DNA Repair 16 (2014) 66–73

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DNA Repair journal homepage: www.elsevier.com/locate/dnarepair

Both genetic and dietary factors underlie individual differences in DNA damage levels and DNA repair capacity Jana Slyskova a,b,∗ , Yolanda Lorenzo c , Anette Karlsen c , Monica H. Carlsen c , Vendula Novosadova d , Rune Blomhoff c,e , Pavel Vodicka a,b , Andrew R. Collins c,∗ a

Department of Molecular Biology of Cancer, Institute of Experimental Medicine ASCR, Prague, Czech Republic Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University, Prague, Czech Republic c Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway d Department of Gene Expressions, Institute of Biotechnology ASCR, Prague, Prague, Czech Republic e Division of Cancer Medicine, Surgery and Transplantation, Oslo University Hospital, Oslo, Norway b

a r t i c l e

i n f o

Article history: Received 21 November 2013 Received in revised form 21 January 2014 Accepted 28 January 2014 Available online 6 March 2014 Keywords: DNA damage DNA repair capacity Diet Genetic polymorphisms Molecular epidemiology study

a b s t r a c t The interplay between dietary habits and individual genetic make-up is assumed to influence risk of cancer, via modulation of DNA integrity. Our aim was to characterize internal and external factors that underlie inter-individual variability in DNA damage and repair and to identify dietary habits beneficial for maintaining DNA integrity. Habitual diet was estimated in 340 healthy individuals using a food frequency questionnaire and biomarkers of antioxidant status were quantified in fasting blood samples. Markers of DNA integrity were represented by DNA strand breaks, oxidized purines, oxidized pyrimidines and a sum of all three as total DNA damage. DNA repair was characterized by genetic variants and functional activities of base and nucleotide excision repair pathways. Sex, fruit-based food consumption and XPG genotype were factors significantly associated with the level of DNA damage. DNA damage was higher in women (p = 0.035). Fruit consumption was negatively associated with the number of all measured DNA lesions, and this effect was mediated mostly by ␤cryptoxanthin and ␤-tocopherol (p < 0.05). XPG 1104His homozygotes appeared more vulnerable to DNA damage accumulation (p = 0.001). Sex and individual antioxidants were also associated with DNA repair capacity; both the base and nucleotide excision repairs were lower in women and the latter increased with higher plasma levels of ascorbic acid and ␣-carotene (p < 0.05). We have determined genetic and dietary factors that modulate DNA integrity. We propose that the positive health effect of fruit intake is partially mediated via DNA damage suppression and a simultaneous increase in DNA repair capacity. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Individual risk of multifactorial diseases, including cancer, is influenced by genetic, environmental and lifestyle factors and their mutual interplay. Daily exposure to myriad nutritional substances, together with the thousands of genetic variants borne by each individual, is an example of the complex interactions that govern

Abbreviations: BER, base excision repair; Endo III-site, site sensitive to endonuclease III; FFQ, food frequency questionnaire; Fpg-site, site sensitive to formamidopyrimidine DNA glycosylase; NER, nucleotide excision repair; PBMC, peripheral blood mononuclear cell; SB, DNA strand break; SNP, single nucleotide polymorphism. ∗ Corresponding authors at: Department of Nutrition, University of Oslo, PO Box 1046, Blindern, 0316 Oslo, Norway. Tel.: +47 22 85 13 60. E-mail addresses: [email protected] (A.R. Collins), [email protected] (J. Slyskova). 1568-7864/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.dnarep.2014.01.016

our health status [1]. The health-protective effect of plant-based food is believed to be partly mediated through nutrients and phytochemicals with antioxidant properties, and expressed by removal of free radicals, inhibition of oxidative reactions through regulation of transcription, and via additional metabolic effects [2,3]. As a consequence, a phytochemical-rich diet facilitates protection of the human body against harmful oxidations of lipids, proteins and most seriously, DNA. Strand breaks (SBs) and oxidized bases are generally the most frequently occurring damage to DNA, arising as a by-product of cellular metabolism or exposure to external mutagens, and they are commonly linked to aging and cancer development [4]. These lesions are handled efficiently by base excision repair (BER), with additional help of nucleotide excision repair (NER) in eliminating some of the oxidative damage [5]. Both pathways are active irrespective of cell cycle phase and are crucial in maintaining intact DNA structure [6]. Any investigation of DNA repair is driven by the assumption that it is a multifactorial process,

