Mutagenesis vol. 19 no. 6 © UK Environmental Mutagen Society 2004; all rights reserved.
Folate levels determine effect of antioxidant supplementation on micronuclei in subjects with cardiovascular risk
inská1,3lová1
oková1
imírová11Research Base of Slovak Medical University, Institute of Preventive and Clinical Medicine, Limbová 12, Bratislava 83303, Slovakia and 2Institute for Nutrition Research, University of Oslo, PO Box 1046, Blindern, 0316 Oslo, Norway
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Abstract |
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We have investigated the effect of modest supplementation with
-tocopherol (100 mg/day), ß-carotene (6 mg/day), vitamin C (100 mg/day) and selenium (50 µg/day) on oxidative stress and chromosomal damage, and the influence of methylenetetrahydrofolate reductase (MTHFR) genotype on these end-points. Subjects were two groups of middle-aged men differing in cardiovascular risk; 46 survivors of myocardial infarction before age 50 and 60 healthy controls. They were randomly divided into equal groups to receive antioxidants or placebo for 12 weeks. Twenty-eight patients and 58 controls completed the intervention. Micronucleus levels in peripheral lymphocytes and changes seen after intervention were studied in relation to the MTHFR C677T genotype, basal homocysteine and plasma folate levels. Ferric reducing ability of plasma and concentration of malondialdehyde were measured to assess the antioxidant effect of supplementation. There was no association of micronuclei with folate, homocysteine or malondialdehyde levels before supplementation. Micronucleus frequencies and plasma folate levels did not vary significantly with MTHFR genotype. Homocysteine levels in subjects with the TT variant genotype were significantly higher compared with CT or CC (P = 0.001), especially in subjects with low folate (P = 0.012). In the placebo control group an increase in micronuclei (P = 0.04) was detected at the end of the intervention period. This effect was not seen in the supplemented group. In antioxidant-supplemented myocardial infarction survivors we found an increase in the ferric reducing ability of plasma (P < 0.001) and a decrease in malondialdehyde (P = 0.001). Micronucleus frequency showed a decrease, strongest in subjects with normal folate levels (P = 0.015). In subjects with low folate levels, a high correlation was found between micronuclei after supplementation and homocysteine, both before (r = 0.979, P = 0.002) and after supplementation (r = 0.922, P = 0.009). Thus, folate deficiency may amplify the effect of other risk factors such as elevated homocysteine levels or variant MTHFR genotype, as well as influencing the ability of antioxidant supplementation to protect against genetic damage. |
Introduction |
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A meta-analysis of major epidemiological studies reported a linear, independent relation between homocysteine concentrations and cardiovascular risk (Boushey et al., 1995
; Eikelboom et al., 1999). Precise mechanisms mediating a pro-oxidant activity of homocysteine or a damaging role on vascular cells or tissues have not yet been fully identified. It is hypothesized that homocysteine impairs endothelial function (Tawakol et al., 1997; Chambers et al., 1998, 1999), enhances smooth muscle cell proliferation (Tsai et al., 1994), increases reactive oxygen radical production and, consequently, causes an increase in low density lipoprotein cholesterol oxidation and thrombogenicity (Mayer et al., 1996).
Recently, homocysteine has also been observed to induce DNA damage (Kruman et al., 2000; Oikawa et al., 2003) which might also contribute to the pathogenesis of atherosclerosis (Andreassi et al., 2000; Botto et al., 2001; Andreassi, 2003; Andreassi and Botto, 2003). It is still unclear whether homocysteine is genuinely genotoxic or simply a biomarker of folate deficiency, a known cause of genetic damage (Crott and Fenech, 2001).
A common mutation of MTHFR, the gene for 5,10-methylenetetrahydrofolate reductase gene (EC 1.5.1.20) causes increased thermolability and reduced activity of the enzyme catalysing the reduction of 5,10-methylenetetrahydrofolate (5,10-MTHF) to 5-methyltetrahydrofolate (5-MTHF). 5-MTHF acts as a methyl-group donor in the remethylation of homocysteine to methionine and is the main form of folate in circulation. 5,10-MTHF donates methyl groups for the thymidine synthase-mediated conversion of dUMP to dTMP. The relationship between the C677T polymorphism and cardiovascular risk is not clearly established. Its influence on plasma homocysteine levels has been intensively studied (Kluijtmans et al., 1997). TT homozygosity may be a coronary risk factor (Kluijtmans et al., 1996; Morita et al., 1997) despite many studies with contrary findings (Schmitz et al., 1996; van Bockxmeer et al., 1997; Schwartz et al., 1997; Brattstrom et al., 1998). Meta-analysis of studies on the risk of coronary heart disease related to the C677T polymorphism (Klerk et al., 2002) found significantly higher risk with low folate status in TT homozygotes.
