Mutagenesis Advance Access originally published online on May 21, 2007
Mutagenesis 2007 22(4):281-285; doi:10.1093/mutage/gem014
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Intake of fruits and vegetables and polymorphisms in DNA repair genes in bladder cancer
1Unità di Epidemiologia dei Tumori, Dipartimento di Scienze Biomediche e Oncologia Umana, Università di Torino and CPO-Piemonte, Torino, Italy 2ISI Foundation, viale Settimio Severo 65, Torino 10100 Italy 3Dipartmento di Genetica, Biologia e Biochimica, University of Torino, Italy 4University of Dartmouth, New Hampshire, USA 5Divisione di Urologia 2 6Divisione di Urologia 1 7Divisione di Urologia 3, Ospedale S. Giovanni Battista, Torino 8Imperial College London, UK
The objective is to investigate the relationships between fruit and vegetable intake, DNA repair gene polymorphisms and the risk of bladder cancer. We have analyzed a hospital-based case–control study of 266 individuals with incident, histologically confirmed bladder cancer diagnosed between 1994 and 2003. Controls (n = 193) were patients treated for benign diseases recruited daily in a random fashion from the same hospital as the cases. All cases and controls were interviewed face-to-face for major risk factors, along fruit and vegetable consumption. Odds ratios (ORs) for fruit and vegetable intake and DNA repair gene polymorphisms were adjusted for age and smoking status, using unconditional logistic regression. A statistically significant decreased risk was observed for fruit and vegetable intake above median (versus below the median) [unadjusted OR 0.61, confidence interval (CI) 95% 0.50–0.96 and OR 0.54, CI 95% 0.39–0.80, respectively]; the decreased risk persisted after adjustment for age and cigarette smoking (OR 0.73, CI 95% 0.49–1.01 and OR 0.86, CI 95% 0.56–1.08, respectively). The fruits and vegetables associated with decreased risks included leafy green vegetables, cruciferous vegetables, apples and citrus fruits. We did not find any interactions between DNA repair gene polymorphisms and fruit and vegetable intake. This study found a reduced risk associated with fruit and vegetable intake. No interaction was observed between fruit and vegetable consumption and DNA repair gene polymorphisms.
 |
Introduction |
|---|
 Top Introduction Materials and methodsResultsDiscussionReferences |
|---|
Bladder cancer is the most common cancer of the urinary tract and is the ninth most common cancer among men, accounting for
330 000 new cases per year in the world (1
). The incidence of bladder cancer varies considerably among countries (2); in general, the highest incidence rates for bladder cancer are in Western Europe, North America and Australia. Bladder cancer incidence increases with age, occurring rarely before the age of 40 years. Since the developed countries' population is ageing, the burden of bladder cancer will increase in the next decades. Therefore, bladder cancer prevention is a high-priority public health issue.
Some important causes of bladder cancer, such as cigarette smoking and occupational exposures, have been known for a long time (3). At present, the evidence on the role of dietary factors is still controversial. As for other cancers, it has been suggested that dietary fruits and vegetables may play a protective role (4).
A meta-analysis on diet and bladder cancer (5) (based on seven case–control studies and three cohort studies) concluded that total fruit consumption is probably associated with a small decrease of the risk [smoking adjusted relative risk 0.53, confidence interval (CI) 0.26–0.75, subjects with high fruit consumption versus subjects with low fruit consumption]. However, no association was found for total vegetable intake. With respect to the types of fruits and vegetables consumed, studies have yielded inconsistent results. Yet, at least one cohort study and one case–control study reported reduced risks for a high intake of cruciferous vegetables, with relative risks of 0.49 (CI 95% 0.32–0.72) and 0.79 (0.62–1.01) (7), respectively.
Different mechanisms have been proposed to account for the potential protective effect of fruits and vegetables on several types of cancer, including their antioxidant properties. We hypothesized that individuals with higher exposures to carcinogens, with lower consumption of dietary protective compounds, such as fruits and vegetables, and having DNA repair genotypes with decreased activity could be at higher risk of developing cancer.
