CEBP Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leuratti, C.
Right arrow Articles by Bingham, S. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Leuratti, C.
Right arrow Articles by Bingham, S. A.
Cancer Epidemiology Biomarkers & Prevention Vol. 11, 267-273, March 2002
© 2002 American Association for Cancer Research

Detection of Malondialdehyde DNA Adducts in Human Colorectal Mucosa

Relationship with Diet and the Presence of Adenomas1

Chiara Leuratti2, Mark A. Watson, Eliot J. Deag, Ailsa Welch, Rajinder Singh, Elke Gottschalg, Lawrence J. Marnett, Wendy Atkin, Nicholas E. Day, David E. G. Shuker3 and Sheila A. Bingham

Medical Research Council Toxicology Unit, University of Leicester, Leicester LE1 9HN, United Kingdom [C. L., E. J. D, R. S., E. G., D. E. G. S.]; Norfolk and Norwich Health Care Trust, NR4 7U4, United Kingdom [M. A. W.]; Strangeways Research Laboratory, European Prospective Investigation on Cancer (EPIC), University of Cambridge, Cambridge, CB1 4RN, United Kingdom [A. W., N. E. D.]; Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232 [L. J. M]; Imperial Cancer Research Fund Colorectal Cancer Unit, St. Mark’s Hospital, Middlesex, HA1 3UJ United Kingdom [W. A.]; and Medical Research Council Dunn Human Nutrition Centre, Cambridge, CB2 2X4 United Kingdom [S. A. B]


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Colorectal biopsies from normal mucosa of participants in the United Kingdom Flexible Sigmoidoscopy Trial and European Prospective Investigation on Cancer (EPIC; n = 162) were analyzed for the presence of malondialdehyde-deoxyguanosine (M1-dG), a DNA adduct derived from lipid peroxidation. The aim was to investigate whether dietary factors can modulate M1-dG levels and whether M1-dG in normal mucosa is a risk factor for colorectal adenomas. Samples were analyzed using a sensitive immunoslot blot assay. This study has shown for the first time that M1-dG is present in human colorectal tissue. M1-dG levels ranged from undetectable (n = 13) to 12.23 per 107 total bases. Mean levels were 4.3 ± 3 and 4.6 ± 2.9 per 107 total bases in men and women, respectively. In men, there were positive associations of adduct levels with height and age, and inverse associations with body mass index. Legumes, fruit, salad, and whole meal bread were inversely associated with M1-dG adducts, whereas consumption of offal, white meat, beer, and alcohol were positively associated with elevated levels. In women, there was an inverse association of the adduct with the ratio of polyunsaturated:saturated fatty acids (P = 0.019) and a weak positive correlation with saturated fat (P < 0.061). When levels of adducts were compared in individuals with and without adenomas, there was a trend for higher levels in individuals presenting with adenomas especially in the highest category of M1-dG adducts (P < 0.005).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CRC4 is the second most common cause of death from malignant diseases in Western Europe and the United States, both in men and in women. Approximately 94% of CRC is sporadic in nature (1) , and it is estimated that 75–80% might be attributable to environmental causes (2 , 3) . CRC develops through a multistep sequence of dysplastic morphological change, most commonly including an adenoma, characterized by an accumulation of genetic defects (4 , 5) . The risk of developing cancer rises with the number and size of adenomas and villous histology.

Diet is regarded as the most important environmental influence on CRC (6 , 7) . Increased risk of CRC has been associated with high intake of red meat and total fat (6, 7, 9) . High consumption of vegetables and fruit has been shown to reduce the risk (reviewed in Refs. 6 and 7 ). In addition, Lee et al. (10) and Slattery et al. (11) reported that individuals who maintained high levels of physical activity throughout their lives were at a lower risk for developing colon cancer.

Dietary fat, lipid peroxidation, and arachidonic acid metabolism have all been implicated in colorectal carcinogenesis (9 , 12 , 13) . Lipid peroxidation is initiated by free-radical attack of membrane lipids, generating large amounts of reactive products, which have been implicated in tumor initiation and promotion. Because modification of DNA is believed to be an important early step in carcinogenesis, endogenous DNA adducts derived from oxidative stress, lipid peroxidation, or other endogenous processes have been proposed as contributors to the etiology of human cancer (14) .

MDA is a major genotoxic carbonyl compound generated by lipid peroxidation (14 , 15) . It is also a by-product of the arachidonic acid metabolism in the synthesis of prostaglandins (16) . Both of these endogenous processes are modulated by dietary factors. For example, lipid peroxidation is stimulated by the presence of high levels of {omega}-6 PUFAs and is inhibited by dietary antioxidants. Increased levels of MDA, together with increased levels of PUFAs and prostaglandins, e.g., PGE2, have been reported in tumor tissues of CRC patients as compared with their normal mucosa (13) .

MDA is mutagenic in bacterial and mammalian systems (17) . It reacts with DNA to form adducts with deoxyguanosine, deoxyadenosine and deoxycytidine (18) . The adduct formed on reaction with deoxyguanosine, the highly fluorescent cyclic pyrimidopurinone, M1-dG, was originally detected in liver DNA from healthy individuals, at levels of 5–11 adducts per 107 total bases (19) . Since then, M1-dG has been detected in several human tissues at levels ranging from 0.02 to 21 per 107 normal bases (20, 21, 22, 23, 24) . Very interestingly, it has been shown that M1-dG can also be formed after direct oxidative attack on DNA (25) . M1-dG is a premutagenic lesion and can induce guanine-to-thymine transversions and guanine-to-adenine transitions in DNA (26 , 27) . The adduct is repaired by bacterial and mammalian nucleotide excision repair and by bacterial mismatch repair pathways (15) .