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with an implicit influence on predisposition to malignant diseases. Indeed, some genetic variants and phenotypic defects of both BER and NER pathways have been linked to risk of cancer [7–9]. The investigation of low-penetrance variants (single nucleotide polymorphisms, SNPs) in association with DNA repair capacity or DNA damage, often performed on small study groups, does not usually bring consistent results, with the exception of the BER gene 8-oxoguanine DNA glycosylase, OGG1 [10–15] and the NER gene XPA [16–19]. Only recently has the regulation of human DNA repair capacity by exogenous factors, including diet, begun to be seriously studied. It was previously considered that a function as important as DNA repair was probably constitutive, or at least unlikely to be significantly affected by diet. However, the various modes of repair show extents of inter-individual variation that can hardly be solely explained by genetic polymorphisms, and at the same time experimental evidence is accumulating for modulation of both BER and NER by dietary factors [20]. Indeed, studies on monozygotic twins showed that DNA repair is a phenotype with a heritability estimate in the range of 48–75%, the rest being attributed to environmental and lifestyle factors acting by several possible mechanisms [21], such as regulation of gene expression via DNA methylation [22], modulation of RNA processing by microRNAs [23], or protein function modification [24]. Several observational studies have focused on the characterization of gene–environmental interactions in the modulation of DNA repair capacity, in which, however, the influence of diet was either not observed or not considered at all [16,25–29]. An indication that DNA repair might be affected by nutrients comes rather from in vitro and human intervention studies [20]. An even larger contribution from environmental factors (around 60%) was extrapolated in a large study of 44,788 pairs of twins, focused on the estimation of cancer risk as an endpoint [30]. An attempt to characterize some of genetic–environmental interactions in relation to cancer risk has also been made [31–33]. In the present study, we have investigated to what extent intake of vegetable and fruit, in interaction with genetic background, may affect DNA repair capacity and DNA damage levels – representing intermediate markers of cancer risk. In 340 individuals a large palette of markers of DNA integrity and its maintenance were assessed, namely (i) SBs, (ii) oxidized purines, (iii) oxidized pyrimidines, (iv) the sum of the previous three markers as total DNA damage, (v) BER capacity and (vi) NER capacity. Additionally, the participants were genotyped for SNPs in relevant BER and NER genes. Habitual food intake was estimated using a food frequency questionnaire (FFQ) and a variety of biomarkers of antioxidant status were quantified in fasting blood samples. Our aim was to characterize the inter-individual differences of DNA integrity in the general population and to identify dietary and genetic factors that underlie this variability.

2. Materials and methods

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temporarily interrupted between June and August. The study was conducted according to Declaration of Helsinki and was approved by the Regional Ethics Committee for Medical Research, Oslo, Norway. Written informed consent was obtained from all subjects.

2.2. Habitual diet and nutritional parameters Study participants received the FFQ with written instructions by mail and were asked to complete it at home. FFQs were used to estimate total intake of energy, vegetable and fruit. Intake of total fat, saturated fat, proteins, total carbohydrates, fiber and alcohol were also estimated from the FFQ as listed in Table 1. The validity of the FFQ has been demonstrated by comparison to 7 day weighed food records, plasma carotenoid concentrations and amounts of flavonoids in 24 h urine samples using the methods of triads [34].