The relationship between this mutation and DNA damage was studied (Andreassi et al., 2003; Botto et al., 2003). A positive association was found between aberrant methylation and the 677T allele (Paz et al., 2002). Subjects with the TT genotype had a lower level of DNA methylation compared with those with the CC wild-type genotype. Deficiency of folate may result in the disruption of genomic integrity and alteration of DNA methylation (Fenech, 2001), thus linking nutrition with modulation of gene expression. Friso et al. (2002) reported that genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine levels, however, when the data were analysed according to folate status, the TT subjects with low levels of folate alone accounted for the diminished DNA methylation.
Diet is an important variable in determining DNA damage biomarkers, specifically micronuclei (MN) (Fenech, 2002). Folic acid supplementation decreases homocysteine levels (Mattson et al., 2002; Moat et al., 2004) and reduces chromosomal damage (Blount and Ames, 1995; Fenech et al., 1998). A positive effect of antioxidant supplementation on the spontaneous MN frequency in peripheral human lymphocytes has been documented (Gaziev et al., 1996; Schneider et al., 2001), although in other studies antioxidants (vitamins E and C) had little effect on MN (Fenech et al., 1997a; Record and Jannes, 2000).
This study (part of a larger study of effects of seasonal variations in dietary antioxidant intake in Slovakia) investigates the hypothesis that modest antioxidant supplementation can decrease oxidative stress during the winter period, when intake of antioxidants is low, and thus decrease chromosomal damage in human peripheral lymphocytes. We expected different effects of antioxidant supplementation to be manifested in subjects differing in cardiovascular risk factors and lifestyle and, therefore, have studied survivors of myocardial infarction (MI) together with a rural control (RC) group.
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Materials and methods |
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Design of study
Altogether 106 male subjects were chosen for the trial. Forty-six subjects were survivors of MI by the age of 50. The diagnosis of MI was based on typical clinical symptoms, ECG changes and enzyme elevations. The MI patients were selected from cardiovascular clinics in Bratislava. The control group (60 subjects) was randomly chosen among the inhabitants of a rural area from the databases of patients of three primary care physicians in and around the small town of Pezinok. They did not suffer from overt cardiovascular disease, however, the level of lipids was not considered as a factor in selection. These subjects represent a normal Slovak population. General exclusion critieria (applying to both groups) were: MI or a surgical operation within the previous 2 months; serious illness, including diabetes mellitus, secondary hyperlipidemia or malignancy. Informed consent was obtained from all subjects and the Ethics Committee of the Institute of Preventive and Clinical Medicine approved the study.
To examine the effects of supplementation, groups were divided randomly in two; half received a placebo capsule (0.5 g of glucose), the other a supplement (100 mg -tocopherol, 6 mg ß-carotene, 100 mg vitamin C and 50 µg selenium) daily for 12 weeks. At the end of the supplementation period, 28 (61%) MI patients (16 supplemented and 12 placebo) and 58 (97%) RC subjects (31 supplemented and 27 placebo) remained in the study. Clinical assessment of subjects was performed twice, first in February/March of 1999 and second after 12 weeks of supplementation (May/June). During examination subjects provided information on personal and family history and completed a dietary questionnaire. A physical examination and measurements of body weight and height, ECG and blood pressure were carried out under the supervision of a physician.
Methods
Blood samples were obtained in EDTA after an overnight fast. Blood count, sedimentation and urine analysis were carried out on all subjects in a clinical laboratory by standard methods.
Whole blood for MN analysis was incubated with cytochalasin B (Fenech and Morley, 1985). Briefly, cytochalasin B (Sigma) (final concentration 6 µg/ml) was added 44 h after the start of culture and at 72 h cells were centrifuged, resuspended in 0.075 M KCl and immediately centrifuged again and fixed twice with fixative (1:3 acetic acid:methanol). The fixed cells were dropped onto slides, air dried and stained with 5% aqueous Giemsa solution for 5 min. Cytochalasin B inhibits cytoplasmic cleavage without preventing mitosis. Thus, cells that have divided are readily identified by the presence of two nuclei. MN analysis was performed on 2000 binucleated lymphocytes with preserved cytoplasm for each subject. MN were accepted only when they were morphologically identical to, but smaller than, normal nuclei, had a diameter between 1/16 and 1/3 of the main nuclei, were non-refractile and were not linked to the main nuclei via a nucleoplasmatic bridge, although they might sometimes overlap the boundaries of the main nuclei (Fenech, 1993).
