Oxidative DNA damage in human lymphocytes: correlations with plasma levels of
-tocopherol and carotenoids
Franca Bianchini4,
Sölve Elmståhl1,
Carmen Martinez-Garciá2,
Anne-Linda van Kappel,
Thierry Douki3,
Jean Cadet3,
Hiroshi Ohshima,
Elio Riboli and
Rudolf Kaaks
International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon Cedex 08, France,
1 Department of Community Medicine, Malmö University Hospital, SE-20502 Malmö, Sweden,
2 Escuela Andaluza de Salud Publica, Ap. Correos 2070, E-18080 Granada, Spain and
3 DRFMC/SCIB, Laboratoire des Lésions des Acides Nucléiques, CEA Grenoble, 17 Rue des Martyrs, F38054 Grenoble Cedex 9, France
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Abstract
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In order to investigate whether oxidative damage is associated with differences in antioxidant intake, we measured the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in lymphocytes and
-tocopherol and several carotenoids in plasma of women with different dietary habits. We found that women from Granada (Spain), a region with a typically Mediterranean diet, had significantly higher levels of 8-oxodGuo compared with Malmö (Sweden), a region with a Northern European dietary intake pattern (2.30 ± 0.78 versus 1.59 ± 1.01 8-oxodGuo/106 deoxyguanosine). Levels of plasma
-tocopherol and carotenoids were higher in Granada and these values were significantly positively correlated with levels of 8-oxodGuo. Our results do not support the hypothesis that a Mediterranean diet rich in
-tocopherol and carotenoids protects cells against oxidative DNA damage. It is possible, however, that consumption of foods other than fruits and vegetables, including fats, are responsible for the higher levels of 8-oxodGuo in Granada. Further studies are warranted to better elucidate the role of antioxidants in the modulation of oxidative stress in vivo.
Abbreviations: dG, deoxyguanosine; GC/MS, gas chromatography/mass spectrometry; 5-HMdUrd, 5-hydroxymethyl-2'-deoxyuridine; 8-oxodA, 8-oxo-7,8-dihydro-2'-deoxyadenosine; 8-oxodGuo, 8-oxo-7,8-dihydro-2'-deoxyguanosine.
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Introduction
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It is now widely accepted that a large proportion of cancer incidence world wide may be due to diet and nutrition-related lifestyle factors (1,2), but there are still many uncertainties regarding the type of biological mechanisms involved. One of the most consistent observations from epidemiological studies is an inverse association between fruit and vegetable consumption and cancers of the digestive, respiratory and urinary tracts (3,4). Similar inverse associations have been found between the incidence rates of these various cancers and estimated intake levels as well as plasma concentrations of vitamin C and ß-carotene, of which fruit and vegetables are the main sources (5,6). Due to the in vitro antioxidant properties of these vitamins, it has been suggested that oxidative stress may play an important role in carcinogenesis and antioxidants present in fruit and vegetables may be beneficial by counteracting the formation or the effects of highly reactive oxygen species. Unfortunately, some chemopreventive trials with antioxidant vitamins and mineral supplements showed, contrary to expectation, an increase in cancer risk in supplemented subjects (7,8). A better understanding of the role of antioxidants in cancer prevention could be obtained with prospective cohort studies in which cancer risk and dietary intake patterns will be related to biologically meaningful markers of oxidative damage as potential intermediate end-points. In this context we conducted a study (EPOX) as part of the European Prospective Investigation into Cancer and Nutrition (EPIC), a large multi-centre prospective cohort study on diet, lifestyle and cancer (9). Our aim was to investigate whether oxidative damage to DNA in lymphocytes was associated with differences in antioxidant levels. 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a commonly used biomarker of oxidative damage to DNA, was measured in human lymphocytes and
-tocopherol and several different carotenoids were analysed in plasma.
Premenopausal non-smoking women aged 4550 were recruited in Malmö (Sweden) and Granada (Spain), regions with a typically Northern European and a typically Mediterranean dietary intake pattern, respectively. Women did not regularly use vitamin supplements and were not affected by diabetes, chronic hypertension or chronic inflammatory diseases. Fasting blood samples were collected, using heparin as an anticoagulant, and used for the preparation of plasma by centrifugation and of lymphocytes by centrifugation on a Ficoll gradient (Lymphoprep tubes; Nycomed Pharma AS, Oslo, Norway). Samples were stored at 80°C until laboratory analysis.