Fang et al. (28) reported that volunteers on a diet containing high amounts of PUFAs had higher levels of M1-dG in leukocyte DNA than individuals on a diet rich in monounsaturated fatty acids. The difference between adduct levels after the two diets was greater in women than in men. These results suggested a role of diet in modulating adduct levels. Results from our group have also suggested a dietary influence on M1-dG levels in human leukocyte DNA (29) .

The United Kingdom Flexible Sigmoidoscopy Screening Trial is a multicenter randomized controlled trial designed to investigate the efficacy and effectiveness of a single flexible sigmoidoscopy, with removal of all polyps observed during screening, in decreasing morbidity and mortality from CRC (30) . The Norfolk branch of the study is unique because one-third of the 3000 participants also independently participated in the EPIC study. As part of EPIC, dietary, health, and life-style data from all of the participants had been collected prospectively through detailed dietary diaries and health checks (31) . During flexisigmoidoscopy, colorectal biopsies were taken from the flat mucosa in previous EPIC participants with adenomas and age-sex-matched polyp-free controls. These biopsies were analyzed for evidence of DNA damage caused by MDA, and the results compared with dietary and other data collected previously.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials.
CT-DNA, proteinase K, RNase A (from bovine pancreas), RNase T1, and propidium iodide were purchased from Sigma Chemical Co. Ltd. (Dorset, United Kingdom). PBS tablets (Dulbecco A) were purchased from Oxoid Ltd. (Hampshire, England). All other reagents and solvents of analytical or HPLC grade were obtained from either BDH or Fisher Scientific Ltd. (Loughborough, Leicestershire, United Kingdom).

Human Study
Patients.
Ethical approval for this study was obtained from the Norwich District Ethics Committee in 1997. Patients (2999) from general practices were screened in Norfolk as part of the United Kingdom Flexible Sigmoidoscopy Study. Among the EPIC patients, at the first health check from 1992–1997, 144 were found to have one or more polyps (adenomatous or metaplastic polyps) and to have completed a detailed diary for 7 days of all food and drink consumed (32) . Food diaries from 144 controls, age-, sex-, and general practitioner-matched, were also available. The food diaries were coded and analyzed for nutrients and foods using methods described elsewhere (33) . Only patients with histologically proven adenomas (83 cases) and polyp free controls (79 controls) were included in the study. Information on diet, smoking status, weight, height, and BMI [weight (kg)/height2 (m2)] was obtained at the initial EPIC health check.

Biopsies and Polyp Identification.
During flexible sigmoidoscopy screening, colorectal biopsies were taken from the normal mucosa of the posterior wall of the rectum, 3 cm above the dentate line. When polyps were present, they were removed at the same time. Biopsies were transferred to cryovials, immersed in liquid nitrogen immediately and stored at -80°C. Patients with multiple or large (>1-m) adenomas or with >20% villous histology were considered high risk.

DNA Extraction.
Biopsies were defrosted and washed in PBS twice to minimize bacterial contamination.

For DNA extraction, biopsies were homogenized using glass mortars and PTFE pestles (Polytron or Fisher). DNA was extracted using Qiagen genomic DNA extraction kit (Qiagen Ltd., Crawley, Sussex, United Kingdom) and was digested using proteinase K (160 units), RNase A (400 units), and RNase T1 (400 units). DNA was dissolved in ultrapure water. DNA purity was assessed by 260/280 nm ratio using a GeneQuant II RNA/DNA calculator (Pharmacia Biotech) and by reverse phase-HPLC after digestion to deoxynucleotides (34) .

ISB
Standard MDA-modified CT-DNA was prepared as described previously (34) . The amount of M1-dG was measured by HPLC-fluorescence using a calibration curve obtained with synthetic M1-dG (35) .

The ISB assay was performed as described previously (34) , with several modifications. MDA-modified CT-DNA was diluted with control CT-DNA (both at a concentration of 0.1 µg/µl) to obtain decreasing amounts (5 fmol to 0.2 fmol) of M1-dG adduct and to generate standard curves for the ISB. Standard CT-DNA and colorectal DNA (3.5 µg) were dissolved in 10 mM dipotassium hydrogen Pi, (pH 7; 100 µl) and PBS (150 µl). DNA samples were sonicated for 20 min in a water bath sonicator, heat-denatured for 5 min in a boiling water bath, cooled on ice for 10 min and mixed with an equal volume of 2 M ammonium acetate. Resulting single-stranded DNA (143 µl containing 1 µg DNA/sample, in triplicate) was loaded onto nitrocellulose (NC) filters (0.1 µm, BA79; Schleicher & Schuell, Dassel, Germany) using a Minifold II, 72-well slot-blot microfiltration apparatus (Schleicher & Schuell). The slots were rinsed with 200 µl of 1 M ammonium acetate. The filters were subsequently removed from the support and baked at 80°C for 90 min to immobilize the DNA. Filters were then bathed in 100 ml of PTM [PBS-Tween 20 (0.1% Tween) + 0.5% fat-free milk powder] for 1 h at room temperature to inhibit unspecific antibody binding. After two 5-min washes with PBS-Tween 20 (0.1% Tween), the filters were bathed in 40 ml of PTM containing the anti-M1-dG monoclonal antibody D10A1 (36) , diluted 1:48,000. Filters were incubated for 2 h at room temperature, followed by overnight incubation at 4°C. After one 1-min and two 5-min washes with PBS-Tween, the filters were incubated for 2 h at room temperature with horseradish peroxidase-conjugated secondary antibody (goat antimouse IgG; Dako A/S, Glostrup, Denmark), diluted 1:4,000 in 32 ml of PTM. After additional washes in PBS-Tween, the filters were incubated for 5 min with the chemiluminescent reagent consisting of 4 ml of luminol/enhancer solution plus 4 ml of stable peroxidase buffer (SuperSignal; West Dura, Pierce, Rockford, IL). An image of the filters was acquired using a Fluor-S MultiImager (Bio-Rad, Hercules, CA) and the following setting: filter: chemiluminescence; integration: manual (time ranging from 5 to 20 min depending on the intensity of the signal); light source: Chemi: no light; scan width: 80 mm; high sensitivity. The intensity of chemiluminescent signal for each band was determined using the image analysis software. Adduct level in each sample was determined from the calibration line generated by MDA- modified DNA (diluted with control DNA) containing known amounts of M1-dG.

