Mutagenesis Advance Access originally published online on September 1, 2006
Mutagenesis 2006 21(5):327-333; doi:10.1093/mutage/gel031
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Effects of folic acid deficiency and MTHFR C677T polymorphism on spontaneous and radiation-induced micronuclei in human lymphocytes
Istituto Superiore di Sanità Rome, Italy 1 Università ‘La Sapienza’ Rome, Italy
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.
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Introduction |
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Folic acid metabolism plays an important role in the maintenance of genomic stability, providing methyl groups for the conversion of uracil to thymine and for the synthesis of S-adenosyl-methionine, required for physiological DNA methylation in mammals (1
,2). A growing body of evidence suggests that a deficient supply of dietary folic acid may be a risk factor for several human diseases, including neonatal malformations, Down syndrome, Alzheimer disease, cardiovascular disorders and cancer (3–5). In vitro studies on human cells show that low levels of serum folate are associated with misincorporation of uracil into DNA and DNA damage (6–8), as well as with aneuploidy of chromosomes involved in tumors and other diseases associated with folate deficiency (9,10).
As mammals are unable to synthesize folic acid de novo, serum levels of this micronutrient are mainly modulated by dietary habits (11). Another source of variability in folate metabolism is represented by the polymorphism of genes implicated in relevant biochemical pathways. Among these, a prominent role is played by methylene-tetrahydrofolate reductase (MTHFR), which catalyzes the conversion of 5,10-methylene-tetrahydrofolate into 5-methyl-tetrahydrofolate, the precursor for methylation of homocysteine to methionine. The C677T polymorphism in MTHFR determines an alanine to valine substitution, with a significant reduction of catalytic activity (12).
Epidemiological studies indicate that the MTHFR TT genotype is associated with a lower risk of acute lymphocytic leukemia (13,14) and colon carcinoma (15), especially in subjects with low-risk diet high in folate (16,17). This protective effect has been related to the diversion of folic acid to thymidine synthesis under suboptimal MTHFR activity, with consequent lower misincorporation of uracil into DNA (17,18). On the other hand, in conditions of folic acid deficiency the MTHFR variant genotype has been shown to be associated with an increased risk of developmental defects in utero and cancer at various sites (colon, breast, gastric, cervical and prostate) (19–23).
The mechanism by which low folate status and MTHFR deficiency interact raising the risk of tumors and misconception is not known. Previous studies on cultured human lymphocytes did not highlight a significant influence of the C677T polymorphisms on genomic instability induced by variation of folic acid concentration within the physiological range (12–120 nM) (24,25). However, it was suggested that the high riboflavin content of the culture medium used in these studies could have improved MTHFR activity above normal level, masking the functional consequences of the polymorphism (25). In any case, whether the MTHFR/folate status may modulate DNA repair capacity and the individual susceptibility to induced genotoxic effects has not been elucidated.
In this work, we have further investigated the effects of folate deficiency on genome stability and radiosensitivity in human lymphocytes, also in relation to the MTHFR C677T polymorphism. To this aim, peripheral lymphocytes of donors with different MTHFR genotype were grown in the presence of various folic acid concentrations and analyzed in a comprehensive micronucleus test, including the analysis of nucleoplasmic bridges (NPBs), as biomarkers of chromosome rearrangements, and nuclear buds (NBuds) as biomarkers of gene amplification (26). In order to get insights on the mechanism responsible for micronucleus formation, micronuclei were characterized for centromere content by immunostaining with CREST antibody. The degree of genomic DNA methylation was evaluated as well. Moreover, the sensitivity to ionizing radiation in conditions of folic acid deficiency was assessed after in vitro irradiation with gamma rays, in order to probe the combined effect of folate deficiency and MTHFR genotype on DNA repair proficiency.
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Materials and methods |
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TopIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Study subjects
Study subjects were selected from a cohort of healthy non-smoker female donors in collaboration with the Institute of Haematology of Rome University ‘La Sapienza’. Four age-matched subjects for each genotype were enrolled in the study. The average age of the study group was 32 years (SD 6). All subjects gave an informed consent to their participation in the study.