8-OxodGuo was measured by HPLC with electrochemical detection, following DNA extraction, RNase treatment and enzymatic hydrolysis. It is now widely accepted that an overestimation of 8-oxodGuo in biological samples occurs when gas chromatography/mass spectrometry (GC/MS) and 32P-post-labelling are employed (10,11). Measurement by HPLC with electrochemical detection can also, however, give very high values because of the artefactual formation of 8-oxodGuo during DNA processing (12). Particular care, therefore, had been taken in our study to avoid auto-oxidation of guanine. To this end we added 0.1 mM deferoxamine as a chelating agent to all buffers used, we worked on ice and centrifuged samples at 4°C. DNA was extracted by a high salt protein precipitation method. Briefly, lymphocytes were lysed with SDS and digested with protease (Qiagen, Courtaboeuf, France) at 37°C for 1 h. Proteins were precipitated by adding NaCl to 1.5 M and DNA in the supernatant was collected by addition of 2 vol ethanol. The DNA pellet was resuspended in TrisEDTA, incubated with RNases A and T1 at 37°C for 1 h and precipitated again with ethanol. Enzymatic digestion was then performed using nuclease P1 (Boehringer Mannheim, Meylan, France) at 37°C for 2 h and alkaline phosphatase (Boehringer Mannheim) at 37°C for 1 h. Enzymes were precipitated by addition of CHCl3 and the upper layer stored for analysis of 8-oxodGuo at 80°C under N2. The DNA hydrolysate was analysed by HPLC with an electrochemical detector (Coulochem I; ESA Inc., Chelmsford, MA) using a C18 250x46 mm 5 µm Uptishere column (Interchim, Montlucion, France) equipped with a C18 guard column. The eluent was 50 mM ammonium acetate, pH 5.5, containing 9% methanol, at a flow rate of 0.7 ml/min. The potentials applied were 150 mV and 400 mV for E1 and E2, respectively. The retention time of 8-oxodGuo was ~23 min. Deoxyguanosine (dG) was measured in the same run of corresponding 8-oxodGuo with a UV detector (model SPD-2A; Shimadzu, Kyoto, Japan) at 256 nm; the retention time was ~17 min.
With the aim of obtaining a better assessment of oxidative damage, we also attempted to measure two other DNA oxidative modifications, 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA) and 5-hydroxymethyl-2'-deoxyuridine (5-HMdUrd). For this purpose, fractions corresponding to 8-oxodA and 5-HMdUrd were collected consecutive to the HPLC injections for the determination of 8-oxodGuo, dried under vacuum and analysed by HPLC with electrochemical detection and by GC/MS, respectively. Unfortunately, the presence of interfering peaks did not allow quantification of 8-oxodA and 5-HMdUrd in our samples.
Plasma antioxidants were analysed by HPLC with a diode array detector after extraction with hexane, as previously described (13).
The characteristics of subjects are reported in Table I
. No significant difference between the two groups was found in age, height, weight and Quetelet index. The laboratory analyses were performed each day on the same number of samples from Malmö and Granada, blind and in random order. Levels of 8-oxodGuo ranged from 0.41 to 4.40 per 106 dG (Figure 1
). These values are relatively low and of the same order of magnitude as, or even below, most of the data recently reported using the same method of detection (1418). This suggests that the procedure used for sample processing avoids artefactual formation of 8-oxodGuo. Interindividual variation was relatively high, ~10-fold, and this is also consistent with reported data (14,17,19). The mean values were 1.59 ± 1.01 (n = 24) and 2.30 ± 0.78 8-oxodGuo per 106 dG (n = 28) in Malmö and Granada, respectively (Figure 1
). This difference was statistically significant by Wilcoxon's score test (P < 0.01). The protocol used for isolation of lymphocytes was the same in the two centres, although subtle variations in applying the method cannot be completely excluded. DNA extraction, hydrolysis and HPLC analyses were performed in the same laboratory. It seems that artefactual formation of 8-oxodGuo is more related to DNA processing than to lymphocyte preparation, so differences in 8-oxodGuo between Malmö and Granada are unlikely to be due to small differences in the isolation of lymphocytes.

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Fig. 1. Distribution and mean level (± SD) of lymphocyte 8-oxodGuo in women from Malmö and Granada (P < 0.01 by Wilcoxon's score test).
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Levels of
-tocopherol and several carotenoids are reported in Table II
.
-Tocopherol ranged from 19.76 to 46.95 µmol/l, while values of total carotenoids ranged from 0.84 to 6.15 µmol/l, with ß-carotene, lutein and lycopene being the most prevalent.
-Tocopherol, ß-cryptoxanthin, lycopene, zeaxanthin and total carotenoids were significantly higher in Granada compared with Malmö. Lymphocyte 8-oxodGuo and plasma concentrations of the main antioxidants were significantly positively correlated by Spearman's correlation coefficient, either before or after adjustment for Quetelet index (Table III
). Similar positive correlation coefficients were found within each centre separately, although they did not always remain significantly different from 0, especially for Malmö. Figure 2
shows the distribution of the individual values of lymphocyte 8-oxodGuo in relation to the concentration of lutein and lycopene, as well as the linear relationships in the two centres and in Malmö and Granada separately.
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Table III. Spearman's correlation coefficients, adjusted by Quetelet index, between 8-oxodGuo in lymphocytes and -tocopherol and carotenoids in plasma
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Fig. 2. Linear relationships between lymphocyte 8-oxodGuo with plasma lutein (a) and lycopene (b) in the two centres and within Malmö and Granada separately.