Environmental and occupational chemical carcinogens, such as polycyclic aromatic hydrocarbons, aromatic amines and N-nitroso compounds, form DNA adducts repaired primarily through the nucleotide excision repair pathway [e.g. xeroderma pigmentosum-D (XPD) gene and excision repair complementing defective in Chinese hamster, group 1 (ERCC1)]. These agents can produce interstrand cross-links repaired by genes involved in both nucleotide excision repair [e.g. ERCC1 and ERCC4] and homologous recombinational repair [e.g. X-ray repair cross-complementing group 2–3 (XRCC2–3)] pathways. Reactive oxygen species also can induce base damage, abasic sites, single-strand breaks and double-strand breaks: Single-strand breaks are repaired through the base excision repair pathway [e.g. XRCC1 and proliferating cell nuclear antigen (PCNA)], whereas double-strand breaks are corrected by either homologous recombination (e.g. XRCC2–3) or non-homologous end-joining pathways. Hundreds of polymorphisms in DNA repair genes have been identified; however, the effect on repair phenotype and cancer susceptibility remains uncertain for many of these [see also dbDNP database: http://www.ncbi.nlm.nih.gov/SNP/; (1–3)]. We investigated genetic polymorphisms that could be relevant to the bladder carcinogenetic process particularly in individuals with higher exposure and lower consumption of dietary protective compounds.
|
Materials and methods |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Subjects
We conducted a hospital-based case–control investigation at three urology departments of S. Giovanni Battista hospital in Turin, which treat roughly half of the newly diagnosed bladder cancers in the Turin metropolitan area. The case group comprised men, aged 40–75 years, residing in the Turin metropolitan area with newly diagnosed, histologically confirmed bladder cancer treated from 1994 to 2003. Cases were identified by daily contact between a trained interviewer and the urology departments followed by histological confirmation from the pathology departments. Controls were recruited daily in a random fashion (i) from patients treated at the same urology departments for non-neoplastic diseases, mainly prostatic hyperplasia and cystitis (all newly diagnosed), and (ii) from patients treated at the medical and surgical departments for hernias, vasculopathies, diabetes, heart failure, asthma or other benign diseases (none was represented in >10% of controls). Patients with cancer, liver or renal diseases and smoking-related conditions were excluded. As with cases, controls were men aged 40–75 years and living in the Turin metropolitan area.
Before treatment, a trained interviewer used a standard questionnaire to conduct a face-to-face interview with cases and controls on their history of tobacco smoking (including brands and tobacco type), occupational history and a 24-h dietary and medication use recall. Following informed consent, and prior to treatment, blood was collected from cases and controls.
Additionally, we use a 22-item food frequency questionnaire (FFQ) to assess fruit and vegetable consumption. The questionnaire asked for the average number of servings of fruits and vegetables consumed each day, week or month of a specified portion size (e.g. one half dish, a cup, one fruit). To examine food groups, such as total intake of fruits and vegetables, frequency of consumption we summed across all foods belonging to that group. This questionnaire was a simplified version of the European Investigation into Cancer and Nutrition (EPIC) questionnaire that was validated in our population (8). The average Italian servings of endpoint food items have been estimated by the EPIC 24-h dietary recall questionnaire.
Out of the 741 interviewed subjects, DNA was available from 317 cases and 317 controls. However, genotype data do not sum up to the total for all the analyzed polymorphisms due to different assay efficiencies and in some cases exhaustion of DNA samples during the course of the study. The FFQ, introduced only in recent phases of the study, was available for 266 cases and 193 controls.
DNA repair gene polymorphisms.
White blood cell (WBC) DNA was isolated and purified from stored buffy coat samples by enzymatic digestion of RNA and proteins, followed by phenol–chloroform extraction for the first half of the collected samples (9) and by salting-out procedure for the second half of the study (10).
