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Mutagenesis Advance Access originally published online on December 8, 2005
Mutagenesis 2006 21(1):41-47; doi:10.1093/mutage/gei069
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© The Author 2005. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

A comparison of folic acid deficiency-induced genomic instability in lymphocytes of breast cancer patients and normal non-cancer controls from a Chinese population in Yunnan

Xu Wang1,2, Xiayu Wu2, Ziqing Liang2, Yunchao Huang4, Michael Fenech3,* and Jinglun Xue1,*

1State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, People's Republic of China, 2School of Life Sciences, Yunnan Normal University, Kunming, Yunnan 650092, People's Republic of China, 3CSIRO Human Nutrition, PO BOX 10041, Adelaide BC, SA 5000, Australia and 4The Second Affiliated Hospital of Kunming Medical College, Kunming 650101, People's Republic of China


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
We hypothesized that the genomic response to folate deficiency might be different between breast cancer cases and healthy subjects. To test this hypothesis, we performed a comprehensive study on the genotoxic and cytotoxic effects of in vitro folic acid (FA) deficiency on primary human lymphocytes from 19 breast cancer patients and 20 age-matched healthy females from Yunnan, China using the cytokinesis-block micronucleus assay. Lymphocytes from the volunteers were cultured in RPMI1640 medium containing 30, 120 or 240 nM FA for 9 days. The results showed that 30 nM FA was associated with increased frequencies of micronucleated binucleated cell (MNed BNC), nucleoplasmic bridges (NPB), nuclear buds (BUD), apoptosis (APO) and necrosis (NEC) relative to 120 and 240 nM FA (P < 0.001) in lymphocytes of case and control groups in vitro, however there were no significant differences between the 120 and 240 nM FA within each sampling group. The case group showed significantly higher frequencies of MNed BNC than control at 120 and 240 nM FA (P < 0.05–0.001) but not at 30 nM FA (P = 0.052). NEC was significantly higher in breast cancer group than control at all concentrations of FA (P < 0.005). FA concentration explained 60, 39, 39, 52 and 71% of the variance of MNed BNC, NPB, BUD, APO and NEC, respectively compared with breast cancer status which only explained 6 and 7% of the variance of MNed BNC and NEC(Two way ANOVA, P < 0.0001). Difference of difference analysis showed that breast cancer cases were not abnormally sensitive to the genome-damaging effect of folate deficiency. We concluded that (i) increased concentrations of FA abolished the genome-damaging effect of FA deficiency in lymphocytes of both breast cancer patients and controls to a similar extent and (ii) FA concentration is much more important than breast cancer status in determining genomic instability and cell death.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Genome damage impacts on all stages of life. Above average chromosome damage rate, mal-segregation of chromosome leading to aneuploidy and DNA hypomethylation are important cancer-initiating events. Deficiency of certain micronutrients mimics radiation damage to DNA by causing single- and double-strand breaks and oxidized bases such as 8-oxoguanine (1Go,2Go). Genetic damage caused by micronutrient deficiencies themselves may lead to development defects and increased cancer risk (3Go). Folate is an important micronutrient in the prevention of chromosome aberration and hypomethylation of DNA. It is required for the synthesis of dTMP from dUMP and most methylation processes in cells and, therefore, plays a key role in DNA replication and genome stability (Figure 1A). Under conditions of folate deficiency dUMP accumulates and as a result uracil is incorporated into DNA instead of thymine (4Go,5Go). There is a good evidence suggesting that excessive incorporation of uracil in DNA not only leads to point mutation but also results in single- and double-stranded DNA breaks, chromosome breakage, gene amplification (6Go) and micronucleus formation (7Go8Go9Go). Folate deficiency is also associated with low S-adenosyl methionine (SAM) production which may lead to global DNA hypomethylation, a cause of altered gene expression (10Go) and chromosomal malsegregation (11Go). The evidence indicates that folate plays an important role in the maintenance of genome stability.


