Nutritional and lifestyle determinants of DNA oxidative damage: a study in a Mediterranean population
Lisa Giovannelli1,
Calogero Saieva2,
Giovanna Masala2,4,
Giovanna Testa1,
Simonetta Salvini2,
Vanessa Pitozzi1,
Elio Riboli3,
Piero Dolara1 and
Domenico Palli2,4
1 Department of Pharmacology, University of Florence,
2 Molecular and Nutritional Epidemiology Unit, Centro per lo Studio e la Prevenzione Oncologica (CSPO), Istituto Scientifico della Regione Toscana, Florence, Italy and
3 Nutrition and Cancer Unit, IARC, Lyon, France
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Abstract
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In order to evaluate dietary and lifestyle determinants of oxidative DNA damage we used a modification of the comet assay (single cell alkaline gel electrophoresis), with the fpg enzyme (formamidopyrimidine DNA glycosilase), to measure the basal level of DNA oxidation in peripheral lymphocytes donated by 71 healthy adults living in Florence, Italy. Detailed information about dietary and lifestyle habits was collected by two validated and standardized questionnaires; we also measured plasma concentrations of selected micro-nutrients (six carotenoids, retinol,
- and
-tocopherol). DNA damage, measured as percent DNA migrated in the comet tail (mean 4.67%, interquartile range 2.366.62%), was not associated with gender, age, weight, body mass index, physical activity or smoking history. A positive correlation with height and period of blood sampling emerged: DNA damage tended to be higher among taller subjects (P = 0.02) and in samples obtained in summer months (P = 0.02). Multivariate analyses showed a positive association with coffee (P = 0.01) and tomato consumption (P = 0.05). Instead, the consumption of cruciferous vegetables tended to be negatively associated with oxidative damage (P = 0.09). Furthermore, a positive non-significant association between the consumption of total vegetables and fresh fruit and DNA damage emerged (P = 0.08 and P = 0.10, respectively). The estimated intake of simple sugars showed a strong positive association with oxidative DNA damage (P = 0.01), while vitamin E showed a borderline positive association (P = 0.06). The plasma levels of several micro-nutrients did not appear to influence DNA damage. Our results, although based on a relatively small group of subjects, indicate that individual dietary and lifestyle habits only modestly affect the levels of lymphocyte DNA oxidation and suggest that specific dietary patterns, rich in fresh fruit and vegetables, are not clearly related to decreased oxidative damage in peripheral lymphocytes in a Mediterranean population.
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Introduction
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The hypothesis that oxidative DNA damage might play a relevant role in the development of degenerative diseases (2) has enjoyed wide popularity for many years. This idea is based on theoretical considerations about cell function and biology and on empirical observations linking the consumption of foods, such as fruit and vegetables, containing a mixture of natural antioxidant compounds, with better general health and reduced cancer risk (39). However, several intervention studies focusing on the role of specific dietary antioxidants as cancer preventive agents have not provided conclusive evidences. Some studies show a small effect of dietary supplementation on cancer incidence (7), while others show that supplementation with antioxidant vitamins may have an adverse effect on the incidence of cancer and cardiovascular diseases (1,44) or no effect (28,31).
One critical issue in all these supplementation studies is whether dietary habits can actually modify the level of endogenous DNA oxidation. In this respect, some human intervention trials indicate that increasing the intake of individual antioxidants or antioxidant-rich foods can reduce the basal level of DNA oxidation in lymphocytes (20,29,42,50) and increase lymphocyte resistance to oxidative stress (20,27,51). On the contrary, Collins et al. (13) show that dietary supplementation with a mixture of carotenoids did not modify endogenous oxidative damage in lymphocyte DNA, although a negative correlation was found between the basal level of serum total carotenoids and the basal level of DNA base oxidation. In a study of shorter duration, Welch et al. (65) did not find any effect of ascorbic acid, vitamin E, selenium or of their combinations on DNA oxidation in leukocytes of smokers and non-smokers, whereas ß-carotene supplementation increased oxidative DNA damage in leukocytes of smokers and decreased it in leukocytes of non-smokers, with no concomitant change in ß-carotene blood concentration compared with baseline levels.
Overall, it appears that modulating the levels of endogenous DNA oxidation through dietary or supplementation intervention is possible, but a number of variables such as duration of the trial, type and dose of antioxidant, basal level of antioxidant plasma concentrations (in itself a diet-related variable), and smoking, can interfere with the effectiveness of the intervention.
The aim of our study was to evaluate dietary and lifestyle determinants of endogenous DNA oxidative damage in a series of healthy adults residing in Florence (central Italy). The local dietary pattern is characterized by olive oil as the main added fat, wine (mostly red) and a relatively high consumption of fresh and cured meats. A modification of the comet assay (originally described in ref. 59) was used to measure the basal level of DNA oxidation in lymphocytes (10).
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Materials and methods
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Subjects
The study was conducted on a sample of 71 healthy volunteers residing in Florence and previously enrolled in the local section of a large European prospective study on diet and cancer known as EPIC (European Prospective Investigation into Cancer and nutrition) (45,46,53). A subgroup of volunteers were re-invited on a separate occasion to donate blood and provide detailed dietary and lifestyle information. Approximately five volunteers per week were recruited between March and September 1999. An informed consent form was signed by all subjects (48 males, 23 females; age 3564 years) prior to enrolment in the study.
