Molecular and Nutritional Epidemiology Unit and Cancer Risk Factor Branch, Molecular Biology Laboratory Cspo, Scientific Institute of Tuscany, Florence
1 Cancer Epidemiology Unit, Cpo, Turin
2 Genetics Research Institute, Milan
3 Epidemiology Unit, Int, Milan
4 Department of Clinical and Experimental Medicine, Federico Ii University, Naples
5 Registro Tumori, A.O. Civile-M.P. Arezzo, Ragusa, Italy
6 Nutrition and Cancer Unit, IARC, Lyon, France
7To whom correspondence should be addressed Email: d.palli{at}cspo.it
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Abstract |
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Abbreviations: B[a]P, benzo[a]pyrene; BMI, body mass index; GST, glutathione S-transferase; PAH, polycyclic aromatic hydrocarbon.
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Introduction |
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DNA adducts have been widely used in order to identify health hazards and to evaluate the doseresponse relationship in humans exposed to carcinogens and mutagenic compounds and are considered a biomarker of internal dose. DNA adducts tend to be higher among subjects heavily exposed to air pollutants, such as police officers and bus drivers (10,11), and it has been suggested that high levels of DNA adducts might be predictive of cancer risk, reflecting both the exposure to environmental xenobiotics and susceptibility to carcinogens (2,12,13).
Individual susceptibility to cancer can be partly explained by variability in specific enzyme metabolic pathways. Polymorphisms in these metabolic genes have been linked to increased risk of cancer in several casecontrol studies (14). A genetic deficiency in the detoxifying enzyme glutathione S-transferase (GST) M1 (GSTM1 null genotype) has been associated with increased risk of lung (15,16) and bladder cancer (17) and increased polycyclic aromatic hydrocarbon (PAH)DNA adducts (18,19).
Dietary constituents in fresh fruit and vegetables might play a relevant role in DNA adduct formation by inducing or inhibiting enzymatic activities (20,21). Antioxidant micronutrients have been shown experimentally to inhibit DNA damage by PAH and other carcinogens and to alter expression of metabolic enzymes (22,23).
A few human studies evaluated the effects of micronutrients on DNA adducts. Grinberg-Funes et al. (24) reported an inverse association between blood levels of -tocopherol and vitamin C and PAHDNA adducts in lymphocytes: the relationship was found only in those subjects with the GSTM1 null genotype. Mooney et al. (25) reported a similar inverse association between plasma levels of retinol, ß-carotene and
-tocopherol and PAHDNA adducts in subjects lacking the GSTM1 detoxification gene. Another study (26) did not report any association between the plasma levels of ß-carotene and
-tocopherol and level of DNA adducts in lymphocytes. However, it has also been suggested (27) that polymorphisms of selected metabolic genes and plasma ß-carotene may modulate the level of DNA adducts. We have recently published a cross-sectional study on the association of DNA adduct levels with diet and other individual characteristics in 300 healthy adults randomly selected in the Italian EPIC cohorts (28), in which strong inverse associations emerged with the reported frequency of consumption of fresh fruit and vegetables, olive oil and the intake of all major antioxidants, particularly ß-carotene and vitamins C and E.
The epidemiological evidence suggests, therefore, that DNA damage may be modulated by genetic and nutritional risk factors. In this study we aimed to investigate the association between DNA adduct levels and plasma levels of selected micronutrients (six carotenoids, retinol and - and
-tocopherol), measured in the same blood sample, also taking into account the role of metabolic polymorphisms and smoking history in a large series of healthy adults randomly sampled among volunteers enrolled in the Italian EPIC cohort.
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Materials and methods |
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Diet and lifestyle questionnaires
Dietary information on the frequency of consumption of >120 foods and drinks in a 12 month period prior to enrollment was obtained by a self-administered Food Frequency Questionnaire, validated in a pilot phase (30). All individual questionnaires were checked and coded by trained dieticians, computerized and then transformed into estimates of intake for a series of over 30 nutrients according to specifically developed Italian Food Tables (31). The consumption of vitamin supplements was specifically investigated.
A standardized lifestyle questionnaire, representing the Italian translation of a common English model adopted by all European centres (in two separate versions for men and women), was also filled in by each participant. All subjects were originally classified according to smoking history reported at enrollment in three categories: never, former and current smokers. Since DNA adduct levels in smokers have been reported still to be elevated 14 months after quitting smoking (32), we decided a priori to combine former smokers with one of the other categories (never or current) according to a cut-off value of 4 years since cessation. We thus used a dichotomous classification to define smokers (as subjects classified as current smokers at enrollment plus former smokers who reported to have quit smoking in the last 4 years before enrollment) and non smokers (as subjects classified as never smokers at enrollment plus subjects who reported to have quit smoking at least 4 years before enrollment).