Propidium Iodide Staining for Quantitation of DNA Bound to the Nitrocellulose Filter
The nitrocellulose filters were washed overnight in PBS and incubated with propidium iodide (250 µg) in 50 ml of PBS for 3 h at room temperature and in the dark. The filters were then washed with PBS for 90 min. An image of the filter was captured using the Bio-Rad Fluor-S MultiImager with the following settings: filter: 520 nm; integration: automatic; light source: Epi UV light; high resolution. The intensity of fluorescent signal for each band, which was proportional to the amount of DNA bound to the filter (data not shown), was determined using the image analysis software. The level of adducts in each sample was corrected for the amount of DNA bound to the filter (37) . M1-dG levels are given as adducts per 107 total normal bases.

Statistical Methods
Results for DNA adducts were subjected to Kruskal-Wallis ANOVA followed by Mann-Whitney test. The difference between distributions of cases and controls was evaluated using the {chi}2 test and Fisher’s exact test. Minitab (for Windows) was used in the analysis. Associations of food and nutrients with M1-dG were evaluated by Pearson’s correlation analysis. Statistical packages SAS version 6.12 and STATA version 6 were used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Subject Characteristics.
Dietary data and colorectal biopsies from normal mucosa were available from 100 men and 62 women participating in both the United Kingdom Flexiscope Sigmoidoscopy Screening Trial and the EPIC study. Of the 162 participants, 83 were cases with adenomas and 79 were polyp-free controls. Twenty-nine of the cases, 11 women and 18 men, had high-risk adenomas. Average age for men and women was 58 ± 3 years. The mean values for weight, height, and BMI in men and women are summarized in Table 1Citation . There were no significant differences between cases and controls. Women were shorter but had a higher BMI than men. Of all participants, 9.5% were current smokers, 10 and 9% in men and women, respectively.


View this table:
[in this window]
[in a new window]
  		Table 1 Anthropometric characteristics and MDA-adduct levels (M1-dG per 107 total normal bases) for all of the participants, men and women in cases (Polyps) and controls (C)a

 
DNA Adducts.
DNA samples were analyzed using a previously developed ISB assay, which has a limit of detection of ~0.2 adducts per 107 total bases (34) . This assay, which uses a specific monoclonal antibody against M1-dG (36) , requires only 1 µg of DNA/sample (in triplicate). DNA extraction from colorectal biopsies using a Qiagen kit, as described in "Materials and Methods," gave a yield that varied between 5 to ~200 µg of DNA. Enough DNA for ISB analysis was therefore obtained from all biopsy samples. In the present work, adduct levels measured by ISB in each sample, were corrected for the amount of DNA bound to the filter, as determined by propidium iodide staining.

M1-dG adducts were detected in 92% of the samples analyzed. Thirteen samples had adduct levels below the limit of detection of the assay.

Substantial interindividual variation was observed, with adduct levels ranging from undetectable to 12.23 M1-dG per 107 total bases (Fig. 1)Citation . The mean adduct levels were 4.30 ± 3.0 and 4.60 ± 2.9 per 107 bases in men and women, respectively. There was no significant difference between the sexes. A significant positive association between adduct levels and age was observed in men (P < 0.01), whereas women showed only a weak, not-significant correlation (P = 0.5). A negative correlation between adducts and BMI was evident in men (P < 0.02), whereas no correlation was observed in women (Table 1)Citation . In men, M1-dG levels were also significantly correlated with height (P < 0.05). No correlation with smoking status was observed either in men or in women.



View larger version (60K):
[in this window]
[in a new window]
  		Fig. 1. Distribution plot of M1-dG levels in normal colorectal mucosa of cases and controls (men and women). M1-dG levels ranged from undetectable (n = 13) to 12.23/107 total bases (average: 4.42 ± 2.95/107 bases). *, the difference between the distribution of cases and controls is marginally significant (P = 0.065, by {chi}2 test).

 
DNA Adducts and Diet.
Information from the 7-day dietary diaries was computerized and converted into estimates of intake for a series of more than 30 food items and nutrients. Significant associations between various food components and adduct levels were observed, with marked differences between men and women (Table 2)Citation .


View this table:
[in this window]
[in a new window]
  		Table 2 Associations of selected food groups with M1-dG adducts in male and female participantsa

 
In men, M1-dG levels were inversely associated (P <= 0.05) with the reported consumption of legumes, nuts, and whole meal bread. Cereals, fruit, vegetables, salads, and raw tomatoes showed inverse association with adduct levels, but these were not significant (data not shown). There were significant correlations with adduct levels and beer, offal, white meat, and alcoholic drinks.

In women, there were no significant associations between adduct levels and food consumption.

