1 Molecular and Nutritional Epidemiology Unit and Cancer Risk Factor Branch, Molecular Biology LaboratoryCSPO, Scientific Institute of Tuscany, Florence, 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 and 6 Cancer Epidemiology Unit, CPO, Turin, Italy
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Abstract |
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Abbreviations: BMI, body mass index; CYP1A1, human cytocrome P450 1A1 gene; GSTM1, glutathione S-transferase M1; MTHFR, methylene-tetra-hydrofolate reductase; NAT, N-acetyl-transferase 2; PAH, polycyclic aromatic hydrocarbons
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Introduction |
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DNA adducts have been used widely 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 (1315), 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 genetic and acquired susceptibility to carcinogens (1619). We have suggested previously that DNA adduct levels might be modulated by diet (20) and by a group of dietary intake biomarkers (21). In particular, in the latter study, we have shown that individual levels of DNA adducts were modulated by plasma levels of a few selected micronutrients: six specific carotenoids, - and
-tocopherol and retinol were measured in the same blood sample donated at enrollment by a group of 331 individuals (21).
The genetic deficiency in the detoxifying enzyme GSTM1-null genotype has been associated with increased PAH (polycyclic aromatic hydrocarbons)DNA (22) and 4-aminobiphenyl hemoglobin (23) adducts. In a study, PAHDNA adduct levels have been reported to be inversely correlated with vitamin E and also with smoking-adjusted vitamin C in GSTM1-null genotype subjects (24). 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. In contrast, one study (26) did not report any association between the plasma levels of ß-carotene and
-tocopherol and level of DNA adducts in lymphocytes.
Here we further investigated the association between DNA adduct levels and consumption of major food groups and foods, and questionnaire-derived estimates of intake of major macronutrients and micronutrients, taking into account the role of selected metabolic polymorphisms and, particularly, their potential modifying effects. The loci chosen, CYP1A1, NAT2, MTHFR, GSTM1 and GSTT1, have all been implicated in the metabolism of various dietary components and toxicants, and each are polymorphic in the European population. The present study was based on a larger sample of 634 healthy adults, both sexes, aged 3564 years, randomly sampled among a large series of over 47 000 volunteers enrolled in the Italian EPIC (European Prospective Investigation into Cancer and Nutrition) cohort.
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Materials and methods |
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A random sample of approximately 110 subjects, stratified by age, sex and center, was selected from each of the three main geographical study areas, Northern, Central and Southern Italy (21), and added to the previous sample of 308 participants (20), in order to obtain a larger combined sample of volunteers representative of the national cohort. Overall, results were thus available for 634 subjects (216, 208 and 210 adults of both sexes from the three main areas, respectively). In Southern Italy, where one center had enrolled only women, the final sample included 102 men and 54 women from Ragusa (Sicily) and 54 women from Naples.
Diet and life-style questionnaires
Dietary information on the frequency of consumption of more than 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 (29). 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 (30). At enrollment, weight, height, waist and hip circumferences were measured for each participant, according to the international study protocol. A standardized life-style questionnaire, representing the Italian translation of a common English model adopted by all European centers (in two separate versions for men and women), was also filled out by each participant (28).
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 processed by centrifugation in a dedicated laboratory in each center, on the same day of 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 center in Italy. Straws were retrieved and shipped in dry ice to laboratories for DNA extraction, DNA adduct analyses and for metabolic polymorphisms.