2.3. Blood sampling and processing Peripheral blood was sampled into two BD Vacutainer® CPTTM Citrate (Becton Dickinson). One tube was left at room temperature for up to 2 h and centrifuged at 1500 × g. Plasma was collected and frozen at −80 ◦ C. The second tube was centrifuged at 1500 × g and the layer of mononuclear cells (PBMC) was removed. The PBMC were resuspended in 10 mL of ice-cold PBS. Seven milliliters of cell suspension was spun at 700 × g, and buffer A (45 mM HEPES, 0.4 M KCl, 1 mM EDTA, 0.1 mM dithiothreitol, 10% glycerol, pH 7.8) was added to the pellet in a volume of 20 ␮L for every 106 cells to prepare a cell extract for DNA repair analyses. Aliquots of 50 ␮L were frozen at −80 ◦ C. The other 3 mL of cell suspension, destined for DNA damage analysis, was spun at 700 × g and freezing medium (RPMI 1640, 20% FBS, 0.2% ATB, 10% DMSO, Sigma–Aldrich) was added in a volume of 1 mL for every 3 × 106 cells; the suspension was frozen in aliquots of 0.3 mL to −80 ◦ C inside a box of expanded polystyrene to ensure slow freezing.

2.4. Biomarkers of antioxidant status Biomarkers of antioxidant status, as listed in Table 1, were analyzed in plasma isolated from overnight fasted blood samples. For assessment of vitamin C, heparin plasma was acidified at the time of collection using meta-phosphoric acid at a final concentration of 5% and determined by HPLC [35,36]. For the determination of tocopherols, proteins were precipitated by the addition of 3 volumes of isopropanol followed by centrifugation at 3000 × g at 4 ◦ C for 15 min and the supernatant was analyzed by HPLC [37]. The carotenoids ␣-carotene, ␤-carotene, ␤-cryptoxanthin, lutein, lycopene and zeaxanthin were determined in plasma by HPLC as described elsewhere [38]. Total phenolics were analyzed by a modified version of a previously described method [39].

2.1. Study population 2.5. DNA damage The study included a representative sample of 340 Norwegians aged 18–80 years. In total, 4500 randomly selected inhabitants of the Norwegian capital and surrounding area (provided by the National Tax Office/Population Registration Office) received a written invitation to participate in the study, and 504 responded to the invitation. An interview screening was done to check if respondants were eligible to participate. Exclusion criteria were pregnancy, weight loss of more than 5 kg in the 6 months prior to the study, and participation in other research projects. Finally, 346 participants (8% of those invited) were considered to be eligible and agreed to be enrolled in the study. The collection of biological material was carried out between September 2006 and October 2007,

The levels of SBs, oxidized purines (sites sensitive to formamidopyrimidine DNA glycosylase, referred to as Fpg-sites) and oxidized pyrimidines (sites sensitive to endonuclease III, referred to as EndoIII-sites) were measured using the comet assay [40]. Comet Assay IV software (Perceptive Instruments) was used to evaluate % tail DNA of 50 comets per duplicate gel. Each experiment included negative control (NC; PBMC + buffer) and positive control (PC; Ro 19-8022-treated PBMC + Fpg enzyme) prepared using the same batch of control PBMC from a volunteer. The inter-experimental variability, expressed as coefficient of variability between experiments, was 6.7% for NC and 8.5% for PC, respectively.

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Table 1 Distribution of FFQ-estimated habitual diet and blood-measured biomarkers of antioxidant status in all study participants, and in women and men separately.

Dietary intake (in g/day) Energy (kJ/d) Total vegetablea Total fruitb Proteins Fats Saturated fats Carbohydrates Fiber Alcohol

All study subjects (N = 340)

Women (N = 180)

Median

Median

9369 234 270 92 83 28 240 28 9.1

5–95% 5713–16,034 80–598 64–709 56–157 44–156 14–53 141–430 15–55 0–35

8427 252 276 86 75 25 215 27 6.7

Men (N = 160)

p

5–95%

Median

5–95%

5302–15,233 74–674 61–708 54–149 43–144 13–45 132–395 15–60 0–27

10,589 202 251 103 94 33 270 28 11.9

6154–16,954 84–549 63–766 60–161 45–168 16–59 157–490 15–55 0–41

0.000 0.006 0.890 0.000 0.000 0.000 0.000 0.890 0.000

Clinical parameters (in ␮mol/L) Triglycerides 0.92 Cholesterol 5.07 HDL 1.59 LDL 2.96