Figure 1
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  		Fig. 1.. (A) The main metabolic pathways by which folate, vitamin B12 and methionine impact on DNA methylation, synthesis and repair. DHF, dihydrofolate; 5-MeTHF, 5-methyltetrahydrofolate; 5,10-MeTHF, 5,10-methylenetetrahydrofolate; MS, methionine synthase; MTHFR, methylenetetrahydrofolate reductase; SAM, S-adenosyl methionine; THF, tetrahydrofolate; TS, thymidylate synthase; VIT B12, vitamin B12. (B) Photomicrographs of examples of cells scored in the CBMN assay. a, Normal binucleated cells; b, binucleated cell containing a micronucleus; c, binucleated cell with a nucleoplasmic bridge and a micronucleus; d, a binucleated cell containing two BUD on one nucleus and one BUD on the second nucleus; e, a necrotic cell; b, an apoptotic cell. Photomicrographs of mononucleated cells with or without nuclei are not shown, For more details of the scoring criteria refer to Fenech et al. (25Go).

 
Development and progression of breast cancer is characterized by genomic instability (12Go). A consensus is emerging that a crucial early event in carcinogenesis is the induction of genomic instability, which enables an initiated cell to evolve into cancer cell by achieving a greater proliferative capacity and genetic plasticity to overcome host immunological resistance, localized toxic environments and suboptimal micronutrient supply (1Go2Go3Go,13Go). Low intake of folate may increase risk for several cancers, including breast cancer (14Go,15Go). Epidemiological evidence has suggested a protective role of folate and related B vitamins against breast cancer (16Go).

Although a series of studies have confirmed that folate minimizes genomic instability in lymphocytes of non-cancer subjects, the situation in breast cancer is still uncertain (5Go,9Go,11Go,17Go18Go19Go). Study on differences of genomic instability between breast cancer cases and non-cancer control under folate deficiency may offer some explanation about their potential sensitivity to the stress of low folate status and susceptibility to develop breast cancer. Such evidence may reinforce the concept that genomic protection by adequate folate may have a role in breast cancer prevention. The plausibility that breast cancer cases may be more sensitive to folate deficiency-induced genome damage is supported by studies showing that carriers of BRCA1 and BRCA2 mutations and non-familial young breast cancer cases are more susceptible than normal to mutagen-induced chromosome damage (20Go21Go22Go23Go24Go).

In this study we aimed to determine whether there are any genomic response differences under folic acid (FA) deficiency in vitro between lymphocytes of breast cancer patients and healthy controls. The genomic instability phenotype was determined by means of the cytokinesis-block micronucleus assay (CBMN) which provides a comprehensive measure of chromosomal breakage, chromosomal rearrangement, chromosome loss, non-disjunction, gene amplification, necrosis (NEC) and apoptosis (APO) (25Go) and which we have shown previously to be sensitive to small changes in FA concentration between 10 and 120 nM and the effect of a polymorphism that reduces activity of the MTHFR gene and therefore bioavailability of folate for the synthesis of methionine and of dTMP (Figure 1) (26Go).


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Recruitment and sampling
The study was approved by National Natural Science Foundation of China (NSFC) and Yunnan Scientific and technological committee, China. All breast cancer cases were pathologically diagnosed females in the Department of Cardiothoracic Surgery of The Second Affiliated Hospital of Kunming Medical College, Yunnan, China during the study period. Eligibility criteria for the study were as follows: newly diagnosed female breast cancer cases before chemotherapy and no previous history of any cancer. A total of 19 breast cancer cases were identified aged 36–69 years (55.9 ± 8.3). The breast cancer cases were all sporadic and non-familial; their BRCA1 and BRCA2 mutation carrier status was not determined. Controls (N = 20) were non-cancer health females aged 38–61 years (47.8 ± 7.1) without any family history of cancer, randomly selected from Yunnan residents. The blood samples from breast cancer patients and controls were obtained after informed consent. All subjects had donated blood once during the period from April 2004 to February 2005. By venipuncture 5 ml of blood was collected in heparinized tubes from overnight fasted subjects.