MTHFR genotyping
Genomic DNA was isolated from whole blood samples using Instagene Matrix (BioRad) according to manufacturer's instructions. The MTHFR C677T mutation was detected after PCR amplification with the appropriate primers (27). The variant allele was identified by the presence of a 175 bp fragment after digestion with HinfI that leaves uncut the 198 bp wild-type fragment.
Lymphocyte cultures
From each subject a blood specimen (7 ml) was collected by venipuncture in vacutainers containing sodium heparin as anticoagulant (Becton Dickinson). Lymphocytes were isolated using Histopaque-1077 gradients (Sigma) and placed in culture medium without folic acid, with L-glutamine and phenol red (RPMI 1640; GIBCO) added with 25 µl/ml of 1 M HEPES buffer solution (Euroclone), 1% penicillin (5000 U/ml) and streptomycin (5000 µg/ml) solution (Flow), 10% inactivated fetal calf serum (FCS; Euroclone), 0.5% phytohaemagglutinin HA15 (Murex), 10 U/ml interleukin-2 (Roche Diagnostic), 9 µl/ml sodium pyruvate 100 mM (Euroclone). Three folic acid (Sigma) concentrations, 120, 24 and 12 nM, were selected on the basis of literature data (24,25). Triplicate 2 ml cultures, with 1 x 105 lymphocytes/ml, were set up in 15 ml conical tubes (Falcon) and incubated at 37°C and in a humidified 5% CO2 incubator for 9 days. Culture medium was changed at the 3rd and 6th culture day, according to a published experimental protocol (24,25). Eight days after phytohaemagglutinin addition, each culture was split in two 1 ml aliquots, providing a total of six tubes per dose for each subject. Four cultures were used for cytogenetic analyses and immunostaining with anti-kinetochore antibody, two cultures were used for the gamma rays sensitivity test.
Cytokinesis block micronucleus assay
Cytochalasin B 4.5 µg/ml (Sigma) was added to cultures used for cytogenetic analyses on the 8th day of culture. After 28 h cells were spun onto microscope slides using a cytocentrifuge (Shandon). Smears were air-dried, fixed 10 minutes in methanol and stained in 4% Giemsa phosphate buffer. Cells were analyzed in the comprehensive micronucleus test following the criteria set up by Fenech (26). The frequencies of Binucleated cells with Micronuclei (MnBin), Total Micronuclei (MnTot), Buds and Nucleoplasmic Bridges (NPBs) were determined analyzing 1000 binucleate lymphocytes with a well-preserved cytoplasm from two slides. The nuclear division index (NDI), a cell proliferation index, was determined in 1000 cells according to Eastmond and Tucker (28): [mononucleated cells + (2x binucleated cells) + (3x trinucleated cells) + (4x tetranucleated cells)]/1000.
Immunostaining with CREST antibody
Cytocentrifuge slides processed by CREST antibody immunofluorescence technique were blocked in KCM buffer [120 mm KCl, 20 mm NaCl, 10 mm Tris–HCl (pH 8), 0.5 M EDTA, 0.1% Triton X-100] for 10 min at room temperature. After a gentle wash in 1x phosphate-buffered saline (PBS), 50 µl of anti-kinetochore antibody solution (antibodies incorporated, Davies, CA), diluted 1:1 in 1x PBS, was placed on each smear with a coverslip for 1 h at 37°C in a humidified box. After two 3 min washes in 4x SSC/0.05% Tween-20, and a 6 min passage in PN buffer [0.1 m NaH2PO4, 0.1 m Na2HPO4; 0.1% NP-40 (Sigma), pH 8.0] added with 5% non-fat dry milk (PNM), a 50 µl aliquot of FITC goat anti-human IgG (Chemicon International, CA), previously diluted 1:60 in PNM, was applied on each slide and incubated for 1 h at 37°C in a humidified box. Slides were counterstained with propidium iodide (2.5 µg/ml) in antifade (Vector Laboratories, CA) and observed by fluorescence microscopy. Labeled slides were used immediately or stored in the dark at 4°C. One hundred micronuclei per folic acid concentration per donor were immunocharacterized. Based on the number of immunofluorescence signals micronuclei were classified as CREST-negative (no signal) and CREST-positive with 1, 2 or >2 signals.