Blood collection and storage
Informed consent was obtained from all subjects prior to enrollment in the study. The project has been approved by the local Ethical Committee in Florence. Blood samples were collected in citrate tubes and were processed by centrifugation in a dedicated laboratory in each centre, on the same day as collection, divided into 28 aliquots of 0.5 ml each (12 plasma, eight serum, four concentrated red blood cells and four buffy coat), using an automatic aliquoting and sealing machine specifically developed by BICEF, France (Cryo-Bio Straw). The aliquots were stored in liquid nitrogen tanks at -196°C in a local biological bank in each centre in Italy and centrally at IARC, Lyon. Straws were retrieved and shipped in dry ice to laboratories for DNA extraction and DNA adduct analyses and to detect metabolic polymorphisms. Plasma levels of micronutrients were determined at the Nutrition and Cancer Unit of IARC (Lyon).
DNA 32P-post-labelling technique
Leukocyte DNA was isolated and purified from stored buffy coats by enzymatic digestion of RNA and proteins followed by phenolchloroform extractions (28). DNA samples (5 µg) were digested with 0.21 U micrococcal nuclease and 0.174 U spleen phosphodiesterase at 37°C for 4.5 h. After treatment of DNA samples with 5 µg nuclease P1 for 30 min at 37°C, the hydrolysate enriched in adducted nucleotides was then labelled by incubation with 24 µCi carrier-free [-32P]ATP (3000 Ci/mM) and 10 U T4 polynucleotide kinase at 37°C for 30 min in 25 µl of bicine buffer mixture. Resolution of 32P-labelled DNA digests treated with nuclease P1 was carried out on PEIcellulose TLC plates using the contact transfer technique (28). The solvent systems selected were: (D1) 1 M sodium phosphate, pH 6.8; (D3) 4 M lithium formate, 7.5 M urea, pH 3.5; (D4) 0.65 M LiCl, 0.45 M TrisHCl, 7.7 M urea, pH 8.0; (D5) 1.7 M sodium phosphate, pH 5.0. The adduct spots were detected by autoradiography from 72 to 96 h at -80°C using Kodak XAR-5 films and intensifying screens. DNA adduct levels were determined by excising areas of chromatograms and measuring the levels of radioactivity present by Cerenkov counting. The results were expressed as relative adduct labelling = c.p.m. in adduct nucleotides/c.p.m. in total nucleotides. The detection limit of the nuclease P1 modification of the DNA 32P-post-labelling technique was 0.1 adduct per 109 nucleotides, as previously reported (28). The reproducibility of the DNA 32P-post-labelling technique was verified by analysing
20% of DNA samples in a second independent experiment, and the results of the two analyses were in perfect agreement (r = 0.98). All the analyses were carried out blind prior to decoding. One standard was routinely included in the analysis, i.e. benzo[a]pyrene (B[a]P)DNA adducts, from liver of mice treated i.p. with 0.06 mg/kg B[a]P for 24 h. The average level of B[a]PDNA adducts was 5.1 ± 0.1 per 108 nucleotides.
Polymorphism analysis
A multiple PCR method was used to detect the presence or absence of the GSTM1 and GSTT1 genes and polymorphic alleles at the CYP1A1 MspI, NAT2 and MTHFR loci in genomic DNA samples (obtained from stored buffy coats as described above).
GST
This method had both GST primer sets (GSTM1, 5'-AACTCCCTGAAAAGCTAAAGC and 5'-GTTGGGCTCAAATATACGGTGG; GSTT1, 5'-TCCTTACTGGTCCTCACATCTC and 5'-TCACCGGATCATGGCCAGCA) in the same PCR which included a third primer set for albumin (5'-GCCCTCTGCTAACAAGTCCTAC and 5'-CCCTAAAAAGAAAATC-GCCAATC) and used 30 cycles with denaturing at 94°C for 1 min, annealing at 64°C for 1 min and extension at 72°C for 1 min.