Laboratory methods
32P-DNA postlabeling technique
Leukocyte DNA was isolated and purified from stored buffy coats by enzymatic digestion of RNA and proteins followed by phenolchloroform extractions (20). DNA samples (5 µg) were digested with 0.21 U of micrococcal nuclease and 0.174 U of spleen phosphodiesterase at 37°C for 4.5 h. After treatment of DNA samples with 5 µg of nuclease P1 for 30 min at 37°C, the hydrolysate enriched in adducted nucleotides were then labeled by incubation with 24 µCi of carrier free [-32P]ATP (3000 Ci/mM) and 10 U of T4 polynucleotide kinase at 37°C for 30 min in 25 µl of bicine buffer mixture. Resolution of 32P-labeled DNA digests treated with nuclease P1 was carried out on PEI-cellulose TLC plates using the contact-transfer technique (20). 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 labeling = c.p.m. in adduct nucleotides/c.p.m. in total nucleotides. The detection limit of nuclease P1 modification of the 32P-DNA postlabeling technique was 0.1 adduct/109 nt, as reported previously (20). The reproducibility of the 32P-DNA postlabeling technique was verified analysing
20% of DNA samples with a second independent experiment and the results of the two analyses were in perfect agreement (r = 0.98). All the analyses were carried out blindly prior to decoding. One standard was routinely included in the analysis, i.e. benzo[a]pyrene DNA adducts, from the liver of mice treated intraperitoneally with 0.06 mg/kg B[a]P for 24 h. The average levels of B[a]P DNA adducts were 5.1 ± 0.1 (SE, standard error) 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 CYP1A1 (human cytocrome P450 1A1 gene) MspI, NAT2 (N-acetyl-transferase 2) and MTHFR (methylene-tetra-hydrofolate reductase) loci in genomic DNA samples (obtained from stored buffy coats as described in the previous section).
GST
This method had both GST primer sets (GSTM1 5'-AACTCCCTGAAAAGCTAAAGC 5'-GTTGGGCTCAAATATACGGTGG and GSTT1 5'-TCCTTACTGGTCCTCACATCTC 5'-TCACCGGATCATGGCCAGCA) in the same PCR included a third primer set for albumin (5'-GCCCTCTGCTAACAAGTCCTAC, 5'-CCCTAAAAAGAAAATCGCCAATC) 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.
CYP1A
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, Boston, MA). DNA samples were amplified using the following primers: 5'-CTGACTGGCTTCAGCAAGTT and 3'-TAGGAGTCT TGTCTCATGCCT. 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. PCRs 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 of each primer, 200 µM of dNTPs, 1.5 U Taq polymerase (Amplitaq, Perkin-Elmer), 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 (Carlsbad, CA). Rapid acetylator genotypes were defined as wild-type (*4) allele homo- or heterozygotes, while slow acetylator genotypes included all those with any two of the three slow acetylator alleles (*5, *6 or *7).
MTHFR (677 C > T)
Primer sequences are 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. The amplified product was then digested with Hinf1 before electrophoresis.
Statistical analysis
To investigate the relationship between DNA adduct levels and dietary habits and metabolic polymorphisms, we compared adduct values for different levels of food group consumption and nutrient intake according to selected polymorphic metabolic genes. Negative samples (those <0.1 adduct/109 normal nt, the threshold of detection of the 32P-post DNA labeling method) were arbitrarily assigned a value of 0.1. In order to carry out statistical analyses, we first divided all available subjects into tertiles of food consumption and nutrient intake. Geometric mean levels of DNA adducts across tertiles of consumption of foods or food groups and intake of nutrients were compared by analysis of covariance, introducing into each model terms for age, sex, center, smoking history (never, former and current), period and year of blood drawing, BMI (body mass index) and total caloric intake. Post hoc Dunnett tests were performed for multiple comparisons among nutrient tertiles. The tests for linear trends were calculated by including ordered variables in each covariance model with log transformed adduct values. The covariance analysis was performed separately according to selected polymorphic metabolic genes.
Multivariate logistic analyses were carried out to evaluate the existence of possible interactions between selected polymorphic metabolic genes and dietary variables in predicting high levels of DNA adducts (based on a dichotomous variable: above/below the median value). The statistical significance of these interactions was evaluated on a multiplicative scale, including an interaction term into a model containing the previously identified potential confounders and the two main effect variables (the metabolic polymorphic gene and each dietary variable); such an interaction means a departure from a multiplicative effect for the two factors combined. All the analyses were performed by the statistical package SAS. A P value <0.05 was considered statistically significant.
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Results |
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DNA adducts
DNA adducts were detected in 76.0% of the samples, with some variation between centers (lowest in Ragusa 65.4%, and highest in Naples 88.9%) but not between genders (76.3 in males vs 75.8% in females). The crude mean DNA adduct level was 7.82/109 nt (SE ± 0.40). Differences among subjects from the five participating centers were evident: the geometric-adjusted DNA adduct levels were 5.84, 3.56, 2.22, 1.62 and 1.20 for volunteers from Naples, Florence, Varese, Turin and Ragusa, respectively. While the 54 subjects (all women) from Naples showed higher levels than the 208 participants from Florence (reference group), samples from Turin and Ragusa showed significantly lower levels than those from Florence. Only minor differences were found according to gender and smoking history. DNA adduct values tended to vary according to BMI, with lower values among overweight subjects.