Treatments with gamma rays
To test gamma rays sensitivity, lymphocyte cultures were irradiated at Day 8 with 0.5 Gy from a 137Cs source at a dose rate of 1 Gy/min. After irradiation, lymphocytes were centrifuged, medium replaced and Cyt-B was added at the final concentration of 4.5 µg/ml. Cells were harvested 28 h later using a cytocentrifuge. For each experimental point micronuclei, nucleoplasmic bridges and buds were scored in 1000 binucleated cells from two slides. Baseline levels in unirradiated cells were subtracted to calculate radiation-induced chromosome damage for further statistical analysis.
DNA methylation
DNA methylation was determined using a published method based on the incorporation of [3H]methyl groups by a bacterial CpG methylase in genomic DNA (29). In this assay the ability of DNA to incorporate [3H]methyl groups in vitro is inversely related to endogenous methylation. Briefly, DNA (0.5 µg/sample) was incubated with [3H]methyl S-adenosylmethionine in the presence of the prokaryotic CpG methylase SssI (New England BioLabs), methylated DNA isolated on ion-exchange paper filter and analyzed in a liquid scintillation counter. Incubation conditions and processing of samples followed the original published procedure (30).
Statistical analysis of data
All data were log transformed before statistical analysis. Means of groups with different genotype were compared by two-tailed Student's t-test; mean values of each group at different folic acid concentrations were compared using the matched-pair Student's t-test. The effect of folic acid concentration on genetic biomarkers in the study population (on combined data from all subjects) was evaluated by repeated-measures one-way analysis of variance (ANOVA). Two-way ANOVA was used to analyze the effect of folic acid concentration and genotype and their interaction, and the total variation attributable to these factors. Pearson correlation was used to test the interrelationships among biomarkers of DNA damage. All analyses were performed with the SPSS Statistical Package (Version 11.0).
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Results |
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Cytokinesis block micronucleus assay
Lowering folic acid concentration in culture medium significantly increased the frequency of binucleated cells with micronuclei (MnBin) and total micronuclei (MnTot) in cytokinesis-blocked lymphocytes of all donors (Table I). The effect of folic acid was highly significant (P < 0.001) and accounted for 74 and 72% of total variance of MnBin and MnTot, respectively. At the dose 24 nM, a significantly (P < 0.05) higher incidence of MnBin and MnTot was observed in TT cells compared with both CC homozygotes and CT heterozygotes. However, no significant difference among MTHFR genotypes was observed at the lower folic acid concentration. Overall, the effect of the MTHFR genotype on the incidence of micronuclei was not significant.
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The overall incidence of nucleoplasmic bridges was significantly increased in cells grown with low folic acid supply (P < 0.05). This increase was mainly accounted for by TT cells, which showed significantly more nucleoplasmic bridges compared with CC and CT cells at low folate concentrations (P < 0.01). Both folic acid and the MTHFR genotype significantly affected NPBs (P = 0.002 and 0.006), explaining, respectively, 14 and 39% of the variation in the incidence of the biomarker.
Also nuclear buds were significantly increased at low folic acid concentrations. The effect of folic acid was highly significant (P < 0.001) and explained 38% of total variance. At low folic acid concentrations TT cells displayed slightly higher bud levels compared with CC and CT cells, but the difference did not attain statistical significance nor did the interaction between folic acid and genotype (P = 0.089).
The comparison of NDI values indicated that folic acid deprivation significantly affected the growth of cultured lymphocytes (P = 0.003). Folic acid accounted for 18% of total variance of NDI, while no significant difference was observed among MTHFR genotypes.
Characterization of micronuclei by CREST immunostaining
For each experimental point, one hundred micronuclei were characterized for the content of kinetochore by CREST immunostaining. The results are summarized in Table II. A general prevalence of CREST-positive micronuclei (Mn+) was observed at all folic acid concentrations, with no striking differences among genotypes. Only at the concentration 24 nM, a significant difference in the proportion of CREST-positive and CREST-negative micronuclei was observed between TT and CC subjects (P < 0.05); however, this observation was not confirmed at the lower folic acid concentration (12 nM), where a similar proportions of Mn CREST-positive and CREST-negative were observed.