Intakes of nutrients were also related to adduct levels. Table 3Citation shows associations for those nutrients that would be expected to relate to MDA adducts, e.g., fat, fatty acids, and antioxidant vitamins. All of the associations with adduct levels failed to reach significance, although there was a weak positive association with saturated fatty acids in women (P = 0.061). The ratio of monounsaturated:saturated fatty acids was inversely correlated with M1-dG levels in women, although the correlation was only slightly significant (P = 0.09). In addition, the ratio of polyunsaturated:saturated fatty acids was inversely associated with adduct levels in women (P = 0.019). No association between fatty acids and M1-dG was observed in men.


View this table:
[in this window]
[in a new window]
  		Table 3 Associations of selected nutrient intake with M1-dG levelsa

 
DNA Adducts and Adenomatous Polyps.
Table 1 also shows mean adduct levels in participants found to have adenomas, compared with adenoma-free individuals. Mean differences were not significant, although Fig. 1Citation showed that cases tended to have higher levels. Twelve individuals with polyps had adducts levels above 9 per 107 bases, compared with 1 polyp-free individual. {chi}2 testing of differences in adduct levels between the distributions (Fig. 1)Citation in cases and controls failed to reach statistical significance (P < 0.065). However, Fisher’s exact test, not taking into account that the analysis was data driven, showed that there was a large excess of cases (12) compared with controls (1) in this category (P < 0.005). Five of the 12 samples with M1-dG levels above 9 per 107 bases were from patients presenting with high-risk adenomas.

In females, a trend in relation to the severity of the adenomas was observed (Fig. 2)Citation . M1-dG levels (per 107 total bases) increased from 4.06 ± 2.0 in controls to 4.76 ± 3.6 in low-risk and to 5.40 ± 2.6 in high-risk women. The increase from controls to high-risk women was marginally significant (P = 0.09) by Kruskal-Wallis ANOVA followed by Mann-Whitney U test. There were no differences in men.



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2. M1-dG levels in low-risk (LR) adenomas, high-risk (HR) adenomas, and controls (C) for all of the participants, men and women. Values are means ± SD. *, the difference between controls and high-risk women is marginally significant (P = 0.09) by Kruskal-Wallis ANOVA followed by Mann-Whitney U test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Colorectal biopsies from normal mucosa of 162 participants in both the United Kingdom Flexiscope Sigmoidoscopy Screening Trial and the EPIC study were analyzed for the presence of M1-dG, the major DNA adduct formed by reaction of MDA with DNA. The objectives of the study were to investigate whether this adduct can be regarded as a biomarker of specific dietary intake and whether it can be considered a risk factor for the presence of premalignant lesions in humans.

Samples were analyzed using a quick and sensitive ISB assay, which allows a high throughput of samples in large population-based studies, using only small amounts of DNA.

The present study has shown for the first time that M1-dG is present in the normal colorectal mucosa in humans. The levels measured had an average value, considering all participants, of 4.42 ± 2.95 per 107 total bases. These levels are lower than those detected in our laboratory in human gastric biopsy DNA (23) . It is likely that this difference in M1-dG levels is attributable to tissue-specific differences in adduct formation and removal.

In the present study, there was substantial interindividual variation in adduct levels. Levels in women were slightly, but not significantly, higher than in men. Fang et al. (28) reported higher levels of adducts in WBCs of women as compared with men. Similar findings were reported in the same leukocyte samples for etheno adducts, another product of lipid peroxidation (38) . Similar to our findings for colorectal biopsies, no sex differences in M1-dG levels were reported for gastric tissue (23) .

In general, associations between adduct levels and anthropometric as well as environmental factors were more obvious in men than in women. Here, taller men had higher adduct levels, and there was a significant inverse association with BMI. This was an unexpected finding that is difficult to explain because a positive association would have been predicted from the fact that cohort and prospective studies suggest that excessive body mass is positively correlated with CRC risk (6 , 7) . No correlation was observed in women. Adduct levels increased with age in men but not in women. No relationship with smoking was found, which confirmed other authors findings (23 , 24) .

One objective of our study was to investigate whether M1-dG levels in normal colorectal mucosa were modulated by dietary and life-style habits. The hypothesis was that diets rich in PUFAs, which are precursors of MDA, or poor in fruits and vegetables or other antioxidant sources might be related to higher levels of M1-dG.

The effect of dietary habits, as recorded by carefully collected prospective diaries of all food and drink consumed over a period of 7 days, differed between men and women. A stronger dietary modulation of M1-dG levels was observed in men, with legumes, nuts, whole meal bread, cereals, fruits, vegetables, salads, and raw tomatoes being protective. High consumption of some of these food items has been associated with protection against colon and rectal cancer. Antioxidants present in fruits and vegetables could decrease formation of M1-dG by inhibiting either lipid peroxidation or direct DNA oxidation, which are believed to give rise to M1-dG through the formation of base propenal intermediates (25) . Alternatively, micronutrients and bioactive compounds, such as phenols and flavonoids, could stimulate detoxification pathways and DNA repair mechanisms (6 , 7) .

High intake of beer, alcohol, and white meat was positively correlated with adduct levels in men. Epidemiological studies have reported either an increased risk or no association between beer/alcohol consumption and CRC (7) . In studies of diet and colorectal adenomas, alcohol is usually considered to be a potential confounder, although there is little clear evidence of effects in the literature (39, 40, 41) .

There were no associations between adduct levels and the recorded consumption of about 30 groups of food in women, possibly because of the smaller number of samples analyzed and, therefore, less statistical power. Differences between the sexes in leukocyte adduct levels in response to diet have been reported in a smaller previous study, in which mainly younger and presumably premenopausal women were investigated (28) . In the present study, all of the women were ages 55–65 years, and colonic adduct levels were measured.