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This study represents one of the few attempts up to now to measure the levels of 8-oxodGuo in normal populations with different dietary habits. Women in Granada have a significantly higher mean value of 8-oxodGuo than women in Malmö. This seems to disagree with the hypothesis that a Mediterranean diet, generally rich in antioxidants from fruit, vegetables and vegetable oils, would be protective against oxidative damage. This is striking especially because our data also show higher levels of
-tocopherol and carotenoids in Granada, which have been positively correlated with fruit and vegetable intake (van Kappel et al., manuscript submitted). In addition, the intraindividual level analysis demonstrates a positive association between the levels of 8-oxodGuo and the plasma concentrations of
-tocopherol and several carotenoids, also within the two study centres separately. Consumption of foods other than fruit and vegetables could be responsible for the higher level of 8-oxodGuo in women in Granada and for the positive correlation between levels of antioxidants with oxidative DNA damage. A high fat diet has been shown to increase the level of 5-hydroxymethyluracil, another DNA oxidative modification, in human white blood cells (20). It seems that in Granada high vegetable consumption is accompanied by high fat consumption, as vegetables are generally dressed or fried with oil. The level of fat and the type of fatty acids consumed could be one of the factors determining the higher levels of 8-oxodGuo in Granada.
Measurements of oxidative damage in different European populations have been reported previously (15), but differences in 8-oxodGuo levels between countries were evident only for men and the number of subjects in the study was very low. Different characteristics of the population and the activity of antioxidant or metabolizing enzymes could explain different results. For example, carotenoids have been shown to modulate the effects of smoking and drinking on hepatocellular carcinoma only in GST M1 null subjects (21). Short intervention trials with carotenoids and other antioxidants or vegetable extracts have been conducted in humans, but a protective effect on oxidative damage has not always been demonstrated. Supplementation with ß-carotene did not affect the urinary excretion of 8-oxodGuo (22), and oxidized DNA purines and pyrimidines in lymphocytes, measured with the Comet assay as FPG and endonuclease III sites, respectively, did not change after supplementation with
- or ß-carotene, lutein or lycopene (15,23). Supplementation with vitamin C,
-tocopherol and ß-carotene (24) or carrot juice (25) decreased pyrimidine oxidation, while supplementation with ß-carotene, vitamin C,
-tocopherol, zinc, selenium and copper decreased lymphocyte levels of 8-oxodGuo (26). Correlations between oxidative DNA damage and plasma levels of antioxidants have been measured before, with conflicting results. Levels of serum carotenoids were negatively correlated with oxidized pyrimidines but not with FPG sites, except for lutein in the lutein-supplemented group, in a Spanish population (23). As FPG is the major enzyme repairing 8-oxodGuo, these results do not necessarily disagree with our findings. In addition, no correlation was observed between plasma levels of carotenoids and 8-oxodGuo in lymphocytes from several European populations (15) and total antioxidant capacity (27) or between the average intake of vitamin E and ß-carotene and urinary excretion of 8-oxodGuo (28).
Carotenoids have been shown to exert antioxidant properties in vitro and to have some protective role in tumor promotion and progression (29), but there is little direct evidence that they protect biological structures against free radicals in vivo. On the contrary, it has recently been demonstrated that administration of ß-carotene greatly increases several CYP450 isoforms in rat lung and this increase is positively associated with the overgeneration of superoxide (30). The question of whether carotenoids are significant antioxidants in plasma is difficult to answer. Carotenoids are easily degraded and undergo auto-oxidation. The antioxidant effect of ß-carotene depends on oxygen pressure as a result of competition between two reactions, one producing a chain terminator peroxyl radical and the other producing a chain propagator carotenyl radical in the absence or presence of oxygen, respectively (31,32). It has also been shown that lycopene and ß-carotene protect cells against oxidatively induced DNA damage (as measured by the Comet assay) only at relatively low concentrations, but increase the extent of damage at higher concentrations (33). It is also possible that carotenoids exert an antioxidant effect when other antioxidants, including vitamin C, are low.
In conclusion, our results do not support the hypothesis that a Mediterranean diet protects against oxidative DNA damage or that such protection could be inferred by an increase in plasma concentrations of carotenoids and
-tocopherol. However, the question can be raised as to whether levels of antioxidants in plasma can really modulate levels of 8-oxodGuo in lymphocytes and also whether antioxidants in plasma and 8-oxodGuo in lymphocytes are representative of the situation in the target tissues. Further studies in other populations using other biomarkers of oxidative damage are necessary to confirm our results and to understand the role of antioxidants in the modulation of oxidative stress.
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Acknowledgments
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We wish to thank Carine Biessy, Béatrice Vozar and David Achaintre for technical assistance. This study was supported by the World Cancer Research Fund (grant no. 97A55) and the Europe against Cancer Programme of the European Commission.
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Notes
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4 To whom correspondence should be addressed Email: bianchini{at}iarc.fr

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Received August 10, 1999;
revised October 28, 1999;
accepted October 28, 1999.