0.47–2.22 3.5–6.9 1.0–2.5 1.6–4.6

0.79 5.06 1.76 2.8

0.44–2.02 3.46–6.92 1.09–2.79 1.39–4.39

1.07 5.08 1.41 3.10

0.47–2.61 3.46–6.79 0.9–2.07 1.67–4.72

0.002 0.956 0.000 0.006

Biomarkers of antioxidant status (in ␮mol/L) 221 Total phenolics 55 Ascorbic acid ␣-Carotene 0.1 ␤-Carotene 0.42 0.13 ␤-Cryptoxanthin 0.16 Lutein 0.59 Lycopene 0.04 Zeaxanthin 28.9 ␣-Tocopherol ␤-Tocopherol 0.38 ␥-Tocopherol 1.49 304 Uric acid

164–282 28–84 0.02–0.39 0.14–1.23 0.04–0.41 0.09–0.29 0.19–1.12 0.02–0.08 19.6–46.1 0.13–0.78 0.52–3.65 193–459

212 59 0.13 0.50 0.14 0.16 0.58 0.04 28.2 0.36 1.36 268

157–263 31–86 0.04–0.41 0.16–1.38 0.05–0.47 0.09–0.33 0.2–1.06 0.02–0.08 19.8–46.3 0.12–0.73 0.52–2.97 177–376

228 51 0.09 0.35 0.12 0.15 0.59 0.04 29.5 0.4 1.8 352

176–288 25–81 0.02–0.31 0.13–0.92 0.04–0.37 0.07–0.26 0.16–1.25 0.02–0.08 18.68–46.19 0.15–0.84 0.52–4.38 259–480

0.000 0.000 0.000 0.000 0.005 0.020 0.507 0.959 0.674 0.010 0.000 0.000

Significant differences between sexes are in bold. P-values are adjusted for age and Bonferroni’s correction for multiple comparisons is applied. a Total vegetable intake includes vegetables in mixed dishes. b Total fruit intake includes fruit juices.

2.6. DNA repair capacities

2.7. Genotyping

BER and NER activities were analyzed by an in vitro cometbased repair assay [41]. To prepare DNA substrate to study BER, PBMC were treated with 2 ␮M Ro 19-8022 (Hoffmann-La Roche) for 5 min, and irradiated on ice with a 500 W halogen lamp at 33 cm distance to induce 8-oxoguanines. For NER, PBMC were irradiated with 2 Jm−2 of UVC (20 s at 0.1 J m−2 s−1 ) to generate cyclobutane pyrimidine dimers and 6-4 photoproducts. Untreated PBMC were processed in parallel. Extracts were diluted with four volumes of buffer B (45 mM HEPES, 0.25 mM EDTA, 0.3 mg/mL BSA, 2% glycerol, pH 7.8) before use. For the NER-specific assay, 2.5 mM adenosine-5 -triphosphate was added. Thirty microliters of extract was incubated with substrate DNA in a 12-Gel Comet Assay UnitTM (Severn Biotech) for 20 min for both BER and NER. Levels of incision were evaluated by visual scoring of 50 comets per duplicate gel, on a scale of 0–400 arbitrary units. Each extract sample was incubated with untreated PBMC to control for unspecific nuclease activity of the extract (i.e. specificity control), and with Ro 19-8022- and UV-treated PBMC in parallel. The specificity control value was subtracted from the values from incubation with the treated substrates for BER and NER respectively. Each experiment included NC (untreated PBMC + buffer) and two types of PCs; Ro 19-8022-treated PBMC + Fpg enzyme subtracted by untreated PBMC + Fpg enzyme and UV-treated PBMC + T4 Endonuclease V subtracted by untreated PBMC + T4 Endonuclease V. The interexperimental variability expressed by the coefficient of variability between experiments was 13% for NC and 7.5% and 15.8% for PCs, respectively. To check for repeatibility of the assay, a sub-group of 25 randomly selected samples were re-analyzed. High correlation coefficients between both analyses were observed, being 0.75 for BER and 0.62 for NER, p < 0.001.

SNPs were selected according to their (i) localization within the genes of the pre-incision complex of BER and NER whose activity is detectable by DNA repair assays, (ii) minor allelic frequency >5% and (iii) damaging or deleterious effect on protein function predicted by SIFT or PolyPhen algorithms [42]. OGG1 Ser326Cys (rs 1052133) was analyzed as representative of the BER pathway. Selected SNPs in NER genes were XPA G23A (rs 1800975), XPC Ala499Val (rs 2228000) and Lys939Gln (rs 2228001), XPD Lys751Gln (rs 13181), XPG Asp1104His (rs 17655) and XPF Arg415Gln (rs 1800067). SNPs were detected by TaqMan® SNP Genotyping Assay based on allele-specific TaqMan® MGB probes and PCR primers and analyzed on Applied Biosystems 96-well realtime PCR instrumentation (Life Technologies). For quality control purposes, each run contained non-template control and 3 samples with known sequences, i.e. common allele homozygote, heterozygote and variant allele homozygote for each particular SNP. One in ten samples was randomly re-genotyped with 100% concordance rate (reproducibility observed). All analyzed genotypes were in agreement with Hardy–Weinberg equilibrium. 2.8. Statistical analysis Statistical analysis was performed using IBM SPSS Statistics 18 (Chicago, USA) and SAS 9.3 (NC, USA). Questionnaire-based and blood-measured parameters were not in concordance with Gaussian distribution in the study population, and so non-parametric tests were applied. The correlation analyses were performed by Spearman’s algorithm. Mann–Whitney and Kruskal–Wallis tests were used for median comparisons. All statistical tests were performed at a 5% level of statistical significance and Bonferroni‘s

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Table 2 Distribution of DNA damage and DNA repair capacities in the study population and after stratification for sex and smoking. SBs

Fpg-sites

EndoIII-sites

Total DNA damage

BER

NER

Study population N Median 5–95%

323 0.87 0.11–3.03

322 4.42 1.38–12.42

327 2.05 0.21–6.71

315 7.88 2.75–18.57

309 29.11 4.55–103.03

291 13.33 1.34–55.01

Men N Median 5–95%

147 0.64* 0.08–3.27

150 4.23 1.5–12.68

151 1.91 0.07–6.92

143 7.18 2.75–18.14

138 34.23# 5.99–105.19

133 16.81† 1.79–58.21

Women N Median 5–95%

175 0.99* 0.13–2.86

171 5.03 1.12–12.57

175 2.16 0.29–5.92

171 8.65 2.73–18.73

170 24.65# 4.05–101.49

157 11.24† 0.98–53.63

Non-smokers N Median 5–95%

260 0.89 0.1–2.88

261 4.48 1.33–12.66

264 2.11 0.11–6.53

254 8.2 2.74–18.57

248 27.03 4.99–101.24

240 13.33 1.49–52.14

Smokers N Median 5–95%

57 0.89 0.13–3.66

55 4.1 1.3–13.02

57 1.85 0.28–7.61

55 7.38 2.6–20.41

57 37.58 4.39–105.89

47 11.71 1.05–59.45

Significant differences are shown in bold, with corresponding p-values *p = 0.035, # p = 0.027 and † p = 0.004. P-values are adjusted for age, where appropriate.

correction for multiple comparisons was additionally applied. All analyses were adjusted for age and sex, where appropriate. Experimental data are expressed as median and 5–95 percentiles (5–95%).

3. Results 3.1. Biological and nutritional characteristics of the study population The mean age of the study participants was 46.3 ± 15.1 years with a range of 18–80 years, and the 180 women and 160 men had the same age distribution (46.6 ± 16.1; 18–80 and 45.8 ± 14.1; 18–80 respectively; p = 0.63). Smokers were equally distributed between sexes (17% of smokers among women, 18.6% among men; p = 0.72). BMI differed between sexes with significantly higher values in men (mean ± SD; 26.1 ± 3.6 kg/m2 ) than in women (24.1 ± 3.7 kg/m2 ), p < 0.001. Total energy intake was significantly higher in men than in women (p < 0.001) and correlated positively with BMI (R = 0.19, p < 0.001). Men consumed fewer vegetables compared with women (p = 0.006), while fruit consumption did not differ between sexes. Other differences between sexes in FFQ-estimated habitual food intakes and in levels of antioxidants measured in plasma are displayed in Table 1. FFQestimated total fruit and vegetable intake correlated positively with biomarkers of antioxidant status measured in blood (data not shown).

3.2. Variability in DNA damage and repair in the study population The values of DNA damage and DNA repair in the study population are shown in Table 2. All markers of DNA damage positively correlated with each other, as well as BER capacity correlated with NER capacity (p < 0.001). No correlation was observed between DNA damage and repair parameters. Total DNA damage increased with age in men (R = 0.28 p = 0.0008). Women showed higher levels of SBs and lower levels of BER and NER activities as compared to men (p = 0.035, p = 0.027 and p = 0.004, respectively). Smoking had no effect on either DNA damage, or DNA repair (Table 2).

3.3. DNA damage and repair in relation to genetic variability The distribution of DNA damage and DNA repair in relation to individual genotypes is presented in Table S1. XPG Asp1104His was the only SNP associated with DNA damage and subjects homozygous for the variant C allele showed significantly increased levels of SBs (p = 0.001). This association was significant in the whole group and after stratification for sex, as displayed in Fig. 1. 3.4. DNA damage and repair in relation to diet and individual antioxidants A higher consumption of fruit was associated with lower levels of SBs, Fpg-sites, Endo III-sites as well as total DNA damage in the whole study group and after the adjustment for influence of sex and age (Table 3). Women with fruit intake below the median amount (276 g/day) showed significantly higher levels of total DNA damage (9.4 (5.2–12.3) % tail DNA) than women consuming fruit above the median amount (7.7 (4.5–10.7) % tail DNA, p = 0.03). Men showed no significant difference in DNA damage after stratification for ‘low’ or ‘high’ fruit intake: median values were 7.8 (5.6–11.4) and 6.4 (4.1–10.4) % tail DNA, respectively (p = 0.1). None of the DNA damage parameters was associated with vegetable intake. The bivariate correlations between markers of DNA damage and levels of individual antioxidants are presented in Table 3. DNA damage markers negatively correlated with ␤-cryptoxanthin and ␤-tocopherol plasma levels in the whole study group. Higher consumption of fruit was also associated with higher NER capacity in the whole study group, but the relationship was of borderline significance (p = 0.066), and was not seen in men or women when they were analyzed separately. NER capacity positively correlated with the plasma levels of ascorbic acid and ␣-carotene in the whole study group (Table 3). 4. Discussion For developing surrogate diet-related endpoints for use in intervention studies, the focus should be on molecular changes that precede the appearance of clinical signs of disease, including neoplasia [1]. We assume that such molecular targets might be

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Fig. 1. Effect of Asp1104His polymorphic site in XPG on the level of SBs in all study subjects, and in women and men. The values may not correspond to those reported in Table S1 due to the lack of biomarker data for some subjects.

represented by DNA damage status and by individual DNA repair efficiency. Therefore we have conducted a comprehensive analysis of various types of DNA damage and capacity of two major DNA repair pathways in a group of 340 healthy individuals. Our data characterize the range of DNA damage and repair in a general population, and describe the impact of plant-based food and genetic variants on their levels. An excellent review by Møller reports median values of various types of DNA damage pooled from a total of 125 publications [43]. Among Norwegians in the present study, levels of all types of damage were lower than pooled medians stated in the review–SBs were 10-fold lower, Fpg-sites 2-fold lower and EndoIII-sites 5-fold lower. Quite notably, a sub-analysis of DNA damage from European countries showed a negative correlation with latitude [43]

and positive association with solar irradiance [44]. Our data are consistent with this picture; DNA damage in our study population was below the European median, but it was comparable with published data from populations in Finland and Denmark, as reviewed in [43]. However, practical differences in the performance of the comet assay or quantitation by image analysis could also contribute to the variation [45]. Concerning DNA repair, a comparison of data across studies is not as straightforward as for DNA damage. While protocols for measurement of DNA damage are to a large extent comparable between laboratories, this is not the case for DNA repair protocols. The level of repair efficiency, however, was demonstrated to be a characteristic parameter with only minor variation over time for each individual, but largely variable across the population [26,46]. Here we confirm that the range of inter-individual

Table 3 Correlations of DNA damage and DNA repair with habitual diet and with biomarkers of antioxidant status in the whole study population.

Total vegetable Total fruit Total phenolics Ascorbic acid ␣-Carotene ␤-Carotene ␤-Cryptoxanthin Lutein Lycopene Zeaxanthin ␣-Tocopherol ␤-Tocopherol ␥-Tocopherol Uric acid

R P R P R P R P R P R P R P R P R P R P R P R P R P R P

SBs

Fpg-sites

EndoIII-sites

Total DNA damage

BER

NER

−.017 .776 −.156 .013 −.053 .406 −.080 .207 −.049 .444 −.010 .876 −.212 .000 −.080 .207 .015 .813 −.053 .405 −.002 .974 −.180 .004 −.074 .243 .009 .886

−.017 .787 −.132 .035 −.026 .681 .023 .717 .010 .873 .102 .108 −.036 .565 .008 .901 .015 .170 −.002 .978 −.043 .493 −.032 .611 −.043 .499 .053 .402

−.026 .681 −.165 .008 −.063 .318 −.114 .072 .002 .970 .049 .438 −.157 .013 −.001 .990 .064 .316 −.031 .623 −.028 .657 −.082 .194 −.074 .245 −.064 .315

−.029 .649 −.194 .002 −.063 .322 −.037 .560 .002 .980 .080 .205 −.125 .048 −.006 .920 .109 .086 −.018 .779 .045 .477 −.089 .161 −.076 .228 −.011 .861

.099 .116 .051 .417 .011 .868 0.064 0.316 −.013 .843 −.013 .842 .026 .677 −.008 .895 .035 .578 .000 .990 −.033 .601 .059 .354 −.005 .932 .031 .630

.079 .207 .115 .066 .043 .500 .141 .025 .126 .047 .109 .085 .110 .080 .016 .800 −.031 .624 .057 .369 −.032 .610 .009 .886 −.009 .885 .123 .053

Table shows Spearman’s correlation coefficient (R) and p-value (P) adjusted for an influence of age and sex.

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differences between healthy individuals is substantial (coefficients of variation for BER and NER being 84% and 90%, respectively) and we describe some of the factors influencing this variability. From all investigated polymorphic sites in DNA repair genes, Asp1104His in XPG was associated with DNA damage levels. Among both sexes, carriers of two variant alleles had higher levels of SBs than carriers of none or one variant allele only. Endonuclease XPG is essential for repair of bulky adducts recognized by global NER, but also takes part in the repair of oxidative damage within transcription-coupled NER and by an interaction with the BER system [5]. Although a recent meta-analysis suggests that it is unlikely that the XPG Asp1104His polymorphism contributes to individual susceptibility to cancer risk [47], the His/His genotype was shown to be a negative prognostic factor in melanoma [48] and lung cancer [49]. Our observation shows that nucleotide change at 1104 position of both alleles might affect a biological activity of the XPG protein, while heterozygotes exert activity comparable with common allele homozygotes, thus pointing to probable haplosufficiency of the wild type allele. The same association of 1104His with level of SBs was observed before [50]. However, the level of SBs does not reflect only still unrepaired lesions but involves also transient breaks generated by endonucleases during the repair; so this observation needs to be further explained. The elevated level of SBs thus might reflect higher endonuclease activity, as well as a higher rate of break accumulation due to a lower rate of their elimination. Our results indicate that smoking is not a determinant of DNA damage or repair, unlike sex that significantly modulated levels of both parameters. Differences between sexes were observed also in the relationship between DNA damage and age; the number of lesions increased with age in men only. The relationship between age and DNA damage accumulation was reported earlier [43]. This observation follows the hypothesis that biological aging is driven by an accumulation of damage to macromolecules, including DNA, which might be accompanied by a reduction in DNA repair capacity. However, an effect of aging on DNA repair was not observed and outcomes of other studies do not support a common conclusion [51]. Women in our study group were characterized by relatively healthier food habits; their ratio of plant-based foods to total food intake was higher than in men. This effect was caused by a higher intake of vegetables. Nevertheless, women exhibited significantly higher levels of SBs and lower activities of both BER and NER. It is noteworthy that DNA damage was not influenced by vegetable intake, but was associated with fruit intake, which was equal between sexes. Individuals consuming higher amounts of fruit expressed lower DNA damage and this effect was particularly pronounced in women. After stratification of women according the ‘low’ and ‘high’ fruit intake, we observed that the high intake group had a 20% decrease of DNA damage as compared to the low intake group. Thus, levels of DNA damage among women consuming more than 276 g of fruits per day approached the (lower) levels of DNA damage characteristic for men. Among men, lower levels of damage were not further influenced by diet. We can argue that women, due to some as yet unknown biological reasons, generally bear more DNA lesions than men, but can avert this trend by “healthier” food habits. However, the positive effect of fruit consumption on DNA damage levels was seen in the whole study group, irrespectively on sex and age. Such an association was not observed between DNA repair capacities and total intake of plant-based food. At the level of individual micronutrients measured in plasma of study participants, several antioxidants exerted positive effects on DNA damage, meaning a decrease, or on DNA repair, meaning an increase. Individuals with higher plasma levels of ␤-cryptoxanthin (pro-vitamin A) and ␤-tocopherol (vitamin E) are more likely to have lower DNA damage. ␤-Cryptoxanthin is known to be a good marker of fruit intake and was reported as decreasing DNA damage

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in in vitro models and human intervention studies [52,53]. We further performed a quartile analysis and observed that a plasma level of ␤-cryptoxanthin above 0.13 ␮mol/L significantly decreased the level of DNA damage, by almost 20% (p = 0.03; data not shown). Bringing this value into the context, a large European multicenter study on 3089 individuals reported the mean value of plasma ␤cryptoxanthin as being 0.29 ␮mol/L [54]. It is suggested by us that a level not higher than the general population average may be beneficial in decreasing DNA damage. It is usually ␣- and ␥-tocopherols that are attributed to beneficial biological activities of vitamin E and there is a lack of studies focused on the ␤ isomer [55]. Here, we provide evidence that ␤-tocopherol may also be considered a potent form of vitamin E, having an impact on genome integrity. Tocopherols are most commonly found in nuts and vegetable oil, and carotenoids are contained in yellow or orange fruits and vegetables, and these might be referred to as a “functional” foods in this context. Although the relationship between DNA repair and total fruit or vegetable intake was not detected, we could observe that NER capacity, in particular, was significantly increased with increased plasma levels of ascorbic acid and ␣-carotene. The supporting evidence for NER being induced by diet is very scarce in the literature, since the majority of studies investigating the influence of micronutrients on DNA repair capacity were focused predominantly on the BER pathway [20]. Our study thus sheds new light on the modulation of NER by vitamin C and pro-vitamin A and suggests that a positive health effect of antioxidant-rich food might be mediated also via induction of a DNA damage response. 5. Conclusions We have determined genetic and dietary factors that modulate DNA integrity. Higher intake of plant-rich food is associated with a lower risk of cancer. We propose that this positive influence is in part driven by a stimulation of the DNA repair capacity with an associated reduction in DNA damage. Our results support the notion that DNA damage and repair are of multifactorial and complex nature, with many factors underlying their variability. By identifying the impact of some of them, we have however explained only a small percentage of this variability. Thus, our data indicate that many more factors determine the large inter-individual differences in DNA damage and repair. Conflicts of interest The authors declare no conflicts of interest. Acknowledgement This project was supported by CZ:GACR:GAP 304/12/1585. Ro 19-8022 was supplied by Hoffmann-La Roche. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.dnarep. 2014.01.016. References [1] J.C. Mathers, J.E. Hesketh, The biological revolution: understanding the impact of SNPs on diet–cancer interrelationships, J. Nutr. 137 (2007) 253S–258S. [2] S.K. Bohn, M.C. Myhrstad, M. Thoresen, M. Holden, A. Karlsen, S.H. Tunheim, I. Erlund, M. Svendsen, I. Seljeflot, J.O. Moskaug, A.K. Duttaroy, P. Laake, H. Arnesen, S. Tonstad, A. Collins, C.A. Drevon, R. Blomhoff, Blood cell gene expression associated with cellular stress defense is modulated by antioxidant-rich food

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