncology.

[17]  M. Fenech,et al.  Folate deficiency induces aneuploidy in human lymphocytes in vitro-evidence using cytokinesis-blocked cells and probes specific for chromosomes 17 and 21. , 2004, Mutation research.

[18]  G. Rampersaud,et al.  Folic acid supplements and fortification affect the risk for neural tube defects, vascular disease and cancer: evolving science. , 2003, The Journal of nutrition.

[19]  C. Ulrich,et al.  5,10-Methylenetetrahydrofolate reductase polymorphisms and leukemia risk: a HuGE minireview. , 2003, American journal of epidemiology.

[20]  J. Boer,et al.  A common variant of the methylenetetrahydrofolate reductase gene (1p36) is associated with an increased risk of cancer. , 2003, Cancer research.

[21]  M. Fenech,et al.  Nucleoplasmic bridges are a sensitive measure of chromosome rearrangement in the cytokinesis-block micronucleus assay. , 2003, Mutagenesis.

[22]  A. Aro,et al.  Plasma homocysteine concentration is decreased by dietary intervention* , 2003, British Journal of Nutrition.

[23]  E. Giovannucci.  Epidemiologic studies of folate and colorectal neoplasia: a review. , 2002, The Journal of nutrition.

[24]  S. Duthie,et al.  Impact of folate deficiency on DNA stability. , 2002, The Journal of nutrition.

[25]  Sang-Woon Choi,et al.  Folate status: effects on pathways of colorectal carcinogenesis. , 2002, The Journal of nutrition.

[26]  M. Fenech,et al.  Micronuclei, nucleoplasmic bridges and nuclear buds induced in folic acid deficient human lymphocytes-evidence for breakage-fusion-bridge cycles in the cytokinesis-block micronucleus assay. , 2002, Mutation research.

[27]  J. Beilby,et al.  A polymorphism in the methylenetetrahydrofolate reductase gene predisposes to colorectal cancers with microsatellite instability , 2002, Gut.

[28]  J. Trepel,et al.  Impact of extracellular folate levels on global gene expression. , 2001, Molecular pharmacology.

[29]  B. Ames,et al.  The effect of folic acid deficiency and MTHFR C677T polymorphism on chromosome damage in human lymphocytes in vitro. , 2001, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology.

[30]  B. Ames,et al.  Methylenetetrahydrofolate reductase C677T polymorphism does not alter folic acid deficiency-induced uracil incorporation into primary human lymphocyte DNA in vitro. , 2001, Carcinogenesis.

[31]  M. Fenech.  The role of folic acid and Vitamin B12 in genomic stability of human cells. , 2001, Mutation research.

[32]  M. Fenech.  The in vitro micronucleus technique. , 2000, Mutation research.

[33]  R. Rozen,et al.  Polymorphisms in genes involved in folate metabolism as maternal risk factors for Down syndrome. , 2000, American journal of human genetics.

[34]  G. Morgan,et al.  Polymorphisms in the methylenetetrahydrofolate reductase gene are associated with susceptibility to acute leukemia in adults. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[35]  M. Durante,et al.  Chromatid break rejoining and exchange aberration formation following gamma-ray exposure: analysis in G2 human fibroblasts by chemically induced premature chromosome condensation. , 1999, International journal of radiation biology.

[36]  S. Duthie,et al.  DNA instability (strand breakage, uracil misincorporation, and defective repair) is increased by folic acid depletion in human lymphocytes in vitro , 1998, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[37]  A. Natarajan,et al.  Kinetics of the formation of chromosome aberrations in X-irradiated human lymphocytes, using PCC and FISH. , 1998, Mutation research.

[38]  M. Hande,et al.  Telomere length, chromatin structure and chromosome fusigenic potential , 1997, Chromosoma.

[39]  C. Wagner,et al.  DNA methylation in folate deficiency: use of CpG methylase. , 1993, Biochemical and biophysical research communications.

[40]  T. de Lange,et al.  Shelterin: the protein complex that shapes and safeguards human telomeres. , 2005, Genes & development.

[41]  M. Fenech,et al.  Methylenetetrahydrofolate reductase C677T polymorphism, folic acid and riboflavin are important determinants of genome stability in cultured human lymphocytes. , 2004, The Journal of nutrition.

[42]  J. Mason,et al.  Folate and carcinogenesis: an integrated scheme. , 2000, The Journal of nutrition.

[43]  R. Matthews,et al.  A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase , 1995, Nature Genetics.

[44]  J D Tucker,et al.  Identification of aneuploidy‐inducing agents using cytokinesis‐blocked human lymphocytes and an antikinetochore antibody , 1989, Environmental and molecular mutagenesis.