Here, there was a weak positive association between rectal biopsy M1-dG levels and saturated fatty acid intake in women and an inverse association with the standard dietary ratio of PUFA:saturated fat. The effect of saturated fat on M1-dG adduct levels has not been investigated elsewhere, although the previous study had shown higher levels in leukocytes with PUFAs than with monounsaturated fatty acids (28) . To investigate the effect of saturated fatty acids in leukocytes, a randomized controlled trial is under way at the Dunn Human Nutrition Unit (Cambridge, England), comparing the effects of saturated fatty acids, polyunsaturated {omega}-6 fatty acids, and vitamin E on M1-dG.

The present results could be explained by the fact that different tissues have been analyzed, compared with a Fang et al. study (colorectal mucosa versus leukocytes), and that there was no differentiation between {omega}-6 and {omega}-3 fatty acid intake. However, because of the complexity of the dietary data collected in EPIC and only limited food composition data on specific fatty acids in the United Kingdom food tables (32 , 33) , we are not yet able to accurately differentiate between {omega}-6 PUFAs and {omega}-3 PUFAs. Whereas {omega}-6 PUFAs have been shown to enhance colon tumorigenesis in rodents, {omega}-3 PUFAs can act as growth inhibitors of initiated cancer cells (42 , 43) . {omega}-3 PUFAs could, therefore, have had a confounding effect in the present analysis. However, no associations with fatty fish consumption (the major source of {omega}-3 fatty acids) were shown.

The other objective of our study was to analyze M1-dG levels in relation to the presence or absence of adenomatous polyps, to examine whether adducts could be related to disease outcome. A trend toward higher levels of adducts in cases than in controls was observed for both men and women, although the difference failed to reach significance. The difference in adduct levels between women presenting with high risk adenomas and polyp-free controls was statistically significant, although only marginally (Fig. 2)Citation . This is probably attributable to the wide interindividual variation in adduct levels and consequent high SDs from the adduct means. In addition, the number of women in the high-risk group was quite small (n = 11).

At present, the relationship between MDA-DNA damage and the risk for adenomatous polyps remains unclear. What is probably very important is the location of M1-dG in DNA, i.e., which genes are modified, and the rate to which the damage is converted into mutations. DNA repair rate and fidelity are also extremely important. Moreover, other endogenous as well as exogenous DNA damage has been reported in human colorectal tissue (44, 45, 46, 47) . A previous study (48) has demonstrated increased levels of bulky and/or aromatic DNA adducts associated with increased CRC risk. It is likely that the contribution of DNA damage derived by both exogenous and endogenous sources, together with host susceptibility factors such as polymorphisms in relevant genes (49) , could be of importance in colorectal carcinogenesis.

In conclusion, M1-dG was detected in the normal colorectal mucosa of participants in the United Kingdom Flexiscope Sigmoidoscopy Screening Trial and the EPIC study. Food items that, in epidemiological studies, were associated with modulation of CRC risk, modulated M1-dG levels in men. Saturated fat was correlated to increased adduct levels in women. Cases showed higher adduct levels than did controls, although the difference was not statistically significant. Results from the present study raise the possibility that M1-dG plays a role in human colorectal carcinogenesis, certainly in combination with other genetic and environmental factors.


   Acknowledgments
 
We thank Mike Mulheran for part of the statistical analyses and Ricky Sharma for critically reading the manuscript. The EPIC Norfolk Study is funded by the Cancer Research Campaign, the Medical Research Council, the British Heart Foundation, the Food Standards Agency, the Department of Health, and the Europe Against Cancer Program of the Commission of the European Communities. The United Kingdom Flexible Sigmoidoscopy Screening Trial is funded by the Medical Research Council, National Health Service R&D, and the Imperial Cancer Research Fund. We are grateful to all of the participants and general practitioners who have helped with this study, and to the nurses, technicians, and staff of the EPIC coordinating center.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by a contract from the United Kingdom Food Standards Agency (to C. L., E. J. D., R. S., and D. E. G. S.) and NIH Grant CA-77839 (to L. J. M.) Back

2 To whom requests for reprints should be addressed, at MRC Toxicology Unit, University of Leicester, Leicester LE1 9HN, United Kingdom. Phone: 44-116-2525172; Fax: 44-116-2525616; Email: chiara_leuratti{at}hotmail.com Back

3 Present address: Chemistry Department, The Open University, Milton Keynes, MK7 6AA United Kingdom. Back

4 The abbreviations used are: CRC, colorectal cancer; MDA, malondialdehyde; PUFA, polyunsaturated fatty acid; M1-dG, 1,N2-malondialdehyde-deoxyguanosine; EPIC, European Prospective Investigation on Cancer; CT-DNA, calf thymus DNA; HPLC, high-pressure liquid chromatography; BMI, body mass index; ISB, immunoslot blot; PTM, PBS-Tween 20 + fat-free milk powder. Back