Plasma folate levels were measured by radioimmunoassay for the quantitative determination of vitamin B12 and folate in serum (DRG Instruments GmbH, Germany).
Ferric reducing ability of plasma (FRAP) was detected by a spectrophotometric assay (Benzie and Strain, 1996).
Homocysteine concentrations in the plasma were measured by HPLC using fluorescence detection (Vester and Rasmussen, 1991). The internal standard was 2.5 mM acetylcysteine (Sigma), with a homocyst(e)ine standard (Sigma). The interassay coefficient of variation of the method was 5%.
Malondialdehyde (MDA) levels in plasma were measured by a HPLC method (Richard et al., 1992).
Selenium was measured in serum (after dilution with 0.2% Triton X-100, 0.1% Pd and 1% ascorbic acid) by electrothermal atomic absorption spectrometry (Ursínyová and Hladíková, 1998).
Plasma total cholesterol and triglycerides were determined enzymatically. High density lipoprotein cholesterol (HDL) was measured by dextran sulfate precipitation, followed by enzymatic determination of cholesterol (Warnick et al., 1982). Low-density lipoprotein cholesterol (LDL) was calculated using the Friedewald formula (Friedewald et al., 1972).
Plasma ascorbic acid levels were measured by an ion-pairing HPLC method (Ross, 1994), and ß-carotene and -tocopherol by reverse phase HPLC (Hess et al., 1991).
Leukocyte DNA for analysis of the MTHFR C677T genotype was extracted by phenol extraction (Brown, 1995). Forward primer 5'-TGAAGGAGAAGGTGTCTGCGGGA and reverse primer 5'-AGGACGGTGCGGTGAGGAGGTG were used for PCR amplification and HinfI for RFLP analysis (Goyette et al., 1994). Electrophoretically separated fragments on visigel (Stratagene, La Jolla, CA) were visualized under UV light after ethidium bromide staining.
Statistical analysis
SPSS 11.5 software was used for statistical analysis of data. Normality of distribution was tested by the Kolmogorov–Smirnoff test. For variables with normal distributions Pearson's and for non-normally distributed variables Spearman's coefficients of correlation were calculated. P < 0.05 was defined as significant. Differences in frequencies were tested by 
2 test. Paired samples t-test or the Wilcoxon signed ranks test (for non-normally distributed data) were used for paired values testing. For comparisons of non-paired data we used the independent samples t-test in the case of normally distributed data and the Mann–Whitney U-test for data not normally distributed. All t-tests were two-tailed. Differences between more than two groups were tested by one way analysis of variance (ANOVA) and by Bonferroni's test if equal variance was assumed or Tamhane's test if equal variances were not assumed (as a post hoc procedure when needed). Multifactorial analysis of variance and linear regression analysis were applied to identify the combination of factors or variables with the best predictive value for each parameter studied.
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Results |
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Biochemical characteristics and antioxidants
Table I summarizes clinical profiles and differences between MI and RC subjects in biochemical characteristics, antioxidants and other measured parameters before supplementation. MI patients consumed 80% of supplement capsules issued while RC subjects consumed 84.0%. MI subjects were older and had higher diastolic and systolic blood pressure and MDA levels. They had lower HDL, glycemia, FRAP and folate levels. They consumed less alcohol and only 20% smoked cigarettes, compared with 58.6% of RC subjects. A lower than normal plasma folate level was defined as
3.4 ng/ml, equivalent to the lowest quartile of the range of folate levels in the normal (RC) subjects. Normal levels are defined as levels >3.4 ng/ml. A total of 40.7% of the MI group but only 10.4% of the RC group were below this level; a significant difference (P = 0.002).
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After controlling for age (because of the significant difference between the MI and RC subjects), we found correlations before supplementation between folate levels and FRAP (r = 0.342, P = 0.003, n = 70) and between homocysteine and body mass index (BMI) (r = 0.297, P = 0.007, n = 80). Correlations of homocysteine and MDA with age for either MI or RC group were not significant.