CBMN assay
Lymphocytes were isolated using lymphocyte separation medium (Shanghai Huajing Biotech Company, China). The RPMI 1640 medium was custom made in the laboratory to achieve the required concentration of FA. All other constituents of the medium were standard as commercial RPMI 1640. Lymphocyte cultures were prepared at a concentration of 0.5 x 106 cells/ml in 1 ml custom made RPMI 1640 medium containing either 240 or 120 and 30 nM FA (Sigma), 5% dialysed FBS (HyClone, USA), 10 U/ml interleukin-2 (Shenzhen Xinjier Pharmaceutical, China), 2 mM L-Glutamine (Sigma), 100 U/ml penicillin G and 100 µg/ml streptomycin (Haerbing Pharmaceutical, China). The concentrations of FA chosen were based on previous extensive dose-response studies with lymphocytes and intended to investigate effects within the deficiency and sufficiency concentration range for which we had already shown a 78% decrease in micronucleated cell frequency in lymphocytes at 120 nM FA relative to 30 nM FA (9Go,27Go). Mitogenesis was stimulated by the addition of phytohaemagglutinin (45 µg/ml) (PHA; Murex Bioteck, Kent, UK) and cultures were incubated at 37°C and 5% CO2 in a humidified incubator. After 3 days, cell number and viability were determined using a haemocytometer and Trypan blue exclusion. The cultures were continued in 0.9 ml fresh medium and 0.1 ml ‘conditioned’ medium from the previous 3 day culture at 0.5 x 106 viable cells/ml. The components of medium were same as earlier but without PHA. This process of counting and re-culturing cells was repeated 6 days post-PHA treatment and a final viable cell count was measured on day 9.

After 8 days post-PHA treatment, Cytochalasin B (4.5 µg/ml; Sigma Chemical Co.) was added to each tube and ~28 h later cells were harvested. Cell suspensions were centrifuged at 1000 r.p.m. for 5 min and supernatant removed. FBS (20–30 µl) containing 5% DMSO was added into the cell aliquots for 5 min at room temperature. The cell suspensions were pipetted onto clean dry microscopic slides and smeared. Slides were then air dried, fixed with cold methanol for 10 min and stained with 5% Giemsa for 5 min. Coded slides were scored for the frequency of micronucleated binucleated cell (MNed BNC), MNed MONO, nuclear buds (BUD), nucleoplasmic bridges (NPB), APO and NEC (Figure 1B). The nuclear division index (NDI) was determined as previously described (5Go). 1000–1500 binucleated cell (BNC) were scored per culture to determine frequency of MNed BNC, NPB and BUD. To determine NDI and frequency of NEC and APO 500 cells were scored per culture. MNed MONO cell frequency was determined by scoring 500 MONO cells. The scoring criteria used were those described by Fenech (25Go).

Statistical analysis of data
One-way ANOVA and post hoc multiple comparison tests were performed for cancer case and control separately to determine the significance of differences in the parameters measured in relation to FA concentration within the sampling group. Two-way ANOVA was employed to determine the percentages of variation attributable to breast cancer status and FA concentration for all the biomarkers. One-way and two-way ANOVA analysis was performed using SPSS11.5 (SPSS Inc.) and PRISM 4 (GraphPad Software), respectively. Means of results for the control and breast cancer case groups under FA deficient (30 nM FA) or FA replete (pooled results for 120 and 240 nM FA) conditions were compared using the Newman–Keuls ANOVA post test (two-tailed). The sensitivity differences to FA-deficiency-induced genotoxicity and cytotoxicity between breast cancer and control group were determined using difference of difference analysis by subtracting the pooled observed frequencies of biomarkers at 120 and 240 nM FA from those observed at 30 nM FA for each individual and then comparing differences for these results between groups using a two-tailed Student's t-test (28Go). Parametric tests were used because distribution of data analysed was Gaussian. Significance was accepted at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Results are presented in Table I and Figure 2. A correlation matrix for the various biomarkers measured is presented in Table II and Table III. Variation attributable to FA concentration and breast cancer status is shown in Table IV.


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  		Table I.. Genomic instability at different folic acid concentrations measured by CBMN assay in lymphocytes of breast cancer patients and controls

 

Figure 2
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  		Fig. 2.. Comparison of MNed BNC and necrotic cell frequency measured in the CBMN assay among breast cancer cases and controls. Results are shown as scatterplots of results for each individual. Results for 120 and 240 nM FA were pooled as they were not significantly different.

 

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  		Table II.. Pearson correlation matrix for data of controls (n = 20)

 

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  		Table III.. Pearson correlation matrix for data of breast cancer cases (n = 19)

 

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  		Table IV.. Percentages of variation attributable to FA concentration and breast cancer status on biomarkers*

 
One-way ANOVA showed that increasing FA concentration from 30 to 120 nM or 240 nM significantly minimized MNed BNC, NPB, BUD formation and the frequencies of APO and NEC in both breast cancer and control groups. There were no significant differences of above effects between 120 and 240 nM FA within each group (Table I). Negative correlations between FA concentration and all markers of DNA damage and cytotoxicity were found in the two sampling groups. The r-value for correlation of FA concentration and MNed BNC, NPB, BUD, MNed MONO, APO and NEC were –0.578, –0.396, –0.545, –0.270, –0.506 and –0.766 (P < 0.05–0.0001) in the breast cancer group and –0.813, –0.608, –0.560, –0.248, –0.742 and –0.763 in the control group (P < 0.001 for MNed BNC, NPB, BUD, APO, NEC; P > 0.05 for MNed MONO), respectively. A significant positive correlation between MNed BNC, NPB and BUD (r = 0.50–0.78, P < 0.001) was found as well (Tables II and III).