Diet and lifestyle information
Dietary information on the frequency of consumption of >120 foods and drinks in a 12-month period prior to enrolment was obtained by a self-administered Food Frequency Questionnaire, validated in a pilot phase (47). All individual questionnaires were checked and coded by trained dieticians, computerised and then transformed into estimates of intake for a series of over 30 nutrients, according to a specifically developed program (45), linked to recently updated Italian Food Tables (55). A specific form was developed to collect information about the type of olive oil consumed. Subjects were asked to report the frequency of consumption of olive oil directly obtained from local olive oil mills (here defined as high quality), or commercially distributed extra-virgin olive oil and other types of olive oil (here combined as low/medium quality). Since the food frequency questionnaire did not include details about the types of olive oil consumed, we used the information provided in the specific olive oil form to weight the olive oil consumption and estimated the frequency of consumption of the two main olive oil types. Weight and height were measured for each participant.
A standardised lifestyle questionnaire (in two separate versions for men and women) was completed. Each participant provided detailed information on reproductive history, physical activity, smoking and alcohol drinking history, exposure to environmental tobacco smoke, medical history, occupation, education and other socio-economic variables.
Physical activity at work was classified as light (sedentary and mainly standing occupations) and heavy (manual and heavy manual occupations). Hours of leisure time activities and hours of work were assigned appropriate metabolic equivalents (MET) (16,67) and multiplied by individual basal metabolic rates (67) to obtain an estimate of energy expenditure per day (56). We also computed scores of the various types of activity and a cumulative activity score obtained by summing up all specific activity scores. This cumulative score was categorised into three levels (low, medium and high).
Laboratory methods
Comet assay.
Venous blood samples were obtained using EDTA-treated vacutainer tubes, and mononuclear cells were isolated utilizing Lymphoprep separation medium (Gibco Biology Research Laboratories, Inchinnan, Scotland, UK). Blood samples of 3 ml were diluted 1:1 with phosphate-buffered saline (PBS) and layered on an equal volume of Lymphoprep medium in a centrifuge tube. After centrifugation at 1000 g for 20 min, gradient-separated lymphocytes were recovered, resuspended in PBS and counted in a Neubauer chamber. Vitality was assessed by means of the Trypan Blue exclusion method. Aliquots of the lymphocyte suspension containing about 200 000 cells were further centrifuged at 250 g for 5 min, and the resulting pellets were resuspended in melted agarose (low melting point, Fisher Scientific, Leics, UK), layered on microscopic slides, letting them solidify and run through the comet assay as previously described (25). Briefly, the slides with the agarose-embedded lymphocytes were subjected to a lysis step (1 h incubation at 4°C in 1% N-lauroyl-sarcosine, 2.5 M NaCl, 100 mM Na2EDTA, 1% Triton X-100, 10% dimethylsulfoxide). After the lysis step, slides were washed three times in enzyme buffer (40 mM HEPES-KOH, 100 mM KCl, 0.5 mM EDTA, 0.2 mg/ml bovine serum albumin, BSA, pH 8.0) and then incubated at 37°C for 45 min with 50 µl of the E.coli enzyme formamidopyrimidine DNA glycosilase (fpg, 1:1000) for purine oxidation detection (kindly provided by Dr A.R.Collins, Rowett Research Institute, Aberdeen, Scotland, UK) or enzyme buffer only. Each experimental point was run in duplicate, thus four slides were run for each subject on the same day. All the slides of the experiment (usually 20) were placed in an ice-cold electrophoresis chamber containing alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA) for 20 min to allow DNA unwinding. The electrophoresis was subsequently conducted for 20 min at 25 V and 300 mA. At the end of the electrophoresis the slides were washed with neutralisation buffer (40 mM TrisHCl, pH 7.4), stained with ethidium bromide overnight and analysed the following day.
Microscopical analysis was carried out by means of a Labophot-2 microscope (Nikon, Tokyo, Japan) provided with epifluorescence and equipped with a rhodamine filter (excitation wavelength 546 nm; barrier 580 nm). The images of 50 randomly chosen nuclei per slide were captured and analysed using a custom-made imaging software coupled with a CCD camera (model C5985, Hamamatsu, Sunayama-Cho, Japan).
The amount of broken DNA migrated in the tail was expressed as percent of total fluorescence for each nucleus (% DNA in tail). This value was then averaged over the 50 nuclei measured per slide, and the duplicate values were further averaged. Detection of oxidative DNA base damage was carried out by means of the fpg enzyme, which introduces breaks at sites of oxidised purines such as 8-oxo-2'-deoxyguanosine (8-oxodG), likely the most abundant and mutagenic base oxidation product (18,23), and formamidopyrimidine lesions (11). Thus, the value of DNA damage obtained in the buffer-incubated slides estimated the basal number of single strand breaks (SSBs), whereas specific oxidative damage on purines was assessed for each patient by subtracting the basal number of breaks (buffer-incubated slides) from the number of breaks obtained incubating the slides with fpg.
Micro-nutrient analyses.