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Based on the total number of micronuclei detected (MnTot, as shown in Table I) and the results of CREST hybridization (Table II), the incidence of MN+ and Mn– induced by folic acid deprivation was calculated. As shown in Table III, both CREST-positive and CREST-negative micronuclei were largely increased at 24 and 12 nM folic acid compared with 120 nM. The effect of folic acid concentration on the incidence of both types of micronuclei was highly significant (P < 0.001), while no significant influence of the MTHFR genotype was disclosed by the statistical analysis of data. Folic acid concentration explained 51 and 54% of the variation of Mn+ and Mn–, respectively.
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The interrelationships among biomarkers of genetic damage under different folic acid levels were analyzed by Pearson correlation (Table IV). Data highlight a significant positive correlation between the incidence of buds and nucleoplasmic bridges at both 12 and 24 nM folic acid. Buds were also significantly correlated with MnTot at 24 nM, but not at the lowest folic acid concentration. No significant correlation was observed between the incidences of the biomarkers at 120 nM folic acid and their respective frequencies at lower folate concentrations.
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DNA methylation
Three wild-type MTHFR 677CC and three homozygous TT variant subjects were selected for a preliminary assessment of the influence of folic acid concentration in culture medium on global DNA methylation in cultured lymphocytes. The results obtained are shown in Figure 1. In conditions of folic acid deficiency, a decrease in the incorporation of tritiated methyl groups was observed in all subjects, with no significant differences among genotypes. Folic acid level in culture medium explained 43% of the total variance (P < 0.001) in the incorporation of labeled methyl groups in DNA. As the acceptance of exogenous methyl groups is inversely related to the degree of endogenous methylation, the results obtained indicate that the conditions of folic acid deprivation applied in this work are associated with global DNA hypermethylation.
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Treatments with gamma rays
Lymphocyte cultures grown under different folic acid concentrations were irradiated with 0.5 Gy and analyzed in the micronucleus test. Treatments could not be performed on two subjects (one CC and one CT). The results obtained are summarized in Table V. Treatments induced a distinct increase of MnBin in all cultures. Folic acid significantly affected the incidence of radiation-induced micronuclei (P = 0.003), with relatively greater responses at 24 and 12 nM folic acid compared with 120 nM. The increased susceptibility to irradiation associated with folic acid deficiency was mainly accounted for by the greater response observed in TT and CT cells. At 12 nM folic acid, the incidence of induced micronuclei was significantly higher in CT cells compared with CC wild types (P < 0.05), whereas the difference between TT and CC was not statistically significant (P = 0.3). Folic acid levels explained 24% of the variation in the incidence of radiation-induced micronuclei, whereas the influence of genotype MTHFR was not significant.
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Treatment with ionizing radiations increased the incidence of nucleoplasmic bridges in all cultures grown with 120 nM folic acid (compare with data in Table I). In CC cells, the frequency of NPBs induced by irradiation was slightly higher at low folic acid concentrations, even though the difference with 120 nM did not attain statistical significance. On the other hand, TT cells grown with 12 nM folic acid were apparently resistant to the induction of NPBs by irradiation, showing significantly less induced NPBs than at 120 nM (P = 0.006), and significantly less induced NPB than CC cells at 12 nM folic acid (P = 0.009). The interaction of genotype and folic acid affecting NPB levels was statistically significant (P = 0.02). No consistent effect of irradiation on nuclear buds was detected (data not shown); therefore, data on buds in irradiated cells were not further analyzed. Finally, the NDI of irradiated cultures was generally lower compared with unirradiated cultures (as shown in Table I) and was not significantly affected by folic acid levels or MTHFR genotype.
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Discussion |
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TopIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Epidemiological investigations indicate that the risk of birth defects and cancers associated with folic acid deficiency is modulated by the MTHFR C677T polymorphism (31
–33). The mechanism underlying the interaction between dietary supply of folic acid and MTHFR polymorphism is not elucidated. In fact, while the role of folic acid in the maintenance of genomic stability is well established, the function of the MTHFR polymorphism is less clearly defined. On one hand, the lower methylene-tetrahydrofolate reductase activity associated with the MTHFR variant allele can make available more 5,10-methylene-tetrahydrofolate for the conversion of uracil to thymidine, minimizing uracil misincorporation in DNA. On the other hand, under conditions of severe folic acid deficiency other pathways of folate metabolism, e.g. the maintenance of DNA methylation, may get a prominent role, and the functional consequences of the C677T polymorphism may be different.