Comparison of the breast cancer and control group for DNA damage markers and cytotoxic effects showed that the rate of MNed BNC was higher in the breast cancer group for both 30 nM FA cultures (P < 0.01) and for the pooled results of the 120 and 240 nM FA cultures (P < 0.05). The breast cancer group also showed significantly higher percentage of NEC than the control group in both the FA replete (pooled results for 120 and 240 nM cultures) and the FA deficient (30 nM) cultures (both P < 0.001) (Figure 2).

Two-way ANOVA analysis revealed that FA concentration explained 60, 39, 39, 52 and 71% of the variance of MNed BNC, NPB, BUD, APO and NEC, respectively (P < 0.0001). However, breast cancer status only explained 6 and 7% of the variance of MNed BNC and NEC (P < 0.0001), respectively. There was no interaction between FA concentration and breast cancer status for all the measured biomarkers (Table IV).

Because breast cancer cases and non-cancer controls had different baselines of genome damage and cytotoxicity, the genotoxicity and cytotoxicity sensitivity differences to FA deficiency between breast cancer and control group were determined using difference of difference analysis after subtracting the pooled scores for 120 and 240 nM FA from scores for 30 nM FA. The analyses showed that there were no significant differences in sensitivity to the genotoxic and cytotoxic effects of FA deficiency between cases and controls with respect to all the measured biomarkers (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Folate plays a critical role in maintaining genomic stability, it prevents chromosome breakage and hypomethylation of DNA (5Go). Because it is required for the synthesis of dTMP from dUMP and DNA methylation (Figure 1), the folate metabolic pathway is particularly relevant to the control of genome stability. Folate deficiency results in uracil incorporation in DNA which causes point mutation, single and double-strand DNA breaks, chromosome breakage and aneuploidy (7Go,8Go,11Go), all of which may induce the genome instability phenotype. Genome instability can adversely affect processes such as DNA repair and recombination, checkpoint control of the cell cycle and transcription, which are fundamental for the prevention of cancer. It is generally acknowledged that a crucial event in the initiation and evolution of cancer is the acquisition of the genomic instability phenotype that is manifested by the generation of breakage-fusion-bridge-cycles that are identifiable by the presence of NPB, BUD and micronuclei (17Go). NPB can be generated from dicentric chromosomes produced as a result of either mis-repair of chromosome breaks which also results in micronucleus formation or, alternatively, due to chromosome end fusions caused by telomere shortening; assymmetrical breakage of NPB and subsequent rejoining in the next cell cycle can lead to gene amplification; elimination of amplified genes is mediated by the nuclear budding process (18Go). Micronutrient deficiencies that cause genome damage may themselves cause developmental defects or increased risk of cancer.

It is important to know whether the lymphocytes from breast cancer patients and non-cancer controls exhibit different genetic response to FA deficiency because this may mean that folate requirements in cancer-prone individuals could be different from normal individuals. Investigation of the genetic response of non-cancer cells of cancer patients to FA deficiency may help our understanding of the sensitivity of cancer-prone individuals to genotoxic factors. Current evidence suggests that the genetic background of breast cancer patients may make them more susceptible to the genotoxic events because of inherited defects in DNA repair and/or cell cycle checkpoint proteins (20Go,22Go).

We employed the CBMN assay to investigate different genomic response of lymphocytes from breast cancer patients and controls under FA deficiency because this assay has already been used successfully to demonstrate defects in DNA repair and the genome damaging effect of folate deficiency (3Go,5Go,17Go,25Go).