Overall, DNA adduct values tended to decrease during the 5-year study period, the geometric mean being, respectively, 3.15 in the first 2-year period of enrollment (19931994), 2.65 in the period 19951996 and 1.78 in the period 19971998 (P for trend = 0.05) (Table I).
No statistically significant differences were evident (overall and according to study center) in the mean level of DNA adducts between the first and the second group of approximately 300 subjects sampled from the EPIC Italy cohorts. Within each group the dates of enrollment of volunteers were randomly distributed across the 5-year period. Overall, the geometric mean values of DNA adduct levels were 2.3 and 2.47/109 nt, respectively, in the first and the second group of samples.
DNA adducts and diet
In the analyses carried out in the whole sample of 634 EPIC volunteers a negative association of DNA adduct levels was found with the reported frequency of consumption of raw leafy vegetables and fresh fruit other than citrus fruit (Table II). Negative associations emerged also with the estimated intakes of potassium (P for trend = 0.01), niacin (P for trend = 0.04) and ß-carotene (P for trend = 0.05) (Table III).
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Discussion |
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In this study, carried out in a large sample of healthy adults, both sexes, enrolled in the Italian EPIC cohorts, we evaluated the association between DNA adduct levels and consumption of selected foods and intake of macronutrients and micronutrients, taking into account the effect of metabolic polymorphisms. When analyses were carried out considering the whole series of 634 subjects, we found an inverse association of DNA adduct levels with high consumption of leafy raw vegetables and non-citrus fruit. An inverse association with high intakes of potassium, ß-carotene and niacin was also evident considering the whole dataset. In addition, we did not observe an independent effect of GSTM1-null genotype or any other selected metabolic polymorphic genotype on DNA adduct levels.
Our results thus show a clear modulating role of GSTM1 polymorphic genotype on the effects of diet on DNA adduct levels, in agreement with our previous findings (21). An interaction between GSTM1-null genotype and high consumption of cruciferous vegetables has been described in relation to development of adenomas (31) and colorectal cancer (11,12). This geneenvironment interaction seems to be quite complex considering that active compounds identified (including isothiocyanates and indoles) are believed to inhibit phase I activating enzymes and induce phase II detoxification enzymes and this mechanism could explain the protective effect of high consumption of these vegetables through an effect on the metabolism of pro-carcinogens, a mechanism that has been suggested also for other cancer sites. On the other hand, GST enzymes also metabolize isothiocyanates, and subjects with low or no activity of GST enzymes could metabolize at a lower rate these compounds, thus reducing the risk in this subgroup of subjects (enhancing their protective effect). In a prospective study carried out in men in Shangai, subjects with detectable isothiocyanates in the urine collected at the enrollment were at decreased risk for lung cancer; this effect, however, was primarily evident in subjects with homozygous deletion of GSTM1 (32).
Although understanding the mechanism of GSTM1 polymorphism/dietary interaction will require further research into specific patterns of DNA adducts found in wild-type and null individuals, we can speculate that our data are consistent with a model in which GSTM1 and some components in dietary vegetables both act to reduce DNA adduct formation. GSTM1-null individuals would suffer an increase in levels of toxic metabolites (which cannot be removed by GST) and therefore of adducts, unless they consume sufficient amount of vegetables to block adduct formation.
Both diet and metabolic polymorphisms can affect DNA adduct levels. Antioxidant compounds such as vitamins C, E and ß-carotene may act directly to inhibit oxidants and hence reduce DNA damage, while GST can catalyze the conjugation reaction between glutathione and substrates with electrophilic sites, increasing the detoxification of several procarcinogens.
We have shown previously that the consumption of fresh fruit and vegetables and the intake of several related micronutrients (including all major anti-oxidants) are relevant determinants of DNA adduct levels in a population exposed to genotoxic agents commonly present in the environment (20). Dietary constituents in fresh fruit and vegetables might play a relevant role in DNA adduct formation by inducing or inhibiting enzymatic activities (33,34). Anti-oxidant micronutrients have been shown experimentally to inhibit DNA damage by PAH and other carcinogens and to alter the expression of metabolic enzymes (35,36). Animal studies have shown that natural compounds present in human diet are able to induce GST activities and reduce DNA adduct formation by PAH (37).