The interaction between MTHFR C677T polymorphism and folic acid deficiency was the object of previous in vitro studies with human lymphocytes cultured with low concentrations of folic acid. The results obtained in these studies, using a culture medium with high riboflavin and methionine content, did not highlight a major effect of these polymorphisms on uracil misincorporation (25) and DNA damage induced by folic acid deficiency (24). However, a possible interaction was suggested in a later study with culture medium containing lower physiological concentrations of riboflavine and methionine in which lower incidences of nuclear buds were observed in TT cells compared with CC cells under low folic acid supply (34).
In this work the genetic effects of folate deficiency in relation to the MTHFR C677T polymorphism has been further investigated in a comprehensive micronucleus test coupled with centromere detection and in challenge assays with gamma rays. Overall, the results of micronucleus assays confirmed the effect of folic acid deficiency on genome stability observed in previous studies. The CREST analysis of induced micronuclei performed in this work demonstrated the induction of both kinetochore-positive and kinetochore-negative micronuclei in conditions of folic acid deficiency. This result implies the simultaneous impairment of chromosome integrity and segregation in cells grown with an insufficient folic acid supply. The aneugenic effect of folate deficiency in human lymphocytes and in a lymphoblastoid cell line was described in recent studies in which the malsegregation of specific chromosomes was investigated (9,10). Herewith we provide a measurement of the overall aneugenic effect of folic acid deficiency, affecting all chromosomes, which allows a direct comparison with the clastogenic potential exerted in the same experimental conditions. Based on the results presented it can be concluded that, in the experimental conditions of this work, folic acid deficiency affected to the same extent chromosome integrity and segregation.
A preliminary assessment of the influence of the MTHFR C677T polymorphisms on the studied end-points was also undertaken. The results obtained should be interpreted with caution, bearing in mind the relatively small number of subjects analyzed, and the high riboflavin content of culture medium, which may have lowered the phenotypic expression of the polymorphism (34,35). Anyway, no clear modifying effect of the C677T polymorphism on micronucleus induction or centromere content was observed. On the other hand, a significant influence of the MTHFR genotype on nucleoplasmic bridges was detected. Despite the small number of subjects, the prevalence of bridges in TT cells with low folic acid was clearcut, suggesting a true modifying effect of the C677T polymorphism and confirming similar findings reported by others (34).
The different modifying effect of the MTHFR C677T polymorphism on various cytogenetic markers may indicate that under folic acid deprivation micronuclei and bridges arise through different mechanisms. CREST-negative micronuclei may represent mainly chromosome breaks originating from catastrophic repair of uracil, i.e. from the simultaneous removal of adjacent uracils on opposite strands. As uracil is misincorporated under folate deficiency independently on the MTHFR genotype (6,18), this polymorphisms is not expected to affect the incidence of CREST-negative micronuclei. On the other hand CREST-positive micronuclei, representing chromosome loss, are mainly ascribed to altered methylation of critical CpG sequences, e.g. in satellite DNA or in the promoter of mitotic spindle check-point genes (8–10). The influence of the MTHFR genotype on DNA methylation in relation to folic acid supply has not been studied in detail. However, the preliminary evaluation of global CpG methylation performed in this study did not highlight significant differences among MTHFR genotypes, showing a paradoxical hypermethylation of CpG in all cultures at the lowest dose of folic acid, possibly due to the compensatory upregulation of CpG methyltransferase (35). Indeed folate deficiency affects DNA methylation in a complex way, with site-specific hypomethylation and hypermethylation (27), and it is conceivable that C677T could only affect the methylation of specific target sequences. In this respect, the demethylation of H3 histone at telomeric sequences, with chromatin remodeling and associated chromosome fusigenic potential, could be hypothesized to explain the elevated incidence of NPBs in TT cells (36,37). In fact the hypomethylation of subtelomeric regions and associated proteins is known to lead to telomere elongation (38), which is associated with high incidences of end-to-end chromosome fusion and recombination (39,40), leading to chromosome rearrangements visualized as NPBs.