The results of the present study showed that frequencies of MNed BNC, BUD and NPB were increased at 30 nM FA relative to 120 and 240 nM in both breast cancer and control groups, and that there was no further reduction in these DNA damage end-points beyond 120 nM FA suggesting that this concentration is optimal for genome health maintenance. The results of the study also show no genotoxic effect of FA concentration at 120 and 240 nM which is relevant in addressing concerns relating to the potential toxic effects of supra-physiological concentration of vitamins. The significant differences between breast cancer group and non-cancer control with respect to the frequencies of MNed BNC suggests an increased chromosomal instability that in the case of MNed BNC may be independent of NPB and BUD and could be related to increased chromosomal mal-segregation leading to chromosome loss and aneuploidy. Other studies in cells from breast cancer cases or cells defective in BRCA1 and BRCA2 genes reported higher base-line rates of micronucleus frequency, chromosome breakage, chromosome loss and aneuploidy relative to controls possibly due to defects in DNA repair, centrosome replication or cytokinesis (29Go30Go31Go32Go33Go34Go). The elevated NEC rate in breast cancer cases could be indicative of a propensity to death by this pathway rather than APO which could be due to altered redox status or insufficient ATP to complete APO. Whether NEC rate and MNed BNC frequency are directly related remains to be tested. The difference of difference analysis clearly shows that those individuals who develop breast cancer do not benefit more from increased FA than those who do not develop breast cancer. Results from a recent study of breast cancer cases who were verified carriers of BRCA1 and BRCA2 pathological mutations suggest that genes that predispose to breast cancer such as BRCA1 or BRCA2 may not be involved in the repair of folate deficiency induced genome damage (35Go). However, two-way ANOVA still revealed that both FA concentration and breast cancer status contributed significantly (albeit to different extents) to the observed variations of measured biomarkers. MNed BNC, NPB, BUD, APO and NEC were markedly modified by FA concentration, but MNed BNC and NEC were only marginally affected by breast cancer status. This result suggests that FA is more important than breast cancer status in determining genomic instability and cell death and highlights the possibility that nutritional factors may be more important to genome maintenance than genetic factors that might predispose to breast cancer although this was not tested directly in our study.

The results of this study showed that NPB and BUD were negatively correlated with FA concentration but strongly correlated with MNed BNC (r = 0.546–0.739, P<0.001) which is in agreement with previously published results using the same system (9Go,27Go). With regards to the mechanisms of NPB and BUD formation (9Go,17Go,18Go), it is probable that FA deficiency causes genomic instability and gene amplification by the initiation of breakage-fusion-bridge cycle both in breast cancer cases and controls (18Go). The strong correlation between MNed BNC, NPB and BUD induced by FA deficiency in both breast cancer and control groups is consistent with this explanation.

It cannot be excluded that the response of mammary epithelial cells to FA deficiency may be different to the response of lymphocytes as there are no published studies on folate metabolism in mammary cells. In addition, it is possible that folate metabolism in mammary cells may be different to lymphocytes. Clearly it would have been ideal to use primary mammary cell cultures from donors with BRCA1 and BRCA2 mutations but these cells are not readily available other than from mastectomies. This could be a possible investigation for the future. Therefore until comparative studies are done between lymphocytes and mammary epithelial cells the relevance of our results in lymphocytes to mammary cells in vivo or ex vivo remains questionable.

In conclusion, the study shows that moderate FA deficiency induced genomic instability in lymphocytes of breast cancer patients and non-cancer controls. The results do not support the hypothesis that lymphocytes of breast cancer cases are abnormally sensitive to the genome damaging effects of moderate folate deficiency when compared to non-cancer controls. However, lymphocytes of breast cancer case appear to have higher base-line of genome damage and NEC rates which may be associated with enhanced risk of cancer development. It is apparent from the results of this study that moderate FA deficiency within the physiological range has a much stronger effect on genome stability than breast cancer status. Further research is needed to investigate other dietary factors that may also contribute to the genetic instability in cultured lymphocytes and mammary epithelial cells from breast cancer cases and to establish whether mutations in BRCA1 or BRCA2 genes increases sensitivity to genome damage caused by folate deficiency.


   Acknowledgments
 
This research was supported by National Natural Science Foundation of China (Project #30560061) and the International Scientific Cooperation Project of Yunnan Province. We are very thankful to the volunteers who participated in the study.


   Notes
 
* To whom correspondence should be addressed. Michael Fenech, Tel: +618 8303 8880; Fax: +618 8303 8899; Email: michael.fenech{at}csiro.au Correspondence may also be addressed to Jinglun Xue, Tel/Fax: +8621 6564 9899; Email: jlxue{at}fudan.ac.cn


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received on August 5, 2005; revised on November 9, 2005; accepted on November 17, 2005.


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