Mutagenesis vol. 18 no. 4 pp. 371-376,
July 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Nutritional supplementation with antioxidants decreases chromosomal damage in humans
inská2
imírová
oková
olováInstitute of Preventive and Clinical Medicine, Limbova 14, 83301 Bratislava, Slovak Republic and 1Institute for Nutrition Research, University of Oslo, POB1046 Blindern, N-0316 Oslo, Norway
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Abstract |
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In order to investigate the effects of antioxidant supplementation on chromosome damage, a 3 month antioxidant supplementation trial was conducted on groups of 28 myocardial infarction survivors and 57 rural controls, all male. The supplement consisted of vitamin C (100 mg/day), vitamin E (100 mg/day), ß-carotene (6 mg/day) and selenium (50 µmg/day). Dietary antioxidants in plasma were measured, as well as the ferric reducing ability of plasma (a measure of total plasma antioxidant status) and the concentration of malondialdehyde as an indicator of oxidative stress. Lymphocytes collected at the beginning and end of the supplementation period were stimulated to proliferate and metaphases accumulated for scoring of chromosome aberrations: per cent aberrant cells and chromatid and chromosome breaks. Supplementation with antioxidants was associated with a decrease in the percentage of cells with chromosome aberrations in the group of rural controls (0.63% before compared with 0.27% after supplementation; P = 0.03). The largest effect of supplementation was seen in smokers in this group (0.12% aberrant cells in supplemented compared with 0.81% in placebo group; P > 0.001). The results support the hypothesis that antioxidants decrease genetic damage.
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Introduction |
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Supplementation of the human diet with antioxidants, or an increased intake of fruits or vegetables rich in antioxidants, has been shown in numerous studies to decrease the level of oxidative DNA damage measured in peripheral lymphocytes ( Duthie et al., 1996
; Pool-Zobel et al., 1997; Collins et al., 2001). Since DNA damage is the initiating event in carcinogenesis, these results lend support to the idea that fruits and vegetables protect against cancer by preventing free radical attack on DNA. Oxidative stress is also a major risk factor for atherosclerosis. Low density lipoprotein oxidation leads to atherogenic changes in the arterial wall and may contribute to the coronary event. Cancer and atherosclerosis may thus entail common biological mechanisms (Andreassi et al., 2000) and be subject to a common mode of protection via dietary antioxidants.
DNA damage, as measured in lymphocytes, is generally considered to be a marker of exposure to potential carcinogens, rather than of their effect. Most of the damage is rapidly repaired, and only the damage that is not repaired when the cell replicates its DNA will have a lasting effect. Little is known about individual variation in repair rates or about intrinsic or extrinsic factors that modulate repair activity. It is important to examine other biomarkers, such as chromosome aberrations and micronuclei, that relate to the damage still present after cellular processing. These are more likely to correlate with cancer risk and, indeed, prospective studies indicate a positive correlation between frequency of chromosome aberrations and subsequent cancer incidence (Hagmar et al., 1994; Bonassi et al., 1998).
Gaziev et al. (1996) found that 4 months supplementation with a mixture of vitamins A, C and E, ß-carotene and folic acid caused a significant decrease in the frequency of spontaneous micronuclei in stimulated lymphocytes from a group of aged subjects (56–83 years old) but not in cells from younger subjects. In this study, we have measured chromosome aberrations in groups of healthy male volunteers and survivors of myocardial infarction, who received daily for 12 weeks either a placebo or a supplement of ß-carotene, vitamin C, vitamin E and selenium. As a marker of oxidative stress, we assayed plasma levels of malondialdehyde (MDA).
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Materials and methods |
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Design of supplementation study
Two groups of male volunteers participated in this study: 40 patients living in or near Bratislava who were receiving treatment following myocardial infarction, average age 53 (range 35–66) (MI group); 60 healthy controls living in or around the rural town of Pezinok, average age 44 (range 34–56) (rural controls, RC group). Smoking status was ascertained from a questionnaire.
At the end of February 1999, the volunteers were randomly assigned to receive placebo capsules containing glucose or supplement capsules containing 100 mg of vitamin C, 100 mg of vitamin E, 6 mg of ß-carotene and 50 µg of Se. Supplementation continued for 12 weeks, at the end of which further blood samples were taken. Twenty-eight of the MI group completed the trial (16 supplemented, 12 placebo) and 58 of the RC group (31 supplemented, 27 placebo).
Lymphocyte culture
Samples of whole blood were taken from all subjects at 0 and 12 weeks. Short-term lymphocyte cultures were set up by adding 0.5 ml of whole blood to 4.5 ml of RPMI medium with L-glutamine (Gibco) supplemented with 20% foetal calf serum (Gibco) and antibiotics (penicillin and streptomycin). Lymphocytes were stimulated with 0.18 mg/ml phytohaemagglutinin (Murex) and incubated at 37°C with 5% CO2. Two cultures were set up from each sample.
Chromosome aberrations
Lymphocytes were harvested at 48 h following stimulation; 0.75 µg/ml colchicine (Sigma) was added 2 h before harvest. They were centrifuged and subjected to a hypotonic shock in 75 mM KCl for 20 min at 37°C. The lymphocytes were fixed in acetic acid/methanol (1:3) and air dried preparations were made. Slides were stained with 5% aqueous Giemsa solution for 10 min. One-hundred well-spread metaphases per person were examined. Chromosomal abnormalities (chromatid and chromosome gaps, breaks and exchanges) were recorded. Because of the controversy over their classification, gaps were not included in the analysis. Chromosomal damage was expressed as per cent aberrant cells and as number of breaks per cell.
Measurement of individual plasma antioxidants and ferric reducing ability of plasma (FRAP)
Plasma vitamin C was measured by an ion-pairing HPLC method (Ross, 1994) and carotenoids and tocopherols (vitamin E) by reverse phase HPLC (Hess et al., 1991). The total antioxidant capacity of plasma was estimated by measuring the ferric reducing activity in a spectrophotometric assay (Benzie and Strain, 1996).
Measurement of malondialdehyde
Following hydrolysis of plasma lipoperoxides in dilute phosphoric acid, the conjugation product of MDA with thiobarbituric acid was measured by HPLC with fluorescence detection at 525/550 nm (Wong et al., 1987).
Statistical analysis
The first step in data analysis was descriptive statistics. To test for significant differences in chromosome aberration frequencies between groups we used the Mann–Whitney U-test and the 
2 test. For paired values we used the paired samples t-test (comparison of paired values before and after supplementation) or the Wilcoxon matched pairs signed ranks test when values were not normally distributed (chromosome aberration values). For comparison of two different groups (such as supplemented/placebo, smokers/non-smokers) we used the independent samples t-test if data were normally distributed and the Wilcoxon rank sum W-test for data not normally distributed.
For the comparison of supplementation-dependent differences between groups (MI/RC, supplemented/placebo), a new parameter was created which represents the difference in paired values before and after supplementation for each individual parameter (vitamin C, retinol, 
-tocopherol, -tocopherol/cholesterol, -carotene, ß -carotene, Se, FRAP and MDA). Multivariate analysis was applied to this parameter (i.e. the difference), with supplementation and group (RC or MI) as determinant factors (see Table III). If a determinant was significant in a valid model, we performed an independent samples t-test to identify significant differences between groups.
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For logistic regression analysis of chromosome aberration data, we created another new parameter for all subjects, describing whether: (i) they showed an apparently beneficial effect of supplementation, i.e. they showed a decrease in aberration frequency (or scored 0 before and after supplementation and so could not show a decrease); (ii) they showed no beneficial effect or even an adverse effect, i.e. the number of aberrations was increased or (if positive before supplementation) did not decrease. FRAP and MDA were treated as covariates, and supplementation, smoking status and group (RC, MI) as categorial covariates.
All tests were performed at a significance level = 0.05.
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Results |
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Effects of supplementation on antioxidant levels and oxidative stress
Mean plasma levels of vitamin C,
-tocopherol, -tocopherol/cholesterol and Se increased in the MI supplemented group, while the RC supplemented group saw significant increases in vitamin C, ß-carotene and Se; retinol went down in both + and –RC groups (Tables I and II). In addition to the antioxidants listed in Tables I– III, we also measured 
-tocopherol, lycopene and ß-cryptoxanthin; no significant effects of supplementation were seen. Total antioxidant status of plasma, assessed using the FRAP assay, showed an increase during the supplementation period, significant for all groups except the MI placebo group (Tables I and II). Conversely, MDA, a product of lipid peroxidation and indicator of oxidative stress, decreased in all MI subjects during the 3 months, but significantly only in supplemented subjects. There were no significant decreases in MDA in RC subjects receiving either antioxidants or placebo (Tables I and II).
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Table III describes the results of multivariate analysis of the differences in paired values (before and after supplementation) of vitamin C, retinol,
-tocopherol, -tocopherol/cholesterol, -carotene, ß -carotene, Se, FRAP and MDA. Where significant models were identified, the independent sample t-test was used to assess the significance of differences between groups, which were as follows. The increase in ß-carotene in supplemented subjects (MI and RC groups combined) was significant (P = 0.007) compared with all placebo subjects. The decrease in MDA levels in the supplemented MI group was significant (P = 0.004) compared with the placebo MI group. The increase in Se was higher in MI supplemented compared with RC supplemented subjects (P = 0.027), but there was no difference between placebo groups.
Effects of supplementation on chromosome damage
Chromosome aberrations (chromatid and chromosome breaks) were monitored in the MI and RC groups before and after 3 months of supplementation with antioxidants or placebo. Before supplementation, there were no differences between the two groups and aberration frequencies were within what is regarded as the normal range (Bavorová and Oadlíková, 1989).
Table IV shows frequencies of cells showing chromosome aberrations and frequencies of chromosome/chromatid breaks before and after supplementation with antioxidants or placebo. In the MI group, no significant effects of supplementation were detected. A significant decrease in the per cent aberrant cells was seen in RC subjects after antioxidant supplementation compared with before, and also in comparison with RC subjects who received placebo. The largest effect was on the number of chromatid breaks; the difference between placebo- and antioxidant-supplemented groups was significant at P = 0.005.
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We analysed the chromosome aberration data for the RC subjects according to smoking habit (Table V). Changes in non-smokers were not significant. At the end of the supplementation period the per cent aberrant cells in samples from smokers was very significantly different when antioxidant- and placebo-supplemented groups were compared (P < 0.001) (Figure 1), and this difference was mirrored in the numbers of chromatid breaks (P = 0.0002) (Figure 2).
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In order to circumvent the problem that few chromosome aberrations were detected in subjects, we created a parameter to describe, for each subject, whether supplementation resulted in a beneficial effect or otherwise at the level of the chromosomes (see Materials and methods for details). A statistical model was devised, using logistical regression analysis. Group (MI or RC), supplementation (antioxidants or placebo), smoking habit, FRAP and MDA levels were the covariates incorporated in the model to explain changes in chromosome aberration frequencies. Supplementation, smoking status and FRAP had significant influences, and the model as a whole was highly significant (P = 0.004). Supplemented smokers had the highest probability of showing a beneficial effect on aberration frequency (Table VI).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Epidemiological studies overwhelmingly support the conclusion that consumption of fruit and vegetables helps to prevent degenerative diseases such as cardiovascular disease and certain cancers. Fruits and vegetables contain different classes of compounds, including vitamin C, vitamin E, carotenoids and flavonoids, that can act in vitro as antioxidants, and it is widely assumed that they are effective in vivo at preventing oxidative damage to biomolecules and thus preventing disease. Experimental evidence is sparse and conflicting. Antioxidant supplements decrease oxidative DNA damage in humans (Duthie et al., 1996
), as do antioxidant-rich foods (Pool-Zobel et al., 1997; Mitchell and Collins, 1999; Collins et al., 2001). On the other hand, in two large-scale, long-term supplementation trials, ß-carotene was linked to substantial increases in the incidence of lung cancer (Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study Group, 1994; Omenn et al., 1996). We have studied the effect of supplementing the human diet with vitamin C, vitamin E, ß-carotene and Se on the occurrence in lymphocytes of chromosome aberrations, the only biomarker so far shown to have predictive value in terms of cancer risk (Hagmar et al., 1994; Bonassi et al., 1998).
Supplementation was effective in raising plasma levels of the corresponding antioxidant micronutrients. During the supplementation period, MDA concentrations decreased significantly in the MI supplemented group. Total antioxidant status as indicated by the FRAP value increased in both supplemented and placebo groups, suggesting a possible contribution from the normal seasonal variation in dietary intake of antioxidants (Duinská et al., 2002).
Antioxidant supplementation was associated with a striking decrease in the percentage of aberrant cells and the number of chromatid as well as chromosome breaks. This effect was seen only in the RC group, and within that group only in smokers. MI patients showed no effect of supplement on chromosome aberrations, even though they showed substantial increases in plasma antioxidants, particularly vitamin C, -tocopherol and Se, after supplementation. Almost all of the MI patients were current non-smokers, and the non-smokers in the RC group also showed no effect of antioxidants on chromosome aberrations. We previously reported that a similar mix of antioxidants (but without Se), given to middle-aged men, decreased endogenous oxidation in lymphocyte DNA (Duthie et al., 1996). In this study, too, smokers were more affected by supplementation than non-smokers.
The role of antioxidants in protecting us from genetic damage is confirmed by the results of logistic regression analysis (Table VI). Taken together with the evidence that the level of chromosome aberrations is a predictor of cancer risk, these studies uphold the antioxidant hypothesis in the face of contrary claims that vitamin C is a pro-oxidant (Podmore et al., 1998) and that ß-carotene may contribute to the incidence of lung cancer (Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study Group, 1994; Omenn et al., 1996). It is plausible that positive protective effects depend on receiving a mixture of antioxidants (as is found in normal foods) rather than a high dose of a particular compound. The possibility that antioxidant supplements are more effective in smokers than in non-smokers at preventing chromosome damage, as suggested by our results, requires further investigation.
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Acknowledgements |
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We thank S.Wood for technical help with antioxidant analyses and K.Gaval’ová and Z.Ro
táová for help with cytogenetic analysis. Dr P.Blaíek kindly carried out MDA analyses. We also thank B.Vallová, M.Drliková, A.Gaiová, A.Morávková and N.Arvaiová for their excellent technical help with sampling. This work was supported by EC contract IC15-CT96-1012, the Ministry of Health of the Slovak Republic and the Scottish Executive Environment and Rural Affairs Department.
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Notes |
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2To whom correspondence should be addressed. Email: dusinska{at}upkm.sk
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Received on December 23, 2002;
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