CYP1A1
DNA was amplified in a total reaction volume of 50 µl containing 1.2 mM dNTP, 1.2 µM oligonucleotide primers and 2.5 U Taq polymerase (AmpliTaq; Perkin-Elmer). DNA samples were amplified using the primers 5'-CTGACTGGCTTCAGCAAGTT and 3'-TAGGAGTCTTGTCTCATGCCT. PCR was performed for 45 cycles with denaturing at 95°C for 1 min, annealing at 56°C for 1 min and extension at 65°C for 2 min. PCR products were digested with excess MspI (New England Biolabs) for 3 h and then electrophoresed through 1.8% agarose and visualized by ethidium bromide staining.
NAT2
Three known slow acetylator alleles (NAT2*5, NAT2*6 and NAT2*7) were identified. PCR was carried out in a total volume of 50 µl using primers 5'-TGACGGCAGGAATTACATTGTC and 3'-ACACAAGGGTTTATTTTGTTCC. The PCR mixture contained 5 µl DNA, 50 pM each primer, 200 µM dNTPs, 1.5 U Taq polymerase (AmpliTaq), 10 mM TrisHCl buffer, pH 8.3, 50 mM KCl and 1.5 mM MgCl2. PCR was performed for 35 cycles with denaturing at 94°C for 1 min, annealing at 56°C for 1 min and extension at 72°C for 2 min. PCR products were incubated with restriction enzymes KpnI, TaqI and BamHI from Gibco. Rapid acetylator genotypes are wild-type (*4) allele homo- or heterozygotes, slow acetylator genotypes are those with any two or the three slow acetylator alleles (*5, *6 or *7).
MTHFR (677 CT)
Primer sequences were 5'-TGAAGGAGAAGGTGTTCTGCGGGA and 5'-AGGACGGTGCGGTCAGAGTG. Amplification was performed using initial denaturation at 95°C for 2 min followed by 29 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 30 s, with a final extension at 72°C for 10 min. Amplified product was digested with Hinf1 before electrophoresis.
Plasma levels of micronutrients
Aliquots of plasma were extracted from the central EPIC biological bank in 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 were analysed for six carotenoids (- and ß-carotene, zeaxanthin, ß-cryptoxanthin, lutein and lycopene), retinol and
- 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. (33). Samples were extracted with 800 µl of hexane after 30 min and the mobile phases were enriched with 1 ml/l 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 v.6.4; Hewlett Packard) but controlled one by one by three different laboratory technicians. Peaks for carotenoids that were under the detection limits were set at 0.
Statistical methods
The correlation between the micronutrients measured in the same blood sample was investigated by Spearman rank correlation coefficient.
To investigate the relationship between DNA adduct levels and plasma concentration of selected micronutrients, we compared adduct values across tertiles of plasma levels. Negative samples (those below 0.1 adduct per 109 normal nucleotides, the threshold of detection of the DNA 32P-post-labelling method) were assigned a value of 0.1. In order to carry out statistical analyses, we used log transformed data for micronutrients and first divided all available subjects into tertiles of plasma levels of each nutrient, and the mean levels of DNA adducts across tertiles were compared by analysis of covariance, introducing into each model terms for age, sex, centre, smoking habit (never, former and current), body mass index (BMI), year and period of blood drawing and total caloric intake. The covariance analysis was also performed separately for non-smokers and smokers and separately for each genotype according to selected polymorphic metabolic genes. Post hoc Dunnett tests were performed for multiple comparisons among tertiles of plasma levels for each carotenoid and selected micronutrient. All analyses were performed on log transformed data. The tests for linear trends were calculated by including ordered variables in each covariance model with log transformed adduct values.
A P value 5% was considered significant. All the analyses were performed by the statistical package SAS.
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Results |
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Among participants, never smokers represented the largest group (127/331 or 38.4%), whereas 34.7% (115/331) reported at enrollment to be former smokers and 26.9% (89/331) current smokers. According to our dichotomous classification, non-smokers and smokers were 69.8 and 30.2%, respectively.
The consumption of vitamin supplements in our sample was reported at blood drawing by only a few subjects (13/331, 3.9%).
DNA adducts
DNA adducts were detected in 78.3% of the samples (259 subjects), with some variation between centres (lowest in Ragusa, 68.7%, and highest in Naples, 100%), but not between genders (79.1% in males and 77.4% in females).
The crude mean of the DNA adduct level was 7.46 (±0.48) per 109 nucleotides, with relevant differences between subjects from the five participating centres: crude mean values were 14.8, 9.2, 6.8, 5.1 and 4.8 for volunteers from Naples, Florence, Varese, Turin and Ragusa, respectively (data not shown).