As far as the treatment with gamma rays is concerned, it was recently shown that folate status is an important modifier of the sensitivity to radiation, with higher incidences of radiation-induced micronuclei at low folate concentrations (10). These findings were confirmed in the present investigation. In addition, this study suggested a modifying effect of the MTHFR genotype. Interestingly, at the lowest folic acid concentration the incidences of micronuclei and NPBs in irradiated cells showed an inverse association with the MTHFR genotype: high micronuclei and low NPBs in carriers of the variant allele (CT + TT) and low micronuclei and high NPBs in wild types. Considering that micronuclei and NPBs may represent, respectively, chromosome fragments and exchanges (41), these results may indicate that carriers of the variant allele process radiation-induced double strand breaks with lower efficiency compared with wild types. In fact the kinetic of radiation-induced chromosome damage shows that chromosome breaks decrease as exchanges appear (42,43), indicating that the latter result from the processing of primary lesions, which, if unrepaired, will show up as micronuclei. The impaired methylation of critical targets, such as chromatin-associated proteins involved in repair of DNA double strand breaks (44), could explain the lower efficiency in the processing of radiation induced damage in MTHFR cells. Admittedly these considerations are based on limited experimental findings and are only proposed as a possible conceptual framework for further studies, rather than as an explanation of the results obtained.
In conclusion, the results obtained indicate that folic acid deficiency induces to a comparable extent chromosome breakage and loss in cultured human lymphocytes. Folic acid deficiency was also associated with an increased sensitivity to micronucleus induction by ionizing radiation. The formation of micronuclei was not significantly affected by the MTHFR C677T polymorphism, which instead exerted a modifying effect on nucleoplasmic bridges induced by irradiation or low folic acid supply. Owing to the limited number of subjects investigated, the possibility that other unmatched polymorphisms in folate metabolism are involved cannot be ruled out. Therefore, these findings are to be regarded as preliminary and will need confirmation in larger studies using physiological concentrations of relevant micronutrients.
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Acknowledgments |
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The authors are grateful to Dr Rossella Galati for her help with genotyping and to Mrs M.C.D'Ascoli for technical assistance.
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Notes |
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*To whom correspondence should be addressed. Tel: +390649902840; Fax: +390649903650; Email: crebelli{at}iss.it
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REFERENCES |
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1. Duthie S.J., Narayanan S., Brand G.M., Pirie L., Grant G. (2002) Impact of folate deficiency on DNA stability. J. Nutr. 132:(Suppl. 8), 2444S–2449S.
2. Fenech M. (2001) The role of folic acid and Vitamin B12 in genomic stability of human cells. Mutat. Res 475:57–67.[Web of Science][Medline]
3. Hobbs C.A., Sherman S.L., Yi P., Hopkins S.E., Torfs C.P., Hine R.J., Pogribna M., Rozen R., James S.J. (2000) Polymorphisms in genes involved in folate metabolism as maternal risk factors for Down syndrome. Am. J. Hum. Genet 67:623–630.[CrossRef][Web of Science][Medline]
4. Bailey L.B., Rampersaud G.C., Kauwell G.P. (2003) Folic acid supplements and fortification affect the risk for neural tube defects, vascular desease and cancer: evolving science. J. Nutr 133:1961S–1968S.
5. Choi S.-W. and Mason J.B. (2000) Folate and carcinogenesis: an integrated scheme. J. Nutr 130:129–132.
6. Duthie S.J. and Hawdon A. (1998) DNA instability (strand breakage, uracil misincorporation, and defective repair) is increased by folic acid depletion in human lymphocytes in vitro. FASEB J 12:1491–1497.
7. Fenech M. and Crott J.C. (2002) 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. Mutat. Res 504:131–136.[Web of Science][Medline]
8. Fenech M. (2005) The Genome Health Clinic and Genome Health Nutrigenomics concepts: diagnosis and nutritional treatment of genome and epigenome damage on an individual basis. Mutagenesis 20:255–269.