An inverse association between blood levels of -tocopherol and vitamin C and PAHDNA adducts in lymphocytes of subjects with the GSTM1-null genotype was found in a cross-sectional study on 63 male heavy cigarette smokers (24). A similar inverse association was reported between plasma levels of retinol, ß-carotene and
-tocopherol and PAHDNA adducts in subjects lacking the GSTM1 detoxification gene in a study carried out on 159 current smokers (25). Another study (26) did not report any association between the plasma levels of ß-carotene and
-tocopherol and level of DNA adducts in lymphocytes. Recently, we have found that plasma levels of antioxidant vitamins may play an important role in inhibiting DNA adduct formation in subjects who do not have the capacity to detoxify carcinogens via the GSTM1 pathway (21). Specific patterns of associations between antioxidant levels and DNA adducts emerged when analyses were carried out stratifying by GSTM1 genotype, with high plasma levels of
- and ß-carotene showing an effect only among GSTM1-null subjects.
A similar effect on DNA adduct levels was also reported by us for high plasma levels of retinol (21). No effect of preformed retinol intake on DNA adduct level was evident in the present study considering either the whole series of 634 subjects or in the analyses carried out according to GSTM1 genotypes. Nevertheless, we have to consider that ß-carotene and other carotenoids are converted to retinol in many tissues and the effect we observed in relation to plasma levels of retinol could be actually related to dietary intake of its precursors.
An inverse association of high dietary level of niacin with DNA adduct levels was found in subjects with GSTM1-null genotype. Niacin is the generic descriptor for NA (nicotinic acid) and NAM (nicotinamide) that are both dietary precursors of NAD+ (nicotinamide adenine nucleotide) the substrate for the enzyme PARP [poly(ADP-ribose) polymerase]-1. This enzyme activated by DNA strand breaks has been shown to be involved in the BER pathway (38). A role of PARP-1 has been also suggested in the repair of bulky DNA adducts (39), but a study that attempted to evaluate the role of niacin supplementation on DNA damage (cytogenetics damage) in a group of smokers did not find evidence of this effect on peripheral blood lymphocytes (40).
Our finding of an inverse association between a high consumption of fish and DNA bulky adduct levels is consistent with previous animal studies reporting an inhibitory effect of fish oil, rich in -3 fatty acids, on DNA adduct formation following dietary exposure to heterocyclic amines (41,42). We have no explanation for the inverse association with white meat, although a high consumption of this source of animal proteins could be a marker of prudent diet, in contrast to consumption of red or preserved meats; in a cross-sectional study within the Italian EPIC cohorts the consumption of poultry correlated with plasma
-3 fatty acids (43).
The results of the assays for the two random samples of approximately 300 subjects (20,21), which were combined for the present study, were compared and the mean values did not differ. In addition, within each group the dates of enrollment of volunteers were randomly distributed across the 5-year period. The decrease of DNA adduct levels during the study period is thus probably due to a parallel decrease in the exposure to vehicle traffic pollutants, following major decisions at the national level during the mid 1990s (including modifications of fuels, large-scale introduction of catalytic exhaust systems, banning of older vehicle models). We have carried out previously a specific analysis for the Florence volunteers because of the availability of additional information on environmental concentrations of pollutants and occupational exposures (15) showing higher levels in traffic-exposed workers in comparison with randomly sampled volunteers. Overall, determinants of DNA adduct levels appear to be strongly related to vehicle traffic and, possibly, photochemical pollution in warmer months.
Overall, our results show an important role of antioxidants in preventing DNA adduct formation when the detoxifying activity of GSTM1 isoenzyme is lacking. Information about this and other metabolic polymorphisms should be included in the design and analysis of molecular epidemiology studies focused on diet and genotoxic damage (or cancer), in order to better understand possible associations. Approximately 50% of the general population is represented by GSTM1-null individuals: thus current dietary guidelines including recommendations to increase the consumption of fresh fruit and vegetables are supported by these findings.
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Notes |
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Acknowledgments |
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References |
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