Received 7/ 6/01; revised 12/ 7/01; accepted 1/ 6/02.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Midgley R., Kerr D. Colorectal cancer. Lancet, 353: 391-399, 1999.[Medline]
  2. Doll R., Peto R. The causes of cancer: quantitative estimates of avoidable risks of cancer in the US today. J. Natl. Cancer Inst. (Bethesda), 66: 1191-1308, 1981.
  3. Willett W. C. Diet, nutrition and avoidable cancer. Env. Health Persp., 103: 165-170, 1995.
  4. Fearon E. R., Vogelstein B. A genetic model for colorectal tumorigenesis. Cell, 61: 759-767, 1990.[Medline]
  5. Fearon E. R., Jones P. A. Progressing towards a molecular description of colorectal cancer development. FASEB J., 6: 2783-2790, 1992.[Abstract]
  6. Potter J. D. Colorectal cancer: molecules and populations. J. Natl. Cancer Inst. (Bethesda), 91: 916-932, 1999.[Abstract/Free Full Text]
  7. World Cancer Research Fund (WCRF) Panel, J. D. Potter (Chair). . Food, Nutrition and the Prevention of Cancer: A Global Perspective, WCRF/American Institute for Cancer Research Washington DC 1997.
  8. Report of the Working Group on Diet, and Cancer of the Committee on Medical Aspects of Food, and Nutrition Policy (COMA). . Nutritional Aspects of the Development of Cancer. Report on Health and Social Subjects, No. 48, Department of Health, Her Majesty Stationery Office London, UK 1998.
  9. Giovannucci E., Goldin B. The role of fat, fatty acids and total energy intake in the etiology of human colon cancer. Am. J. Clin. Nutr., 66 (Suppl.): 1564S-1571S, 1997.[Abstract/Free Full Text]
  10. Lee I. M., Paffenbarger R. S., Hsieh C-C. Physical activity and risk of developing colorectal cancer among college alumni. J. Natl. Cancer Inst. (Bethesda), 83: 1324-1329, 1991.[Abstract/Free Full Text]
  11. Slattery M. L., Edwards S. L., Ma K-N., Friedman G. D., Potter J. D. Physical activity and colon cancer: a public health perspective. Ann. Epidemiol., 7: 137-145, 1997.[Medline]
  12. Ozdemirler G., Pabuccoglu H., Bulut T., Bugra D., Uysal M., Toker G. Increased lipoperoxide levels and antioxidant system in colorectal cancer. J. Cancer Res. Clin. Oncol., 124: 555-559, 1998.[Medline]
  13. Hendickse C. W., Kelly R. W., Radley S., Donovan I. A., Keighley M. R. B., Neoptolemos J. P. Lipid peroxidation and prostaglandins in colorectal cancer. Br. J. Surg., 81: 1219-1223, 1994.[Medline]
  14. Marnett L. J. Oxyradicals and DNA damage. Carcinogenesis (Lond.), 21: 361-370, 2000.[Abstract/Free Full Text]
  15. Marnett L. J. Lipid peroxidation—DNA damage by malondialdehyde. Mutat. Res., 424: 83-95, 1999.[Medline]
  16. Marnett L. J. Generation of mutagens during arachidonic acid metabolism. Cancer Metastasis Rev., 13: 303-308, 1994.[Medline]
  17. Basu A. K., Marnett L. J. Unequivocal demonstration that malondialdehyde is a mutagen. Carcinogenesis (Lond.), 4: 331-333, 1983.[Abstract/Free Full Text]
  18. Marnett, L. J. DNA adducts of {alpha}, ß-unsaturated aldehydes and dicarbonyl compounds. In: K. Hemminki, A. Dipple, D. E. G. Shuker, F. F. Kadlubar, D. Segerback, and H. Bartsch (eds.), DNA Adducts: Identification and Biological Significance, IARC Sci. Publ. 125, pp. 151–163. Lyons, France: IARC, 1994.
  19. Chaudhary A. K., Nokubo M., Reddy G. R., Yeola S. N., Morrow J. D., Blair I. A., Marnett L. J. Detection of endogenous malondialdehyde-deoxyguanosine adducts in human liver. Science (Wash. DC), 265: 1580-1582, 1994.[Abstract/Free Full Text]
  20. Vacal C. E., Fangl J-L., Mutanen M., Valsta L. 32P-postlabelling determination of DNA adducts of malondialdehyde in humans: total white blood cells and breast tissue. Carcinogenesis (Lond.), 16: 1847-1851, 1995.[Abstract/Free Full Text]
  21. Wang M., Dhingra K., Hittelman W. N., Liehr J. G., de Andrade M., Li D. Lipid peroxidation-induced putative malondialdehyde-DNA adducts in human breast tissues. Cancer Epidemiol. Biomark. Prev., 5: 705-710, 1996.[Abstract]
  22. Rouzer C. A., Chaudary A. K., Nokubo M., Ferguson D. M., Reddy G. R., Blair I. A., Marnett L. J. Analysis of the malondialdehyde-2'-deoxyguanosine adduct pyrimidopurinone in human leukocyte DNA by gas chromatography/electron capture/negative chemical ionization/mass spectrometry. Chem. Res. Toxicol., 10: 181-188, 1997.[Medline]
  23. Everett S. M., Singh R., Leuratti C., White K. L. M., Neville P., Greenwood D., Marnett L. J., Schorah C. J., Forman D., Shuker D. E. G., Axon A. T. R. Levels of malondialdehyde-deoxyguanosine in the gastric mucosa—relationship with lipid peroxidation, ascorbic acid and Helicobacter pylori. Cancer Epidemiol. Biomark. Prev., 10: 369-376, 2001.[Abstract/Free Full Text]
  24. Kadlubar F. F., Anderson K. E., Lang N. P., Thompson P. A., MacLeod S. L., Chou M. W., Mikhailova M., Plastaras J. P., Marnett L. J., Haussermann S., Nair J., Velic I., Bartsch H. Comparison of DNA adduct levels associated with oxidative stress in human pancreas. Mutat. Res., 405: 125-133, 1998.[Medline]
  25. Dedon P. C., Plastaras J. P., Rouzer C. A., Marnett L. J. Indirect mutagenesis by oxidative DNA damage: formation of the pyrimidopurinone adduct of deoxyguanosine by base propenal. Proc. Natl. Acad. Sci. USA, 95: 11113-11116, 1998.[Abstract/Free Full Text]
  26. Benamira M., Johnson K., Chaudhary A., Bruner K., Tibbetts C., Marnett L. J. Induction of mutations by replication of malondialdehyde-modified M13 DNA in Escherichia coli: determination of the extent of DNA modification, genetic requirements for mutagenesis, and types of mutations induced. Carcinogenesis (Lond.), 16: 93-99, 1995.[Abstract/Free Full Text]