9. Wang X., Thomas P., Xue J., Fenech M. (2004) Folate induces aneuploidy in human lymphocytes in vitro—evidence using cytokinesis-blocked cells and probes specific for chromosomes 17 and 21. Mutat. Res 551:167–180.[Web of Science][Medline]
10. Beetstra S., Thomas P., Salisbury C., Turner J., Fenech M. (2005) Folic acid deficiency increases chromosomal instability, chromosome 21 aneuploidy and sensitivity to radiation-induced micronuclei. Mutat. Res 578:317–326.[Web of Science][Medline]
11. Silaste M.L., Rantala M., Alfthan G., Aro A., Kesaniemi Y.A. (2003) Plasma homocysteine concentration is decreased by dietary intervention. Br. J. Nutr 89:295–301.[CrossRef][Web of Science][Medline]
12. Frosst P., Blom H.J., Milos R., et al. (1995) A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat. Gene 10:111–113.[CrossRef][Web of Science][Medline]
13. Skibola C.F., Smith M.T., Hubbard A., Shane B., Roberts A.C., Law G.R., Rollinson S., Romanollinson S., Cartwright R.A., Morgan G. (1999) Polymorphism in the methylene-tetrahydrofolate reductase gene are associated with susceptibility to acute leukemia in adults. Proc. Natl Acad. Sci. USA 96:12810–12815.
14. Robien K. and Ulrich C. (2003) 5,10-Methylenetetrahydrofolate reductase polymorphisms and leukemia risk: a HuGE minireview. Am. J. Epidemiol 157:571–582.
15. Strohle A., Wolters M., Hahn A. (2005) Folic acid and colorectal cancer prevention: molecular mechanisms and epidemiological evidence. Int. J. Oncol 26:1449–1464.[Web of Science][Medline]
16. Giovannucci E. (2002) Epidemiologic studies of folate and colorectal neoplasia: a review. J. Nutr 132:(Suppl. 8), 2350S–2355S.
17. Le Marchand L., Wilkens L.R., Kolonel L.N., Henderson B.E. (2005) The MTHFR C677T polymorphism and colorectal cancer: the multiethnic cohort study. Cancer Epidemiol. Biomarkers Prev 14:1198–1203.
18. Choi S.W. and Mason J.B. (2002) Folate status: effects on pathways of colorectal carcinogenesis. J. Nutr 132:(Suppl. 8), 2413S–2418S.
19. Kapiszewska M., Kalemba M., Wojciech U., Milewicz T. (2005) 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. J. Nutr. Biochem 16:467–478.[CrossRef][Web of Science][Medline]
20. Chen J., Gammon M.D., Chan W., Palomeque C., Wetmur J.G., Kabat G.C., Teitelbaum S.L., Britton J.A., Terry M.B., Neugut A.I., Santella R.M. (2005) One-carbon metabolism, Mthfr polymorphisms, and risk of breast cancer. Cancer Res 65:1606–1614.
21. Graziano F., Kawakami K., Ruzzo A., Watanabe G., Santini D., Pizzagalli F., Bisonni R., Mari D., Floriani I., Catalano V., et al. (2006) Methylenetetrahydrofolate reductase 677C/T gene polymorphism, gastric cancer suscptibility and genomic DNA hypomethylation in an at-risk Italian population. Int. J. Cancer 118:628–632.[CrossRef][Web of Science][Medline]
22. Zoodsma M., Nolte I.M., Schipper M., Oosterom E., van der Steege G., de Vries E.G., te Meerman G.J., van der Zee A.G. (2005) Methylenetetrahydrofolate reductase (MTHFR) and susceptibility for (pre)neoplastic cervical disease. Hum. Genet 116:247–254.[CrossRef][Web of Science][Medline]
23. Singal R., Ferdinand L., Das P.M., Reis I.M., Schlesselman J.J. (2004) Polymorphism in the methylenetetrahydrofolate reductase gene and prostate cancer risk. Int. J. Oncol. 25:1465–1471.[Web of Science][Medline]
24. Crott J.W., Mashiyama S.T., Ames B.N., Fenech M. (2001) The effect of folic acid deficiency and MTHFR C677T polymorphism on chromosome damage in human lymphocytes in vitro. Cancer Epidemiol. Biomarkers Prev 10:1089–1096.
25. Crott J.W., Mashiyama S.T., Ames B.N., Fenech M. (2001) Methylenetetrahydrofolate reductase C677T polymorphism does not alter folic acid deficiency-induced uracil incorporation into primary human lymphocyte DNA in vitro. Carcinogenesis 22:1019–1025.