Significant differences between placebo and supplemented subjects in the MI group were found for BMI (26.9 ± 2 for placebo and 29.2 ± 3 for supplemented subjects, P = 0.036) and HDL (1.02 ± 0.3 for placebo and 1.21 ± 0.2 for supplemented subjects, P = 0.035).
The expected changes in the levels of antioxidants were seen in antioxidant-supplemented subjects after intervention (Table II). Increases in ascorbic acid, ß-carotene, selenium and FRAP and a decrease in homocysteine level were detected in the RC supplemented group. Within supplemented MI subjects significant increases occurred in ascorbic acid, -tocopherol, -tocopherol/cholesterol, selenium, FRAP and MDA. Except for supplemented RC subjects, where HDL cholesterol decreased (P = 0.036), no changes were found in lipid parameters after intervention. No changes in individual dietary antioxidants were seen in placebo subjects (Table III), but in RC there was an increase in FRAP.
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Micronuclei
Before supplementation there was no difference between MI and RC groups in mean levels of MN (Table I). Frequencies of cells with MN did not correlate with other measured parameters or with age. Among the RC subjects the placebo group and antioxidant-supplemented group differed significantly in MN (5.06 ± 3.6 for placebo and 7.43 ± 3.5 for antioxidant group, P = 0.014).
MN increased in the RC subjects receiving placebo but did not change in the RC supplemented group. In antioxidant-supplemented MI subjects there was a decrease in MN of borderline significance (P = 0.051).
The group of antioxidant-supplemented subjects (MI and RC combined) was divided according to whether MN showed a decrease or increase after supplementation. Subjects with a decrease in MN (75% of MI and 52% of RC group) also showed a decrease in MDA (1.77 ± 0.98 µmol/l before and 1.18 ± 0.52 after supplementation, P = 0.003) and in homocysteine (13.05 ± 4.12 µmol/l before and 11.60 ± 3.5 after supplementation, P = 0.044). Subjects with an increase in MN did not show significant changes in MDA or homocysteine.
To avoid baseline differences in MN we performed multifactorial analysis for differences between MN values measured before versus after supplementation. The general linear model (P < 0.001) includes interactions of factors (supplementation with antioxidants and folate category, P = 0.01; supplementation and group, P = 0.043; MTHFR genotype and group, P = 0.009) and homocysteine as covariate (P = 0.023) affecting MN changes. We have tested the effect of possible confounding parameters from Table I on MN change after supplementation for each group (MI and RC) according to folate level (low, normal). For MI normal folate (model P = 0.043, n = 16) we found an effect of supplementation (P = 0.009) and glycemia (P = 0.032). For MI low folate (model NS, n = 11) and RC normal folate (model NS, n = 43), we did not find any confounding factors. Because of the small number of subjects in the RC low folate group (n = 5) it was not possible to perform this analysis. All models were adjusted for age, BMI, systolic and diastolic blood pressure, number of cigarettes smoked, uric acid, lipid parameters and supplementation.
MTHFR genotypes
Table IV shows genotype frequency and values of measured parameters for different MTHFR genotypes in the combined groups (MI and RC). MTHFR genotype frequencies analysed by 2 test did not differ significantly between groups. Folate and MDA levels were similar for all genotypes. Homocysteine levels in TT homozygotes were significantly higher compared with CT or CC. Among individuals with normal folate levels there was no significant difference between MTHFR genotypes in homocysteine levels; conversely among subjects with low folate levels there were significantly higher homocysteine levels in TT homozygotes (Figure 1). MN did not vary significantly (either before or after supplementation) with genotype for all subjects combined or for those with low or normal folate levels.
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We did not find any relationship between MTHFR genotype and changes in homocysteine after supplementation (the extent of homocysteine decrease was similar in all genotypes). Homocysteine levels in TT subjects with low folate levels in spite of supplementation remained above the reference value (27.11 ± 3.0 µmol/l before and 24.78 ± 11.0 after, n = 3). Similarly, we did not find a relationship between changes in MN and MDA with genotype.
Folic acid and homocysteine
The decrease in homocysteine after supplementation (in the combined groups) differs according to folate level. Subjects with low folate level are characterized by higher homocysteine (17.49 ± 7.96 µmol/l) with only a slight decrease after supplementation (16.67 ± 8.65 µmol/l). On the other hand, subjects with normal folate level showed a significant decrease in homocysteine (13.07 ± 4.00 µmol/l before and 11.40 ± 3.26 after supplementation).