Overall, differences between centres persisted after adjustment for age, sex, smoking history, BMI, period and year of blood drawing, with samples from Ragusa showing lower mean levels than those from all other centres (Table I). No difference emerged according to smoking history [either in three (current, ex and never smoker) or two categories (smokers and non-smokers)].
Plasma levels of selected micronutrients
Table II shows the plasma levels of the micronutrients measured. Crude mean values together with adjusted mean values from a covariance analysis according to geographical area are reported for six carotenoids, retinol and - and
-tocopherol. Among the six carotenoids, ß-carotene, lutein and lycopene showed the highest concentrations. Mean plasma levels were significantly higher in Southern Italy for total carotenoids (P = 0.009) and particularly for lycopene and ß-cryptoxanthin (P = 0.0001 for both micronutrients), whereas mean levels of
-carotene were higher in Northern Italy (P = 0.0001). In contrast, ß-carotene and lutein levels did not show any significant geographical variation. Differences between geographical areas also emerged for retinol and
- and
-tocopherol, with lower mean levels in Southern Italy (P = 0.0003, P = 0.008 and P = 0.006, respectively).
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The Spearman correlation coefficients between selected micronutrients measured in the same blood sample are available on request.
DNA adducts and plasmatic micronutrients
In the whole series, our multivariate analyses found strong inverse associations between DNA adduct levels and plasma concentrations of retinol (P = 0.02), -tocopherol (P = 0.04) and
-tocopherol (P = 0.03). No association emerged with the plasma levels of carotenoids, except a borderline inverse association (P = 0.08) with ß-carotene (Table III).
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Discussion |
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Our main results are in agreement with previous studies (24,25) showing that plasma levels of selected micronutrients (retinol, ß-carotene and -tocopherol) were inversely associated with DNA adduct levels. Our study is the first to report an inverse association between DNA adduct levels and plasma concentration of
-tocopherol. Several human and animal studies have suggested that plasma concentration of
-tocopherol may be inversely associated with cancer risk, particularly prostate cancer (34,35). We conducted several analyses (typically including comparisons for several nutrients in separate strata) and thus some of the statistically significant results may be due to chance, although our a priori hypothesis was clearly defined. A possible example of a chance finding is the divergent association of ß-cryptoxanthin with DNA adduct levels in the two groups of smokers and non-smokers.
We have recently shown in a cross-sectional study (28) a strong inverse association between DNA adduct levels and reported frequency of consumption of fresh fruit and vegetables, the main sources of micronutrients with antioxidant properties (ß-carotene and vitamins C and E). This strong inverse relationship between fresh fruit and vegetable consumption and DNA adduct formation was also reported in a recent casecontrol study on bladder cancer (36). Antioxidant vitamins (vitamin C, ß-carotene and -tocopherol) derived from fresh fruit and vegetables were significantly inversely correlated with DNA adducts: subjects with higher levels of intake of these foods had lower levels of DNA damage. Several dietary components (flavonoids, polyphenols and other natural compounds) have been shown to inhibit DNA adduct formation in vitro and in vivo (20). These dietary components might modulate DNA adduct formation by antioxidant activity or by interfering with the metabolic pathways of carcinogen activation/detoxification (20). This effect might explain the capacity of reduced cancer risk specifically associated with a dietary pattern rich in fruit and vegetables.
Levels of micronutrients measured in our series are quite similar to those evaluated in other recent studies in Mediterranean populations (3739), and the consumption of vitamin supplements was reported quite rarely by our study participants. Among the six carotenoids measured, ß-carotene, lutein and lycopene showed the highest concentrations, in agreement with series collected in Spain and Sweden (37) and in Italy (38). In our study population, strong geographical trends emerged, confirming different dietary patterns in the three major areas of the country, with higher levels of lycopene (and total carotenoids) in Southern Italy and higher levels of -carotene, retinol and tocopherols in Northern Italy. Samples from Central Italy (Florence) showed intermediate levels for most of the biomarkers of dietary intake of the antioxidants we have measured. This variability, however, did not explain differences in adduct levels between centres, which are probably more related to environmental exposures (11,28).
Overall, no clear association was found between cigarette smoking and DNA adducts, in agreement with the results of our previous study (28). However, the association between DNA adducts and plasma level of selected micronutrients was modulated by smoking history. Non-smokers with higher plasma levels of retinol and -tocopherol (but not
-tocopherol) showed lower levels of DNA adducts. A significant inverse association with
-tocopherol emerged only in smokers, suggesting a possible modulation of the relationship between micronutrients and adduct formation. The total levels of 32P-post-labelled leukocyte DNA adducts may be reflective of different environmental carcinogen exposures, e.g. cigarette smoke, air pollution and dietary carcinogens (2,10). Thus, modulation by cigarette smoking of the relation between micronutrients and formation of DNA adducts may be due to the fact that antioxidant micronutrients play an important role in the antioxidant defense system against DNA damage induced by cigarette smoke, i.e. by quenching oxidants, including PAHs. Alternatively, this may be due to the fact that cigarette smoking has been shown to independently influence the levels of circulating antioxidant micronutrients (40).