  27. Fink S. P., Reddy G. R., Marnett L. J. Mutagenicity in Escherichia coli of the major DNA adduct derived from the endogenous mutagen malondialdehyde. Proc. Natl. Acad. Sci. USA, 94: 8652-8657, 1997.[Abstract/Free Full Text]
  28. Fang J-L, Vaca C. E., Valsta L. M., Mutanen M. Determination of DNA adducts of malondialdehyde in humans: effects of dietary fatty acid composition. Carcinogenesis (Lond.), 17: 1035-1040, 1996.[Abstract/Free Full Text]
  29. Leuratti, C., Singh, R., Deag, E. J., Griech, E., Hughes, R. M., Bingham, S. A., Plastaras, J. P., Marnett, L. J., and Shuker, D. E. G. A sensitive immunoslot-blot assay for the detection of malondialdehyde-deoxyguanosine in human DNA. In: B. Singer and H. Bartsch (eds.), Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis, IARC Sci. Publ. 150, pp. 197–203. Lyons, France: IARC, 1999.
  30. Atkin, W. S., Edwards, R., Wardle, J., Northover, J. M. A., Sutton, S., Hart, A. R., Williams, C. B., and Cuzick, J. Rationale and design of a multicentre randomised trial to evaluate the suitability of flexible sigmoidoscopy as a mass population screening tool to reduce colorectal cancer morbidity and mortality. J. Med. Screen., in press. 2002.
  31. Day N. E., Oakes S., Luben R., Khaw K. T., Bingham S. M., Welch A., Wareham N. EPIC in Norfolk: study design and characteristics of the cohort. Br. J. Cancer, 80 (Suppl. 1): 95-103, 1999.
  32. Bingham S., Welch A., McTaggart A., Mulligan A., Runswick A. A., Luben R., Oakes S., Khaw K. T., Wareham N., Day N. E. European prospective investigation of cancer in Norfolk: nutritional methods and studies. Public Health Nutr., 4: 847-858, 2002.
  33. Welch A. A., McTaggart A., Mulligan A. A., Luben R., Walker N., Khaw K. T., Day N. E., Bingham S. A. DINER (Data into Nutrients for Epidemiological Research) a new data entry program for nutritional analysis in the EPIC-Norfolk cohort. Public Health Nutr., 4: 1753-1765, 2002.
  34. Leuratti C., Singh R., Lagneau C., Farmer P. B., Plastaras J. P., Marnett L. J., Shuker D. E. G. Determination of malondialdehyde-induced DNA damage in human tissues using an immunoslot blot assay. Carcinogenesis (Lond.), 19: 1919-1924, 1998.[Abstract/Free Full Text]
  35. Chapeau M-C., Marnett L. J. Enzymatic synthesis of purine deoxynucleoside adducts. Chem. Res. Toxicol., 4: 636-638, 1991.[Medline]
  36. Sevilla C. L., Mahle N. H., Eliezer N., Uzieblo A., O’Hara S. M., Nokubo M., Miller R., Rouzer C. A., Marnett L. J. Development of monoclonal antibodies to the malondialdehyde-deoxyguanosine adduct, pyrimidopurinone. Chem. Res. Toxicol., 10: 172-180, 1997.[Medline]
  37. Singh R., Leuratti C., Josyula S., Sipowicz M. A., Diwan B. A., Kasprzak K. S., Schut H. A. J., Marnett L. J., Anderson L. M., Shuker D. E. G. Liver lobe-specific increases in malondialdehyde-DNA adduct formation in the livers of mice following infection with Helicobacter hepaticus. Carcinogenesis (Lond.), 22: 1281-1287, 2001.[Abstract/Free Full Text]
  38. Nair J., Vaca C. E., Velic I., Mutanen M., Valsta L. M., Bartsch H. High dietary {omega}-6 polyunsaturated fatty acids drastically increase the formation of etheno-DNA base adducts in white blood cells of female subjects. Cancer Epidemiol. Biomark. Prev., 6: 597-601, 1997.[Abstract]
  39. Breuer-Katschunski B., Nemes K., Marr A., Rump B., Leindecker B., Bruer N., Goebell H. Alcohol and cigarette smoking and the risk of colorectal adenomas. Dig. Dis. Sci., 45: 487-493, 2000.[Medline]
  40. Sandler R., Lyles C., Peipens L., McAuliffe C., Woosely Kupper L. Diet and risk of colorectal adenomas. J. Natl. Cancer Inst. (Bethesda), 85: 884-891, 1998.[Abstract/Free Full Text]
  41. Giovannucci E., Stampfer M., Colditz G., Rimm E., Willett W. C. Relationship of diet to colorectal adenoma in men. J. Natl. Cancer Inst. (Bethesda), 84: 91-98, 1992.[Abstract/Free Full Text]
  42. Torosian M. H., Charland S. L., Lippin J. A. Differential effects of {omega}-3 and {omega}-6 fatty acids on primary tumour growth and metastasis. Int. J. Oncol., 7: 667-672, 1995.
  43. Singh J., Hamid R., Reddy B. S. Dietary fat and colon cancer: modulation of cyclooxygenase-2 by types and amount of dietary fat during the postinitiation stage of colon carcinogenesis. Cancer Res., 57: 3465-3470, 1997.[Free Full Text]
  44. Pool-Zobel B. L., Abrahamse S. L., Collins A. R., Kark W., Gugler R., Oberreuther D., Gunther, Siegel E., Trptow-van Lihaut S, Rechkemmer G. Analysis of DNA strand breaks, oxidised bases, and glutathione S-transferase P1 in human colon cells from biopsies. Cancer Epidemiol. Biomark. Prev., 8: 609-614, 1999.[Abstract/Free Full Text]
  45. Povey A. C., Hall C. N., Badawi A. F., Cooper D. P., O’Connor P. J. Elevated levels of the pro-carcinogenic adduct, O6-methylguanine, in normal DNA from the cancer prone regions of the large bowel. Gut, 47: 362-365, 2000.[Abstract/Free Full Text]
  46. Nagao M., Wakabayashi K., Ushijima T., Toyota M., Totsuka Y, Sugimura T. Human exposure to carcinogenic heterocyclic amines and their mutational fingerprints in experimental animals. Environ. Health Perspect., 104 (Suppl.): 497-501, 1996.
  47. Alexandrov K., Rojas M., Kadlubar F. F., Lang N. P., Bartsch H. Evidence of anti-benzo[a]pyrene diolepoxide-DNA adduct formation in human colon mucosa. Carcinogenesis (Lond.), 17: 2081-2083, 1996.[Abstract/Free Full Text]
  48. Pfohl-Leszkowicz A., Grosse Y., Carriere V., Cugnenc P. H., Berger A., Carnot F., Beaune P., de Waziers I. High levels of DNA adducts in human colon are associated with colorectal cancer. Cancer Res., 55: 5611-5616, 1995.[Abstract/Free Full Text]
  49. Lai C., Shields P. G. The role of interindividual variation in human carcinogenesis. J. Nutr., 129 (Suppl.): 552S-555S, 1999.