We used a variety of genotyping techniques, using the most efficient (i.e. cost effective) approaches for a given single nucleotide polymorphism (SNP). For some SNPs, we genotyped half the sample with a technique and the other half with a different technique because during the years we had the opportunity to access new techniques and set up new methodologies, allowing us also to maximize the DNA use.
PCR–RFLP analysis.
Polymerase chain reaction (PCR) followed by enzymatic digestion was used for genotyping of the XRCC1-28152 (Arg399Gln, 28152G>A), XPD-35931 (Lys751Gln, 35931 A>C) and XRCC3-18067 (Thr241Met, 18067 C>T) polymorphisms on the first half of the study. For details on primers, PCR conditions and other technical aspects, see Matullo et al. (11).
Primer extension/denaturing high-performance liquid chromatography.
Five polymorphisms, i.e. PCNA-6084 (3'-Untranslated, 6084 G>C), XRCC1-26304 (Arg194Trp, 26304 C>T), XRCC1-26651 (Pro206Pro, 26651 A>G), XRCC2-31479 (Arg188His, 31479 G>A) and XRCC3-17893 (IVS6– 14, 17893 A>G) have been analyzed by using the primer extension technique (12) on a denaturing high-performance liquid chromatography (DHPLC) instrument (13) by Varian, Inc. (Walnut Creek, CA, USA) on the first half of the study, and for XRCC3-4541 (5'-UTR, 4541 T>C) on the overall sample. The primer extension technique is based on specific incorporation of the complementary dideoxynucleotide into the base substitution; the rate of correct incorporation completely overwhelms dideoxynucleotide misincorporations, and heterozygotes are easily detectable after separation of the extended primers through a DHPLC column.
5' Nuclease assay (TaqMan). The 5' nuclease assay with fluorogenic Minor Groove Binder probes was used to genotype seven of the polymorphisms (XPD-35931, PCNA-6084, XRCC1-26304, XRCC1-26651, XRCC1-28152, XRCC3-17893 and XRCC3-18067) in the second half of the study, and two polymorphisms (XPD-23591 Asp751Asn, 23591 G>A, and ERCC1-19007 Asn118Asn, 19007 T>C) in the overall sample.
Primer extension/sequencing. A multiplex reaction was performed using a primer extension reaction followed by fragment analysis (SNaPshot technique, Applied Biosystems, Foster City, CA) on an automated sequencer ABI PRISM 310 (Applied Biosystems). Two polymorphisms in the ERCC4 gene were amplified in the same PCR fragment: ERCC4-30028 (Ser835Ser, 30028 T>C) and ERCC4-30147 (Glu875Gly, 30147 A>G); in this analysis, the following extension primers were used: ERCC4-30028 5'-GGCCATTACAGCAGATTC-3', ERCC4-30147 5'-ATTAGCAGCCCTGCACAAGACGA-3'.
DNA typing quality control
Methodological validation included a comparison between the PCR–Restriction Fragment Length Polymorphism, DHPLC and TaqMan assays. Moreover, at least 10% of the genotyping was randomly repeated for each polymorphism. Concordance was in the range between 99 and 100% for all comparisons.
Statistical analyses
Odds ratios (ORs) and the corresponding 95% CIs were computed by unconditional logistic regression methods, and adjusted for age, smoking status (current, former, never) and maximum number of smoked cigarettes. In addition, we analyzed DNA repair genotypes stratified by intake of fruits and vegetables (above/below median value). All analyses were performed using the SAS V8 package for personal computers (SAS, Inc. Version 8.2, Cary, NC).
|
Results |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
We analyzed fruit and vegetable consumption and DNA repair gene polymorphisms for 266 male bladder cancer patients and 193 male hospital controls. Table I shows the distribution of selected variables by case–control status. As expected, smoking was a strong risk factor in bladder cancer with an increasing trend in risk for ex-smokers and current smokers. Higher (i.e. above the median) vegetable and fruit consumption was associated with a reduced bladder cancer risk. The unadjusted ORs for above-median consumption of fruits and vegetables were statistically significant (OR 0.61, CI 95% 0.50–0.96 and OR 0.54, CI 95% 0.39–0.80). However, after adjustment for age, smoking status and smoking behavior in the unconditional logistic regression model, the OR remained below one but was no longer statistically significant (OR 0.73, CI 95% 0.49–1.01 and OR 0.86, CI 95% 0.56–1.08).
|
The ORs (adjusted for age and smoking) for specific vegetables and fruits are shown in Table II. Reduced ORs were observed for above-median intake of leafy green vegetables (spinach, lettuce, chard) (OR 0.76, CI 95% 0.49–1.16), cruciferous vegetables (broccoli, cabbage, cauliflower, brussels sprouts) (OR 0.71, CI 95% 0.40–1.17), apples (OR 0.63, CI 95% 0.39–0.99) and citrus fruits (oranges, tangerines, grapefruit) (OR 0.92, CI 95% 0.59–1.14). In a combined analysis of cigarette smoking and fruit and vegetable consumption, we found a significantly increased risk of bladder cancer among ‘current smokers’ with fruit and vegetable intake below the median (Table III). The risk of bladder cancer among ‘current smokers’ was reduced among individuals with fruit and vegetable consumption above the median. Lastly, the association with fruit and vegetable intake was absent among ‘never smokers’ (P-value of interaction =0.09).
|
|
No interactions were found between fruit and vegetable consumption and DNA repair gene polymorphisms (Table IV). The allele frequencies of the analyzed DNA repair SNPs were comparable to those of other Northern Italian populations, but were not directly comparable to those of other European populations (13
).
|
|
Discussion |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In agreement with previous studies, our study supports a protective effect of fruit and vegetable intake on bladder cancer development (5
). There are several potential mechanisms by which fruits and vegetables could influence bladder cancer risk. These include (i) increased antioxidant activities, (ii) promotion of cell differentiation, (iii) increased activity of detoxifying enzymes, (iv) blocked formation of nitrosamines, (v) effects on DNA methylation, (vi) maintenance of normal DNA repair, (vii) increased apoptosis of cancer cells and (viii) decreased cell proliferation.
Emerging data indicate potential anticarcinogenic effects of specific micronutrients in fruits and vegetables. For instance, some studies have suggested that flavonoids, a class of phytochemicals ubiquitous present in fruits and vegetables, modulate the expression of the CYP family of genes involved in carcinogen activation and detoxication (5,14). Further, carotenoids have shown a wide range of effects in in vitro systems, including enhancement of DNA strand break repair in mouse lymphocytes following 
-ray irradiation, reduced proliferation of aortic smooth cells and suppression of N-myc RNA synthesis in human neuroblastoma cell lines arrested in G0 (14). Antioxidant properties of vitamins C, E and ß-carotene are hypothesized to result from scavenging free radicals, and thus breaking the oxidative chain reaction that can lead to cellular and DNA damage (16). In vivo intervention studies, based on dietary supplements, only rarely find a clear association between intake of a specific nutrient and a biological effect. This may be due to the different doses used in in vitro tests and in vivo dietary interventions. Moreover, in vivo effects may be exerted by the combination of nutrients in food, rather than a single component.
DNA repair capacity can be assessed by mRNA or protein levels or by enzyme activity, where mRNA levels probably reflect an altered cellular state of enhanced DNA repair gene transcription consequent to oxidative stress. At least three studies have found no change in OGG1 mRNA expression after ingestion of kiwi fruit (17), fruits and vegetables (18) or oral supplements containing vitamins C and E (19). Each of these was a long-term study. The short-term effects of antioxidants could include transient upregulation of OGG1 mRNA expression, although this has not been studied as yet. Despite unaltered OGG1 mRNA expression, the repair activity of 8-oxodG lesions appears to be increased by kiwi fruit ingestion (17). Also supplementation of 100 mg/day of coenzyme Q10 for 1 week increased 8-oxodG DNA repair activity. Of interest, dietary supplementation with cooked carrots increased 8-oxodG repair activity in WBCs, whereas the similar amount of 
-carotene and ß-carotene as oral supplements had no effect (20). This supports the hypothesis that whole food products rather than single antioxidants may have beneficial chemopreventive effect.
We previously reported findings of a relationship between fruit and vegetable consumption and DNA-adduct formation in a subsample of this case–control study on bladder cancer (9). The level of WBC DNA adducts decreased with higher levels of fruit and vegetable consumption; additionally, the association between the case–control status and level of adduct formation (below or above the median value) was stronger in subjects who consumed fewer than two portions of vegetables per day (OR 7.80, CI 95% 3.0–20.3) than in those who consumed two or more or three or more portions a day (OR 4.98 for consumers of two portions/day; OR 2.0 for consumers of three or more portions/day).
In the present study, we found a statistically significant reduction of bladder cancer risk among subjects whose vegetable/fruit intake was above the median (versus below the median) for the following items: leafy green vegetables, cruciferous vegetables, tomatoes and citrus fruit. However, we did not find any interaction between fruit and vegetable consumption and DNA repair gene polymorphisms, although we previously did detect a possible role of DNA repair gene polymorphisms in bladder cancer (11,22). We had hypothesized that the protective effects of fruits and vegetables might be greater in subjects with poorer DNA repair capacity, particularly if they were exposed to environmental or endogenous carcinogens. The lack of support for this hypothesis in our study suggests that impairment of the DNA repair response may be less sensitive to DNA damage modulation by dietary antioxidant compounds, or that the DNA repair gene polymorphisms we have considered are not strongly involved in bladder carcinogenesis. Thus, the investigation of more SNPs and more genes might be useful and possibly exploiting a linkage disequilibrium approach by selecting haplotype-tagging SNPs that cover a whole gene region, particularly in the absence of sufficient functional data on the SNPs analyzed.
An important limitation of this study is the lack of an experimental design (i.e. randomized controlled trial). However, intervention studies with continuous ingestion of antioxidants have shown mixed results with respect to effects on oxidative DNA damage in WBCs, possibly due to problems with design, statistical power and period effects (23). In future studies, care should be taken to consider individual differences in effects, i.e. due to gender, genetic variation in defense enzymes as well as DNA repair capacity.
|
Acknowledgments |
|---|
This paper was made possible by a grant from the Compagnia di San Paolo (Turin, Italy; P.V.) and of the Associazione Italiana per le Ricerche sul Cancro (G.M.). P.V. and G.M. are partially funded by Environmental Cancer Risk Nutrition and Individual Suspectibility, a network of excellence operating within the European Union sixth Framework Program, Priority 5: ‘Food Quality and Safety’ (Contract No. 513943).
|
Notes |
|---|
* To whom correspondence should be addressed. Tel: +39 011 6603555; Fax: +39 011 2365601; Email: giuseppe.matullo{at}unito.it
|
References |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
1. Steward BW, Leinhaus KP. World Cancer Report (2003) Lyon: WHO-IARC.
2. Schottenfeld D, Fraumeni JF. Cancer Epidemiology and Prevention (1996) New York: Oxford University Press.
3. Zeegers PA, Kellen E, Buntinx F, Van den Brandt P. The association between smoking, beverage consumption, diet and bladder cancer: a systematic literature review. World J. Urol. (2004) 21:392–401.[CrossRef][Web of Science][Medline]
4. IARC Handbooks of Cancer Prevention. Fruits and Vegetables (2003) Lyon, France: International Agency for Research on Cancer World Health Organization.
5. Steinmaus CM, Nunez S, Smith AH. Diet and bladder cancer: a meta-analysis of six dietary variables. Am. J. Epidemiol. (2000) 151:693–702.
6. Michaud DS, Spiegelman D, Clinton SK, Rimm EB, Willett WC, Giovannucci EL. Fruit and vegetable intake and incidence of bladder cancer in a male prospective cohort. J. Natl Cancer Inst. (1999) 91:605–613.
7. Castelao JE, Yuan JM, Gago-Dominiguez M, et al. Carotenoids/vitamin C and smoking-related bladder cancer. Int. J. Cancer (2004) 110:417–423.[CrossRef][Web of Science][Medline]
8. Pisani P, Faggiano F, Krogh V, Palli D, Vineis P, Berrino F. Relative validity and reproducibility of a food frequency dietary questionnaire for use in the Italian EPIC centres. Int. J. Epidemiol. (1997) 26(Suppl. 1):S152–S160.
9. Peluso M, Airoldi L, Magagnotti C, et al. White blood cell DNA adducts and fruit and vegetable consumption in bladder cancer. Carcinogenesis (2000) 21:183–187.
10. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. (1988) 16:1215.
11. Matullo G, Guarrera S, Carturan S, Peluso M, Malaveille C, Davico L, Piazza A, Vineis P. DNA repair gene polymorphisms, bulky DNA adducts in white blood cells and bladder cancer in a case-control study. Int. J. Cancer (2001) 92:562–567.[CrossRef][Web of Science][Medline]
12. Kellen E, Zeegers M, Paulussen A, Van Dongen M, Buntinx F. Fruit consumption reduces the effect of smoking on bladder cancer risk. The Belgian case control study on bladder cancer. Int. J. Cancer (2006) 118:2572–2578.[CrossRef][Web of Science][Medline]
13. Matullo G, Dunning AM, Guarrera S, et al. DNA repair polymorphisms and cancer risk in non-smokers in a cohort study. Carcinogenesis (2006) 27:997–1007.
14. Chao PDL, Hsiu SL, Hou YC. Flavonoids in herbs: biological fates and potential interactions with xenobiotics. J. Food Drug Anal. (2002) 10:219–228.
15. Ciolino HP, Yeh GC. Inhibition of aryl hydrocarbon-induced cytochrome P-450 1A1 enzyme activity and CYP1A1 expression by resveratrol. Mol. Pharmacol. (1999) 56:760–767.
16. Collins AR. Carotenoids and genomic stability. Mutat. Res. (2001) 475:21–28.[Web of Science][Medline]
17. Collins AR, Harrington V, Drew J, Melvin R. Nutritional modulation of DNA repair in a human intervention study. Carcinogenesis (2003) 24:511–515.
18. Møller P, Vogel U, Pedersen A, Dragsted LO, Sandström B, Loft S. No effect of 600 g fruit and vegetables per day on oxidative DNA damage and repair in healthy human non-smokers. Cancer Epidemiol. Biomarkers Prev. (2003) 12:1016–1022.
19. Moller P, Viscovich M, Lykkesfeldt J, Loft S, Jensen A, Poulsen HE. Vitamin C supplementation decreases oxidative DNA damage in mononuclear blood cells of smokers. Eur. J. Nutr. (2004) 43:267–274.[CrossRef][Web of Science][Medline]
20. Astley SB, Elliot RM, Archer DB, Southon S. Evidence that dietary supplementation with carotenoids and carotenoid rich foods modulates the DNA damage: repair balance in human lymphocytes. Br. J. Nutr. (2004) 91:63–72.[CrossRef][Web of Science][Medline]
21. Tomasetti M, Alleva R, Collins AR. In vivo supplementation with coenzyme Q10 enhances the recovery of human lymphocytes from oxidative DNA damage. FASEB J. (2001) 15:1425–1427.
22. Matullo G, Guarrera S, Sacerdote C, et al. Polymorphisms/haplotypes in DNA repair genes and smoking: a bladder cancer case-control study. Cancer Epidemiol. Biomarkers Prev. (2005) 14:2569–2578.
23. Moller P, Loft S. Interventions with antioxidants and nutrients in relation to oxidative DNA damage and repair. Mutat. Res. (2004) 551:79–89.[Web of Science][Medline]
Received on July 25, 2006; revised on March 13, 2007; accepted on March 13, 2007.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||