Several studies (including cohorts and casecontrol studies) have reported an inverse association between vitamin E intake and lung cancer risk, particularly in smokers (41). Likewise, some studies showed that high intake of vitamin C was significantly associated with reduced risk of lung cancer (42,43), but a modest protective effect or no association was found in other studies (4446). We could not evaluate the effects of plasma concentrations of vitamin C on DNA adduct levels because, according to our original protocol, measurements of this vitamin's levels were not carried out. With regard to vitamin A, several studies have shown contrasting results (45,46). Large intervention trials have also been carried out to test the hypothesis that antioxidant vitamin supplements could reduce lung cancer risk. Two randomized intervention trials aimed to reduce lung cancer risk in heavy smokers were terminated early because preliminary analyses indicated that supplementation with ß-carotene and -tocopherol (ATBC study) or ß-carotene and vitamin A (CARET study) did not protect from lung cancer and might even be harmful (47,48).
Although the negative results of these intervention trials exclude a protective effect of these compounds when used as supplements, epidemiological evidence, including a recent report from the same ATBC study (8), consistently suggests that several dietary components may block carcinogenic pathways by antioxidant activity or by interfering with the binding of carcinogens to DNA. Estimates of intake of -tocopherol, either as dietary intake (measured by a questionnaire) or as plasma level, probably correlate with other natural compounds present in foods. It is interesting that, recently, a positive association between higher DNA adduct levels and increased lung cancer risk in smokers was reported in a prospective study (13), whereas here we report an inverse association between
-tocopherol and DNA adduct levels only among smokers. This provides additional evidence for a possible protective role of
-tocopherol from lung cancer in smokers, although it cannot be excluded that the effects observed in epidemiological studies and attributed to
-tocopherol are actually due to some other natural compound present at high concentantion in the same foods. Possible candidates include phenols, flavonoids, isothiocyanates and other compounds. Residual confounding (due to low accuracy of self-reported smoking histories for both intensity and duration) has also been suggested as a reasonable explanation of the apparent protective effects of dietary antioxidants in epidemiological studies of lung cancer (49), but this is not applicable to our results in smokers versus non-smokers.
The role of genetic polymorphisms of several enzymes involved in the metabolism of chemical compounds was also explored, but differences only emerged on stratifying the analysis by GSTM1 genotype. Frequencies of genotypes were in agreement with expected values. The inverse association between DNA adducts and plasma levels of some micronutrients (- and ß-carotene and retinol) were found only in those subjects with the null genotype. Borderline negative associations also emerged with plasma level of
- and
-tocopherol. These results suggest that plasma levels of antioxidant vitamins may be very important in inhibiting DNA adduct formation in subjects who do not have the capacity to detoxify carcinogens via the GSTM1 pathway. Both antioxidant vitamins and GSTM1 polymorphisms appear to protect against DNA damage (23). Antioxidant compounds such as vitamins C and E and ß-carotene may act directly to inhibit oxidants and hence reduce DNA damage, whereas GST can catalyse the conjugation reaction between glutathione and substrates with electrophilic sites, increasing the detoxification of several procarcinogens. Other studies have already observed a relationship between DNA adducts and blood levels of vitamins in subjects with the GSTM1 null genotype (24,25). On the other hand, an association between DNA adducts and GSTM1 null genotype has been found by some studies (18,19,50).
In conclusion, our results show that DNA damage may be modulated by interactions between genetic, environmental and nutritional risk factors: overall, high plasma levels of selected micronutrients with antioxidant activity are significantly associated with reduced levels of DNA adducts in peripheral white blood cells. Some specific effects are evident only in subjects lacking the ability to detoxify carcinogens via a specific metabolic pathway (GSTM1 null) or among smokers. This could contribute to better explaining the complex interaction between nutritional status, inherited genetic susceptibility to DNA damage and environmental exposures, including smoking.
Further studies are needed to evaluate the effects of dietary antioxidant compounds on human DNA damage and the effect of genetic polymorphisms on these associations.
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Acknowledgments |
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References |
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