26. Fenech M. (2000) The in vitro micronucleus technique. Mutat Res 455:81–95.[Web of Science][Medline]
27. Stempak J.M., Sohn K.-J., Chiang E.-P., Shane B., Kim Y.-I. (2005) Cell and stage of transformation-specific effects of folate deficiency on methionine cycle intermediates and DNA methylation in an in vitro model. Carcinogenesis 26:981–990.
28. Eastmond D.A and Tucker J.D. (1989) Identification of aneuploidy-inducing agents using cytokinesis-blocked human lymphocytes, and an antikinetochore antibody. Environ. Mol. Mutagen 13:34–43.[Web of Science][Medline]
29. Santos-Rosa H., Bannister A.J., Dehe P.M., Géli V., Kouzarides T. (2004) Methylation of H3 Lysine 4 at euchromatin promotes Sir3p association with heterochromatin. J. Biol. Chem 279:47506–47512.
30. Balaghi M. and Wagner C. (1993) DNA methylation in folate deficiency: use of CpG methylase. Biochem. Biophys. Res. Comm 193:1184–1190.[CrossRef][Web of Science][Medline]
31. Heijmans B.T., Boer J.M., Suchiman H.E., Cornelisse C.J., Westendorp R.G., Kromhout D., Feskens E.J., Slagboom P.E. (2003) A common variant of the methylenetetrahydrofolate reductase gene (1p36) is associated with an increased risk of cancer. Cancer Res 63:1249–1253.
32. Shannon B., Gnanasampanthan S., Beilby J., Iacopetta B. (2002) A polymorphism in the methylene-tetrahydrofolate reductase gene predisposes to colorectal cancers with microsatellite instability. Gut 50:520–524.
33. Lucock M. and Yates Z. (2005) Folic acid—vitamin and panacea or genetic time bomb? Nat. Rev 6:235–240.
34. Kimura M., Umegaki K., Higuchi M., Thomas P., Fenech M. (2004) Methylenetetrahydrofolate reductase C677T polymorphism, folic acid and riboflavin are important determinants of genome stability in cultured human lymphocytes. J. Nutr 134:48–56.
35. Powers H.J. (2005) Interaction among folate, riboflavin, genotype, and cancer, with reference to colorectal and cervical cancer. J. Nutr 135:2960S–2966S.
36. Jhaveri M.S., Wagner C., Trepel J.B. (2001) Impact of extracellular folate levels on global gene expression. Mol. Pharmacol 60:1288–1295.
37. Slijepcevic P., Hande M.P., Bouffler S.D., Landsdorp P., Bryant P.E. (1997) Telomere length, chromatin structure and chromosome fusigenic potential. Chromosoma 106:413–421.[CrossRef][Web of Science][Medline]
38. Gonzalo S., Jaco I., Frago M.F., Chen T., Li E., Esteller M., Blasco M.A. (2006) DNA methyltransferases control telomere lenght and telomere recombination in mammalian cells. Nat. Cell Biol. published online 26 March 2006.
39. de Lange T. (2005) Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev 19:2100–2110.
40. Blasco M.A. (2005) Telomeres and human disease: cancer, ageing and beyond. Nat. Rev. Genet 6:611–622.[Medline]
41. Thomas P., Umegaki K., Fenech M. (2003) Nucleoplasmic bridges are a sensitive measure of chromosome rearrangement in the cytokinesis-block micronucleus assay. Mutagenesis 18:187–194.
42. Darroudi F., Fomina J., Meijers M., Natarajan A.T. (1998) Kinetics of the formation of chromosome aberrations in X-irradiated human lymphocytes, using PCC and FISH. Mutat. Res 404:55–65.[Web of Science][Medline]
43. Gotoh E., Kawata T., Durante M. (1999) Chromatid break rejoining and exchange aberration formation following gamma-ray exposure: analysis in G2 human fibroblasts by chemically induced premature chromosome condensation. Int. J. Radiat. Biol 75:1129–1135.[CrossRef][Web of Science][Medline]
44. Huyen Y., Zghelb O., DiTullio O. Jr, Gorgoulis V., Zacharatos P., Petty T.J., Sheston E.A., Mellert H.S., Stavridi E.S., Halazonetis T.D. (2004) Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks. Nature 432:406–411.[CrossRef][Medline]
Received on April 13, 2006; revised on June 22, 2006; accepted on June 23, 2006.
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