Blood samples were immediately processed by centrifugation and divided into several aliquots of each constituent (serum, plasma, red cells and buffy-coat). Aliquots of plasma were shipped on dry ice to the study laboratory at IARC, Lyon. Manipulation and second storage were done in liquid nitrogen (196°C). On the day of analysis, aliquots were rapidly thawed at room temperature, protected from light. Samples (200 µl) were analysed for six carotenoids (
- and ß-carotene, zeaxanthin, ß-cryptoxanthin, lutein and lycopene), retinol,
- and
-tocopherol by reversed-phase high-performance liquid chromatography (HPLC-1100 system, Hewlett Packard, Les Ullis, France) following a method based on that of Steghens et al. (62). Samples were extracted with 800 µl of hexane after 30 min agitation, and mobile phases were enriched with 1 ml/l of triethylamine. One aliquot from a standard quality control sample was analysed and injected at the beginning, middle and end of each series. Chromatograms were integrated automatically by the system (Chemstation version 6.4, Hewlett Packard, Les Ullis, France) but controlled one by one by three different laboratory technicians. Peaks for carotenoids that were under the detection limits were set at zero, while peaks that could not be detected because of technical problems were set at missing value.
Statistical methods
To investigate the relationship between oxidative DNA damage and anthropometric, sociodemographic and lifestyle habits, non-parametric tests, i.e. MannWhitney and KruskalWallis test, were used. In order to carry out statistical analyses for dietary variables, we first classified subjects according to tertiles of food consumption and nutrient intake. Mean levels of oxidative DNA damage across tertiles of consumption of foods or food groups and intake of nutrients were compared by analysis of covariance, introducing into each model terms for age, sex, height, smoking history (never, former and current), period of blood sampling and total caloric intake. Spearman correlation coefficient was used to determine the correlation between oxidative DNA damage values and selected plasma level micro-nutrients. Analysis of covariance was also performed to evaluate mean levels of oxidative DNA damage across tertiles of plasma micro-nutrient. Analyses on both raw and log-transformed data were performed; however, since results did not differ substantially, only results based on original data are presented. Tests for linear trend were based on log transformed oxidative DNA damage, by including ordered variables in each covariance model.
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Results
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The basal level of DNA strand breaks in the analyzed sample was 4.61 ± 0.40 (% DNA in tail, mean ± SE). Figure 1
shows the distribution of net oxidative DNA damage (number of breaks obtained with fpg minus the basal number of breaks) in the series of analysed lymphocyte samples. The median level of DNA oxidation was 3.89%, measured as percent DNA migrated in the comet tail after the addition of the fpg enzyme (mean = 4.67%; geometric mean = 3.47%; range 0.0819.09%; 25th percentile = 2.36%; 75th percentile = 6.62%). The average viability of the isolated lymphocytes, evaluated by the Trypan Blue exclusion test, was 97.5% and the mean baseline level of DNA oxidation in the lymphocytes of our population sample was estimated to be 0.21 8-oxodG 10-6dG according to the method described by Collins et al. (11).

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Fig. 1. Box-plot of the distribution of oxidative DNA damage (%DNA in tail) in 71 healthy volunteers. The net oxidative DNA damage was obtained by subtracting the values of damage measured in the slides without fpg from those of the slides with fpg treatment. Values are presented as %DNA in tail. The upper and lower boundaries of the box are the upper and lower quartiles. The box length is the interquartile distance so the box contains the middle 50% of values (the horizontal line inside the box indicates the median). The vertical lines extend to highest and lower levels that are not outliers.
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The crude mean values of oxidative DNA damage are reported in Table I
according to selected individual characteristics of study participants: two-thirds were males (67.6%), mostly residing in the urban area of Florence (63.4%) and non-smokers (83.1%). Twelve subjects (16.9%) were current smokers with a mean of 16.4 cigarettes/day smoked and 35/71 (49.3%) were ex-smokers reporting a mean interval of 14.0 years since interrupting smoking. Median values for age, weight, height and body mass index (BMI) were respectively 47 years, 72.2 kg, 169 cm and 25.7 kg/m2. The blood samples were uniformly collected between March and September 1999.
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Table I. Crude mean values (and lower and upper quartiles) of oxidative DNA damage (%DNA in taila) according to selected individual characteristics
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The values of oxidative DNA damage were significantly higher among taller subjects (P = 0.02). A relevant seasonal variation was also evident: oxidative DNA damage was higher in the summer months and lower in the midseason (P = 0.02). A subjects smoking history showed no effect on oxidative DNA damage.
Lymphocyte DNA damage was also not affected by subjects type of work or cumulative activity score; on the other hand, oxidative damage showed a non-significant variation with increasing daily energy expenditure and a lower education level (Table II
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Table II. Crude mean values (and lower and upper quartiles) of oxidative DNA damage (%DNA in taila) according to selected individual characteristics
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Table III
shows the associations between oxidative DNA damage and consumption (in tertiles) of several foods or food groups as reported at dietary interview. A strong positive association was found between DNA damage and the reported consumption of coffee (P = 0.01); a borderline positive association with the consumption of tomatoes emerged (P = 0.05). Overall, subjects reporting a higher consumption of all types of vegetables (including leafy vegetables, fruiting vegetables, root vegetables, cabbages, mushrooms, onion, garlic and mixed salad) and fresh fruit showed an increasing trend of oxidative DNA damage (P = 0.08 and P = 0.10, respectively). On the other hand, a non-significant inverse association (P = 0.09) between oxidative DNA damage and consumption of cruciferous vegetables emerged.
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Table III. Adjusted means of oxidative DNA damage according to tertiles of daily consumption of selected food groups or food items
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Table IV
shows the associations with the estimated intake of several macro- and micro-nutrients (in tertiles). The intake of simple sugar (mono- and disaccharides) was strongly and positively associated with oxidative DNA damage (P = 0.01); a borderline positive association with vitamin E intake also emerged (P = 0.06). A similar increasing trend of oxidative damage was evident, although not statistically significant, with increasing intake of ß-carotene and vitamin A (P = 0.20 and P = 0.21, respectively) and potassium, an indicator of total vegetable consumption (P = 0.12).
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Table IV. Adjusted means of oxidative DNA damage according to tertiles of estimated daily intake of various macro and micro-nutrients
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The plasma concentrations of a series of relevant natural antioxidant compounds are shown in Appendix A
. Their effects on lymphocyte DNA damage were estimated by means of Spearman correlation analysis and multivariate covariance analysis (Table V
). In either case no association was found between antioxidant plasma levels and DNA oxidation in lymphocytes obtained from the same blood sample.
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Appendix A. Plasma concentration (crude mean ± SD) of selected micro-nutrients in 71 healthy volunteers residing in Florence, Central Italy
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Table V. Adjusted means of oxidative DNA damage according to tertiles of plasmatic level of selected micro-nutrients and Spearman correlation coefficients between oxidative DNA damage and plasma levels of micro-nutrients
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Discussion
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In our study, the lymphocyte levels of DNA oxidative damage are similar to those reported in previous studies using the comet assay (reviewed in ref. 12), but are lower than those shown by HPLC techniques (5,14,63).
Our results indicate that oxidative DNA damage in lymphocytes was not affected by gender, age, weight or BMI, although age and sex have been suggested as factors associated with a variation in DNA damage (3,14). However, a gender effect on DNA damage is still a matter of controversy (reviewed in ref. 41). Farinati et al. (22) failed to find a correlation between gender and 8-oxo-dG levels in human leukocytes from patients affected by hepatitis C. Comet assay studies of the effect of age on DNA damage in human lymphocytes have also given conflicting results (4,48,60,61). In our sample, the age of subjects varied from 35 to 64 years, and this range might not be wide enough to detect age-related differences in a relatively small population sample, given the lack of young and old subjects.
We did not find an effect of smoking on DNA oxidation, possibly because of the small number of current smokers in our sample (16.9%). Furthermore, although smoking has been repeatedly reported to increase DNA strand breakage and oxidation in human leukocytes (32,36,48) and in urothelial cells (26), a few studies have failed to detect such an effect (63,68).
We found that oxidative DNA damage in lymphocytes was positively correlated with body height and period of blood sampling. Epidemiological evidence indicates that height is one of the anthropometric risk factors for breast cancer incidence (24) and that taller people are in general at higher risk of cancer mortality (57). Seasonal variation in the basal levels of DNA strand breaks in human leukocytes has been reported by various laboratories, with increased DNA damage in the summer, possibly as a consequence of an increased exposure to UV radiation (reviewed in ref. 41). Palli et al. (45,46) have also reported an increased level of DNA bulky adducts in peripheral white blood cells in a group of subjects from Central-Southern Italy during the summer months, suggesting a role for photochemical pollution.
Exercise is supposed to increase DNA oxidation due to higher metabolic rate. In our sample we only found a tendency towards reduced DNA oxidative damage in subjects with lower levels of daily energy expenditure (lowest tertile), but not of physical activity. Again, although the pro-oxidant effects of heavy exercise have often been suggested (reviewed in 35), unchanged levels of DNA base oxidation have been reported immediately after swimming or running with a caloric expenditure of 30004000 kJ (33).
Of the foods reported at interview, only coffee showed a significant positive correlation with lymphocyte DNA oxidation. On the contrary, other studies have reported that coffee consumption is negatively correlated with DNA damage in human leukocytes (63), and caffeine itself has antioxidant properties in vitro (17). Another positive association of borderline statistical significance emerged between oxidative DNA damage and tomato consumption: this result is in contrast with other studies (51,52,54), which indicate that tomatoes enhance lymphocyte resistance to oxidative stress. In our study, overall, the total consumption of vegetables and fresh fruit showed a tendency to be positively associated with oxidative damage, in disagreement with other studies (50). However, an inverse not-significant association between the daily consumption of cruciferous vegetables and oxidative DNA damage emerged, in agreement with other studies (64,69) suggesting that consumption of cruciferous vegetables might result in decreased oxidative DNA damage. The frequent consumption of olive oil, red wine and meats did not seem to influence DNA damage, in contrast with other studies where a relationship emerged between oxidative DNA damage and alcohol consumption (6), meat consumption (19) and olive oil consumption (21).
We did find a strong positive association between oxidative DNA damage and sugar intake, mostly derived from fruit, sugar added to coffee and other beverages, milk, soft drinks and desserts. The oxidative potential of simple dietary carbohydrates is widely documented (58), particularly in diabetic patients (15,30,66) and is generally attributed to glycation and glycoxidation (37). Only one subject in our series reported a medical history of diabetes at interview. A high intake of selected antioxidants (vitamin A, C, E and ß-carotene) tended to be positively associated with oxidative DNA damage, with a borderline statistical significance for vitamin E, in disagreement with several studies (mostly intervention studies) which showed a protective effect of antioxidants with respect to DNA damage.
Plasma concentrations of several antioxidant micro-nutrients were measured and we evaluated their correlation with DNA oxidative damage. The baseline plasma levels of the main carotenoids and vitamin E were similar to those reported in Spanish subjects by Bianchini et al. (5) and are presumably typical of Mediterranean populations, whereas in Northern Europe lower plasma levels of these antioxidants are common (43). None of our statistical analyses showed any correlation between plasma levels of these antioxidants and lymphocyte DNA oxidation, whereas most supplementation studies showed a clear antioxidant effect for carotenoids and vitamins E and C (9,20,34,51), with some exceptions (49).
Intervention studies usually employ antioxidant doses that are much higher than those provided by ordinary healthy diets, resulting in some cases in substantial increases in antioxidant plasma levels (13,51). However, it is not yet clear whether dietary antioxidants are directly involved in protection from oxidative damage, which of them are involved and at what dose. In fact, carotenoid plasma concentration has been reported to be both negatively (13) and positively correlated to leukocyte DNA oxidation (5). It has also been shown that lycopene and ß-carotene protect cells against oxidatively induced DNA damage only at relatively low concentrations, but increase the extent of damage at higher concentrations (40).
Intracellular ascorbate and glutathione levels have been shown to negatively correlate with lymphocyte DNA oxidation (38), but no association was found between vitamin E or vitamin C plasma levels and DNA oxidation in blood (13). It is also possible that dietary antioxidants are involved in protection from oxidative stress rather than in the maintenance of baseline oxidation levels (8).
In conclusion, our results indicate that in a relatively homogeneous group of Italian healthy adults without supplementation of antioxidants, differences in individual dietary habits, with the exception of sugar and coffee consumption, have no effect on lymphocyte DNA oxidation. It is possible that different populations with more marked differences in dietary patterns may show a wider range of diet-associated variation in oxidative damage to their lymphocytes (14). However, within a group of healthy adults with a rather homogeneous lifestyle, these variations in oxidation levels in lymphocytes appear to be modest. We therefore suggest that the association between specific dietary habits and degenerative diseases consistently reported by many studies might be due to mechanisms other than oxidative damage as measured in peripheral cells such as lymphocytes. On the other hand, the seasonal trend of oxidative damage we observed in this study deserves further investigation.
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Notes
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4 To whom correspondence should be addressed Email: d.palli{at}cspo.it 
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Acknowledgments
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The authors wish to thank all study participants and collaborators of the EPIC-Florence Study Group for their cooperation (particularly Maria Martinez and Giovanna Cordopatri), Dr Vittorio Krogh (Milan) for support with the dietary questionnaire and Drs Marco Peluso, Massimiliano Sifone and Chiara Zappitello (CSPO, Florence) for helpful comments and editorial assistance. The authors also wish to thank Dr A.R.Collins for providing the enzyme formamidopyrimidine glycosilase. EPIC-Florence is supported by a generous grant of Associazione Italiana per la Ricerca sul Cancro (AIRCMilan). This work was also supported by the European Community FAIR and Polybind programs, and by the Ministero dellIstruzione dellUniversità e della Ricerca, Italy.
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References
|
---|
- The Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group. (1994) The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. N. Engl. J. Med., 330, 10291035.[Abstract/Free Full Text]
- Ames,B.N. (1983) Dietary carcinogens and anticarcinogens. Science, 221, 12561264.[ISI][Medline]
- Ames,B.N., Shigenaga,M.K. and Hagen,T.M. (1993) Oxidants, antioxidants and the degenerative diseases of aging. Proc. Natl Acad. Sci. USA, 90, 79157922.[Abstract/Free Full Text]
- Betti,C., Davini,T., Giannessi,L., Loprieno,N. and Barale,R. (1994) Microgel electrophoresis assay (comet test) and SCE analysis in human lymphocytes from 100 normal subjects. Mutat. Res., 307, 323333.[ISI][Medline]
- Bianchini,F., Elmstahl,S., Martinez-Garcia,C., van Kappel,A.L., Douki,T., Cadet,J., Ohshima,H., Riboli,E. and Kaaks,R. (2000) Oxidative DNA damage in human lymphocytes: correlations with plasma levels of alpha-tocopherol and carotenoids. Carcinogenesis, 21, 321324.[Abstract/Free Full Text]
- Bianchini,F., Jaeckel,A., Vineis,P., et al. (2001) Inverse correlation between alcohol consumption and lymphocyte levels of 8-hydroxydeoxyguanosine in humans. Carcinogenesis, 22, 885890.[Abstract/Free Full Text]
- Blot,W.J., Li,J.Y., Taylor,P.R., et al. (1993) Nutrition intervention trials in Linxian, China: supplementation with specific vitamin/mineral combinations, cancer incidence and disease-specific mortality in the general population. J. Natl Cancer Inst., 85, 14831492.[Abstract]
- Brennan,L.A., Morris,G.M., Wasson,G.R., Hannigan,B.M. and Barnett,Y.A. (2000) The effect of vitamin C or vitamin E supplementation on basal and H2O2-induced DNA damage in human lymphocytes. Br. J. Nutr., 84, 195202.
- Chen,L., Bowen,P.E., Berzy,D., Aryee,F., Stacewicz-Sapuntzakis,M. and Riley,R.E. (1999) Diet modification affects DNA oxidative damage in healthy humans. Free Radic. Biol. Med., 26, 695703.
- Collins,A.R., Duthie,S.J. and Dobson,V.L. (1993) Direct enzymic detection of endogenous oxidative base damage in human lymphocytes DNA. Carcinogenesis, 14, 17331735.[Abstract]
- Collins,A.R., Dusinska,M., Gedik,C.M. and Stetina,R. (1996) Oxidative damage to DNA: do we have a reliable biomarker? Environ. Health Perspect., 104 (Suppl. 3), 465469.[ISI][Medline]
- Collins,A.R., Cadet,J., Epe,B. and Gedik,C. (1997) Problems in the measurement of 8-oxoguanine in human DNA. Report of a workshop, DNA oxidation, held in Aberdeen, UK, January 1997. Carcinogenesis, 18, 18331836.[Abstract]
- Collins,A.R., Olmedilla,B., Southon,S., Granado,F. and Duthie,S. (1998) Serum carotenoids and oxidative DNA damage in human lymphocytes. Carcinogenesis, 19, 21592162.[Abstract]
- Collins,A.R., Gedik,C.M., Olmedilla,B., Southon,S. and Bellizzi,M. (1998) Oxidative DNA damage measured in human lymphocytes: large differences between sexes and between countries and correlation with heart disease mortality rates. FASEB J., 12, 13971400.[Abstract/Free Full Text]
- Collins,A.R., Raslova,K., Somorovska,M., Petrovska,H., Ondrusova,A., Vohnout,B., Fabry,R. and Dusinska,M. (1998) DNA damage in diabetes: correlation with a clinical marker. Free Radic. Biol. Med., 25, 373377.[ISI][Medline]
- Commission of European Communities (1993) Nutrient and energy intakes for the European Community. Reports of the Scientific Committee for Food, 31st series. Office for Official Publications of the European Communities, Luxembourg.
- Devasagayam,T.P., Kamat,J.P., Mohan,H. and Kesavan,P.C. (1996) Caffeine as an antioxidant: inhibition of lipid peroxidation induced by reactive oxygen species. Biochim. Biophys. Acta, 1282, 6370.[ISI][Medline]
- Dizdaroglu,M. (1991) Chemical determination of free-radical induced damage to DNA. Free Radic. Biol. Med., 10, 225242.[ISI][Medline]
- Djuric,Z., Depper,J.B., Uhley,V., Smith,D., Lababidi,S., Martino,S. and Heilbrun,L.K. (1998) Oxidative DNA damage levels in blood from women at high risk for breast cancer are associated with dietary intake of meats, vegetables and fruits. J. Am. Diet Assoc., 98, 524528.[ISI][Medline]
- Duthie,S.J., Ma,A., Ross,M.A. and Collins,A.R. (1996) Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes. Cancer Res., 56, 12911295.[Abstract]
- Elmadfa,I. and Park,E. (1999) Impact of diets with corn oil or olive/sunflower oils on DNA damage in healthy young men. Eur. J. Nutr., 38, 286292.[ISI][Medline]
- Farinati,F., Cardin,R., Degan,P., De Maria,N., Floyd,R.A., Van Thiel,D.H. and Naccarato,R. (1999) Oxidative DNA damage in circulating leukocytes occurs as an early event in chronic HCV infection. Free Radic. Biol. Med., 27, 12841291.[ISI][Medline]
- Floyd,R.A. (1990) The role of 8-hydroxyguanine in carcinogenesis. Carcinogenesis, 11, 14471450.[ISI][Medline]
- Friedenreich,C.M. (2001) Review of anthropometric factors and breast cancer risk. Eur. J. Cancer. Prev., 10, 1532.[ISI][Medline]
- Giovannelli,L., Testa,G., De Filippo,C., Cheynier,V., Clifford,M.N. and Dolara,P. (2000) Effect of complex polyphenols and tannins from red wine on DNA oxidative damage of rat colon mucosa in vivo. Eur. J. Nutr., 39, 207212.[ISI][Medline]
- Gontijo,A.M., Elias,F.N., Salvadori,D.M., de Oliveira,M.L., Correa,L.A., Goldberg,J., Trindade,J.C. and de Camargo,J.L. (2001) Single-cell gel (comet) assay detects primary DNA damage in nonneoplastic urothelial cells of smokers and ex-smokers. Cancer Epidemiol. Biomark. Prev., 10, 987993.[Abstract/Free Full Text]
- Green,M.H.L., Lowe,J.E., Waugh,A.P.W., Aldridge,K.E., Cole,J. and Arlett,C.F. (1994) Effect of diet and vitamin C on DNA strand breakage in freshly-isolated human white blood cells. Mutat. Res., 316, 91102.[ISI][Medline]
- Greenberg,E.R., Baron,J.A., Tosteson,T.D., et al. (1994) A clinical trial of antioxidant vitamins prevent colorectal adenoma. Polyp. Prevention Study Group. N. Engl. J. Med., 331, 141147.[Abstract/Free Full Text]
- Haegele,A.D., Gillette,C., ONeill,C., Wolfe,P., Heimendinger,J., Sedlacek,S. and Thompson,H.J. (2000) Plasma xanthophyll carotenoids correlate inversely with indices of oxidative DNA damage and lipid peroxidation. Cancer Epidemiol. Biomark. Prev., 9, 421425.[Abstract/Free Full Text]
- Hannon-Fletcher,M.P., OKane,M.J., Moles,K.W., Weatherup,C., Barnett,C.R. and Barnett,Y.A. (2000) Levels of peripheral blood cell DNA damage in insulin dependent diabetes mellitus human subjects. Mutat. Res., 460, 5360.[ISI][Medline]
- Hennekens,C.H., Buring,J.E., Manson,J.E., et al. (1996) Lack of effect of long-term supplementation with beta carotene on the incidence of malignant neoplasms and cardiovascular disease. N. Engl. J. Med., 334, 11451149.[Abstract/Free Full Text]
- Holz,O., Meissner,R., Einhaus,M., Koops,F., Warncke,K., Scherer,G., Adlkofer,F., Baumgartner,E. and Rudiger,H.W. (1993) Detection of DNA single-strand breaks in lymphocytes of smokers. Int. Arch. Occup. Environ. Health, 65, 8388.[ISI][Medline]
- Inoue,T., Mu,Z., Sumikawa,K., Adachi,K. and Okochi,T. (1993) Effect of physical exercise on the content of 8-hydroxydeoxyguanosine in nuclear DNA prepared from human lymphocytes. Jpn. J. Cancer Res., 84, 720725.[ISI][Medline]
- Jenkinson,A.M., Collins,A.R., Duthie,S.J., Wahle,K.W. and Duthie,G.G. (1999) The effect of increased intakes of polyunsaturated fatty acids and vitamin E on DNA damage in human lymphocytes. FASEB J., 13, 21382142.
- Ji,L.L. (1999) Antioxidants and oxidative stress in exercise. Proc. Soc. Exp. Biol. Med., 222, 283292.[Abstract/Free Full Text]
- Kiyosawa,H., Suko,M., Okudaira,H., Murata,K., Miyamoto,T., Chung,M.H., Kasai,H. and Nishimura,S. (1990) Cigarette smoking induces formation of 8-hydroxydeoxyguanosine, one of the oxidative DNA damages in human peripheral leukocytes. Free Radic. Res. Commun., 11, 2327.[ISI][Medline]
- Kristal,B.S. and Yu,B.P. (1992) An emerging hypothesis: synergistic induction of aging by free radicals and Maillard reactions. J. Gerontol. Biol. Sci., 47, B107114.
- Lenton,K.J., Therriault,H., Fulop,T., Payette,H. and Wagner,J.R. (1999) Glutathione and ascorbate are negatively correlated with oxidative DNA damage in human lymphocytes. Carcinogenesis, 20, 607613.[Abstract/Free Full Text]
- Loft,S. and Poulsen,H.E. (1996) Cancer risk and oxidative DNA damage in man. J. Mol. Med., 74, 297312.[ISI][Medline]
- Lowe,G.M., Booth,L.A., Young,A.J. and Bilton,R.F. (1999) Lycopene and beta-carotene protect against oxidative damage in HT29 cells at low concentrations but rapidly lose this capacity at higher doses. Free Radic. Res., 30, 141151.[ISI][Medline]
- Moller,P., Knudsen,L.E., Loft,S. and Wallin,H. (2001) The comet assay as a rapid test in biomonitoring occupational exposure to DNA damaging agents and effect of confounding factors. Cancer Epidemiol. Biomark. Prev., 9, 10051015.[Abstract/Free Full Text]
- Noroozi,M., Angerson,W.J. and Lean,M.E.J. (1998) Effects of flavonoids and vitamin C on oxidative DNA damage to human lymphocytes. Am. J. Clin. Nutr., 67, 12101218.[Abstract]
- Olmedilla,B., Granado,F., Southon,S., et al. (2001) Serum concentrations of carotenoids and vitamins A, E and C in control subjects from five European countries. Br. J. Nutr., 85, 227238.[ISI][Medline]
- Omenn,G.S., Goodman,G.E., Thornquist,M.D., et al. (1996) Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. N. Engl. J. Med., 334, 11501155.[Abstract/Free Full Text]
- Palli,D., Vineis,P., Russo,A., et al. (2000) Diet, metabolic polymorphisms and DNA adducts: the EPIC-Italy cross-sectional study. Int. J. Cancer, 87, 444451.[ISI][Medline]
- Palli,D., Russo,A., Masala,G., Saieva,C., Guarrera,S., Carturan,S., Munnia,A., Matullo,G. and Peluso,M. (2001) DNA adducts levels and DNA repair polymorphisms in traffic-exposed workers and a general population sample. Int. J. Cancer, 94, 121127.[ISI][Medline]
- Pisani,P., Faggiano,F., Krogh,V., et al. (1997) Relative validity and reproducibility of a food-frequency dietary questionnaire for use in the Italian EPIC centres. Int. J. Epidemiol., 26, s152s160.[ISI][Medline]
- Piperakis,S.M., Visvardis,E.E., Sagnou,M. and Tassiou,A.M. (1998) Effects of smoking and aging on oxidative DNA damage of human lymphocytes. Carcinogenesis, 19, 695698.[Abstract]
- Podmore,I.D., Griffiths,H.R., Herbert,K.E., Mistry,N., Mistry,P. and Lunec,J. (1998) Vitamin C exhibits pro-oxidant properties. Nature, 392, 559.[ISI][Medline]
- Pool-Zobel,B.L., Bub,A., Müller,H., Wollowski,I. and Rechkemmer,G. (1997) Consumption of vegetables reduces genetic damage in humans: first results of a human intervention trial with carotenoid-rich foods. Carcinogenesis, 18, 18471850.[Abstract]
- Porrini,M. and Riso,P. (2000) Lymphocyte lycopene concentration and DNA protection from oxidative damage is increased in women after a short period of tomato consumption. J. Nutr., 130, 189192.[Abstract/Free Full Text]
- Rehman,A., Bourna,L.C., Halliwell,B. and Rice-Evans,C.A. (1999) Tomato consumption modulates oxidative DNA damage in humans. Biochem. Biophys. Res. Commun., 262, 828831.[ISI][Medline]
- Riboli,E. and Kaaks,R. (1997) The EPIC Project: rationale and study design. Int. J. Epidemiol., 26 (Suppl.1), S6S14.[Abstract/Free Full Text]
- Riso,P., Pinder,A., Santangelo,A. and Porrini,M. (1999) Does tomato consumption effectively increase the resistance of lymphocyte DNA to oxidative damage? Am. J. Clin. Nutr., 69, 712718.[Abstract/Free Full Text]
- Salvini,S., Parpinel,M., Gnagnarella,P., Maisonneuve,P. and Turrini,A. (1998) Banca Dati di Composizione degli Alimenti per Studi Epidemiologici in Italia, Istituto Europeo di Oncologia, Milan, Italy.
- Salvini,S., Saieva,C., Sieri,S., Vineis,P., Panico,S., Tumino,R. and Palli,D. (2002) Physical activity in the EPIC-ITALY study. In Proceedings of the Nutritional and Cancer Conference. IARC Scientific Publication Series, Lyon, France (in press).
- Samaras,T.T. and Elrick. H. (1999) Height, body size and longevity. Acta Med. Okayama, 53, 149169.[ISI][Medline]
- Shimoi,K., Okitsu,A., Green,M.H., Lowe,J.E., Ohta,T., Kaji,K., Terato,H., Ide,H. and Kinae,N. (2001) Oxidative DNA damage induced by high glucose and its suppression in human umbilical vein endothelial cells. Mutat. Res., 480481 (12), 371378.[ISI]
- Singh,N.P., McCoy,M.T., Tice,R.R. and Scheider,E.L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res., 175, 184191.[ISI][Medline]
- Singh,N.P., Danner,D.B., Tice,R.R., Brant,L. and Schneider,E.L. (1990) DNA damage and repair with age in individual human lymphocytes. Mutat. Res., 237, 123130.[ISI][Medline]
- Singh,N.P., Danner,D.B., Tice,R.R., Pearson,J.D., Brant,L.J., Morrell,C.H. and Schneider,E.L. (1991) Basal DNA damage in individual human lymphocytes with age. Mutat. Res., 256, 16.[ISI][Medline]
- Steghens,J.P., van Kappel.A.L., Riboli,E. and Collombel,C. (1997) Simultaneous measurement of seven carotenoids, retinol and alpha-tocopherol in serum by high-performance liquid chromatography. J. Chromatogr. B Biomed. Sci. Appl., 694, 7181.[Medline]
- van Zeeland,A.A., de Groot,A.J., Hall,J. and Donato,F. (1999) 8-Hydroxydeoxyguanosine in DNA from leukocytes of healthy adults: relationship with cigarette smoking, environmental tobacco smoke, alcohol and coffee consumption. Mutat. Res., 439, 249257.[ISI][Medline]
- Verhagen,H., Poulsen,H.E., Loft,S., van Poppel,G., Willems,M.I. and van Bladeren,P.J. (1995) Reduction of oxidative DNA-damage in humans by brussels sprouts. Carcinogenesis, 16, 969970.[Abstract]
- Welch,R.W., Turley,E., Sweetman,S.F., et al. (1999) Dietary antioxidant supplementation and DNA damage in smokers and nonsmokers. Nutr. Cancer, 34 (2), 167172.[ISI][Medline]
- West,I.C. (2000) Radicals and oxidative stress in diabetes. Diabet. Med., 17, 171180.[ISI][Medline]
- WHO (1985). Energy and Protein requirements, report of a joint FAO/WHO/ONU meeting, WHO Technical Report Series no. 724. WHO, Geneva, Switzerland.
- Wojewodzka,M., Kruszewski,M., Iwanenko,T., Collins,A.R. and Szumiel,I. (1999) Lack of adverse effect of smoking habit on DNA strand breakage and base damage, as revealed by the alkaline comet assay. Mutat. Res., 440, 1925.[ISI][Medline]
- Zhu,C.Y. and Loft,S. (2001) Effects of brussels sprouts extracts on hydrogen peroxide-induced DNA strand breaks in human lymphocytes. Food Chem. Toxicol., 39, 11911197.[ISI][Medline]
Received February 14, 2002;
revised May 13, 2002;
accepted May 20, 2002.