A decrease in MN on supplementation was found only in MI subjects with normal levels of folate (Figure 2). Although in the RC group overall there was no significant change in MN on supplementation, three subjects whose folate levels were <3.4 ng/ml showed an increase in MN, similar to the pattern seen in the placebo group.
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A significant correlation was seen for supplemented MI subjects between homocysteine levels and MN both after supplementation (r = 0.699, P = 0.003, n = 16) and homocysteine levels before and MN after supplementation (r = 0.791, P < 0.001, n = 16). These correlations were extremely high for MI subjects with low folate levels: MN after supplementation and homocysteine both before (r = 0.965, P = 0.002, n = 6) and after supplementation (r = 0.922, P = 0.009, n = 6). For MI subjects with normal folate there was no correlation between homocysteine before and MN after supplementation and only a marginally significant correlation (r = 0.631, P = 0.050, n = 10) between homocysteine and MN both after supplementation.
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Discussion |
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The pathogenesis of atherosclerosis is affected by interactions between genetic and environmental factors (Tall et al., 1997
). Variations in susceptibility of individuals and populations to the development of disease can be explained by the presence of potent genetically determined systems that prevent lipid oxidation, inactivate biologically important oxidized lipids and/or modulate the inflammatory response to oxidized lipids (Berliner et al., 1995).
Our RC group was recruited as apparently healthy subjects from a rural area. In this group we can expect higher white wine consumption, as a result of following a traditional lifestyle and living in a wine producing area (Smolková et al., 2004). Surprisingly, we found a higher proportion of the variant MTHFR genotype in this group compared with our earlier findings for a healthy population (Ralová et al., 2000, 2001). The frequencies in the RC group are similar to those found by Andreassi et al. (2003) for coronary artery disease patients. Multifactorial analysis did not show a significant effect of smoking habit on the parameters studied (MN frequency, homocysteine or folate levels). Therefore, we did not exclude smokers from analysis, although the ratio of smokers in the RC group was higher. A lower frequency of MN among smokers before supplementation (data not shown) is in accordance with many reports (see for example Bonassi et al., 2003). They found a higher frequency of MN only in individuals smoking 
30 cigarettes/day. In our study we analysed the relationship between smoking >20 cigaretts/day and MN because we had only two subjects who smoked 30 cigarettes/day.
We found an increase in FRAP in both supplemented and placebo RC subjects. The intervention period lasted from February/March to May/June (late spring/early summer) and thus a possible explanation could be an increased intake of antioxidants from food according to the season (Duinská et al., 2002). The cause of the increase in mean MN level in the RC placebo group during the intervention period is not known. It is interesting that the increase did not occur in the RC subjects receiving antioxidants, perhaps indicating a modest protective effect. In the antioxidant-supplemented MI group a marginally significant decrease in MN was found.
The MI group represents subjects with clinically proven atherosclerosis who received treatment following MI. We suppose that positive changes in smoking habit and lifestyle occurred after the clinical event. Genotype frequencies were similar to those found for our control population (Ralová et al., 2000, 2001). The increase in FRAP and decrease in MDA after antioxidant supplementation are consistent with a decrease in oxidative stress during the intervention period. Our earlier analysis of MDA and plasma folate levels in summer and winter (Smolková et al., 2004) showed seasonal variability for both; MDA decreased in the summer and increased in the winter period and folate showed a reciprocal pattern in the MI group.
A decrease in MN was found in MI subjects with normal folate levels but not in those with low folate. The positive correlation between homocysteine concentration and MN after supplementation was much stronger for subjects with low folate levels, implying that homocysteine might indeed be directly genotoxic. Fenech et al. (1997b) found a correlation between MN and homocysteine only for subjects with normal folate.
The differences found between the MI and RC groups in clinical characteristics and levels of biochemical parameters measured before supplementation could be partly explained by their different cardiovascular risk. Despite almost double MDA and lower folate levels in the MI group, we did not find any differences between groups in homocysteine levels or MN frequency. Fifty-two per cent of subjects in the MI and RC groups combined have higher values of homocysteine than are considered desirable in terms of cardiovascular disease risk (>12 µmol/l) (Welch and Loscalzo, 1998). Elevated homocysteine levels were found for carriers of the TT mutation, especially in the group with low folate levels. These findings are in agreement with many authors (Jacques et al., 1996; Ma et al., 1996; de Bree et al., 2003). In the same group, interestingly, we did not find elevated MDA levels, indicating that there was no effect on lipid peroxidation in TT homozygotes.
We did not find any relation between MN frequency and homocysteine or folate levels before supplementation and MTHFR genotype. These results are not in accordance with the findings of Fenech et al. (1997a, 1998). They found a correlation between plasma homocysteine and MN index and no correlation between MN and folate status in the general population. Andreassi et al. (2003) reported a relationship between MTHFR polymorphism and MN index in a group of subjects with angiographically proven coronary artery disease. The MN index was significantly higher in TT homozygotes compared with the CC and CT genotypes. In experiments with cultured human lymphocytes, Kimura et al. (2004) found 21% higher MN levels in TT cells than in CC cells and 42% lower levels in high folic acid compared with low folic acid medium. They suggest that the MTHFR C677T polymorphism and riboflavin affect genome instability, however, the effect is relatively small compared with that of folic acid. On the other hand Crott et al. (2001) did not find any effect of MTHFR polymorphisms on chromosome damage using the MN assay in conditions of high methionine and riboflavin in culture medium. When riboflavin and methionine were reduced, there was an effect of MTHFR on genome damage markers in the cytokinesis block MN assay. They found a positive correlation between uracil content in DNA and MN, as did Blount et al. (1997). The optimal concentration of micronutrients for prevention of genome damage is required (Fenech, 2004). Folate deficiency caused a dose-dependent increase in uracil incorporation into lymphocyte DNA in vitro. The role of nutrients, especially folate, in modulating gene expression via DNA methylation has been intensively studied (Friso et al., 2002; Costello and Plass, 2001).
The results of various human supplementation trials on spontaneous MN formation or genomic stability with folate and/or other micronutrients are summarized in Table V.
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Beneficial effects of folates, independent of homocysteine lowering, have been reported in recent years (Verhaar et al., 1998
), especially for cardiovascular morbidity and mortality (Robinson et al., 1998; Loria et al., 2000; Voutilainen et al., 2000). A few studies have mentioned contrary findings (Chasan et al., 1996). The possible antioxidant role of folates, though with a relatively low scavenging potency compared with vitamin C, was emphasized by Verhaar et al. (2002).
As well as nutritional factors, genetic variation may influence folate requirements. According to our data, folate status is important in determining the effectiveness of supplementation at maintaining genomic stability. Supplementation with antioxidants decreases DNA damage in the case of normal folate levels. If folate is low, supplementation is not effective in decreasing MN or homocysteine levels, in spite of an apparent decrease in oxidative stress. The relatively small effect found on MN frequency may be due to the low doses of antioxidant supplementation used. On the other hand, higher, non-physiological concentrations of antioxidants in some studies did not decrease MN frequency (Fenech et al., 1997a) and have been found in human trials to have an adverse effect on disease or clinical event (Omenn, 1998).
Thus folate deficiency may play a critical role by amplifying the effect of other risk factors, such as elevated homocysteine levels or variant MTHFR genotype, especially in subjects with high cardiovascular risk. This should be taken into account when designing nutritional strategies for at-risk populations, especially when attempting to base these strategies on increasing the content of folate in the diet.
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Acknowledgments |
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We thank all participants for their participation. Martina Drli
ková, Bibiana Vallová, Alexandra 
tupáková, Helena Petrovská, Juraj Gaparovi, Alena 
ákoviová, Monika Ursínyová, Vlasta Hladíková, Pavol Blaíek, Kristína Gavalová, Zuzana Rotáová, Anna Morávková and Anna Gaiová carried out the various biomarker assays in Bratislava. Sharon Wood (Rowett Research Institute, Aberdeen, UK) carried out measurements of plasma antioxidants. Natália Arvaiová, Adriana Uheríková and Branislav Vohnout carried out medical investigations of the subjects. Drs Danica Patáková, Mojmír Kvaala and Peter Záruba were responsible for recruiting and examining the rural subjects. We thank Dr Ladislava Wsólová for her excellent advice on statistical analyses. This study was supported by EU contract IC15-CT96-1012. |
Notes |
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3 To whom correspondence should be addressed. Tel: +421 2 59369270; Fax: +421 2 59369270; Email: maria.dusinska{at}szu.sk
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Received on August 20, 2004; revised on October 2, 2004; accepted on October 5, 2004.
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