This article has been cited by other articles:


Home page
 Cancer Res.Home page
M. H. Lewin, N. Bailey, T. Bandaletova, R. Bowman, A. J. Cross, J. Pollock, D. E.G. Shuker, and S. A. Bingham
Red Meat Enhances the Colonic Formation of the DNA Adduct O6-Carboxymethyl Guanine: Implications for Colorectal Cancer Risk
Cancer Res., February 1, 2006; 66(3): 1859 - 1865.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
M. C. Stern, K. D. Siegmund, R. Corral, and R. W. Haile
XRCC1 and XRCC3 Polymorphisms and Their Role as Effect Modifiers of Unsaturated Fatty Acids and Antioxidant Intake on Colorectal Adenomas Risk
Cancer Epidemiol. Biomarkers Prev., March 1, 2005; 14(3): 609 - 615.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
M. A. Murtaugh, C. Sweeney, K.-n. Ma, B. J. Caan, and M. L. Slattery
The CYP1A1 Genotype May Alter the Association of Meat Consumption Patterns and Preparation with the Risk of Colorectal Cancer in Men and Women
J. Nutr., February 1, 2005; 135(2): 179 - 186.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
M. Brink, M. P. Weijenberg, A. F. P. M. de Goeij, L. J. Schouten, F. D. H. Koedijk, G. M. J. M. Roemen, M. H. F. M. Lentjes, A. P. de Bruine, R.A. Goldbohm, and P. A. van den Brandt
Fat and K-ras mutations in sporadic colorectal cancer in The Netherlands Cohort Study
Carcinogenesis, September 1, 2004; 25(9): 1619 - 1628.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
R. A. Sharma and P. B. Farmer
Biological Relevance of Adduct Detection to the Chemoprevention of Cancer
Clin. Cancer Res., August 1, 2004; 10(15): 4901 - 4912.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
C. I.R. Gill, S. Haldar, S. Porter, S. Matthews, S. Sullivan, J. Coulter, H. McGlynn, and I. Rowland
The Effect of Cruciferous and Leguminous Sprouts on Genotoxicity, In vitro and In vivo
Cancer Epidemiol. Biomarkers Prev., July 1, 2004; 13(7): 1199 - 1205.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
L. A. VanderVeen, M. F. Hashim, Y. Shyr, and L. J. Marnett
Induction of frameshift and base pair substitution mutations by the major DNA adduct of the endogenous carcinogen malondialdehyde
PNAS, November 25, 2003; 100(24): 14247 - 14252.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
A. Barbin, H. Ohgaki, J. Nakamura, M. Kurrer, P. Kleihues, and J. A. Swenberg
Endogenous Deoxyribonucleic Acid (DNA) Damage in Human Tissues: A Comparison of Ethenobases with Aldehydic DNA Lesions
Cancer Epidemiol. Biomarkers Prev., November 1, 2003; 12(11): 1241 - 1247.
[Abstract] [Full Text]

Home page
 MutagenesisHome page
U. Nair, H. Bartsch, and J. Nair
Prevention of degenerative diseases; clues from studies investigating oxidative stress, Brussels, 13 November 2002
Mutagenesis, September 1, 2003; 18(5): 477 - 483.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
H. K. Roy, J. M. Gulizia, W. J. Karolski, A. Ratashak, M. F. Sorrell, and D. Tuma
Ethanol Promotes Intestinal Tumorigenesis in the MIN Mouse
Cancer Epidemiol. Biomarkers Prev., November 1, 2002; 11(11): 1499 - 1502.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leuratti, C.
Right arrow Articles by Bingham, S. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Leuratti, C.
Right arrow Articles by Bingham, S. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation