A diet high in fat and meat but low in dietary fibre increases the genotoxic potential of `faecal water'
Martin A. Rieger,
Alexandr Parlesak,
Beatrice L. Pool-Zobel1,
Gerhard Rechkemmer2 and
Christiane Bode3
Department of Physiology of Nutrition, Institute for Biological Chemistry and Nutritional Sciences, Hohenheim University (140), Garbenstraße 28, D-70599 Stuttgart,
1 Department of Nutritional Toxicology, Institute for Nutrition, Friedrich-Schiller-University Jena, Dornburger Straße 25, D-07743 Jena and
2 Institute for Nutritional Physiology, Federal Research Centre for Nutrition, Haid-und-Neu Straße 9, D-76131 Karlsruhe, Germany
 |
Abstract
|
---|
To determine the effects of different diets on the genotoxicity of human faecal water, a diet rich in fat, meat and sugar but poor in vegetables and free of wholemeal products (diet 1) was consumed by seven healthy volunteers over a period of 12 days. One week after the end of this period, the volunteers started to consume a diet enriched with vegetables and wholemeal products but poor in fat and meat (diet 2) over a second period of 12 days. The genotoxic effect of faecal waters obtained after both diets was assessed with the single cell gel electrophoresis (Comet assay) using the human colon adenocarcinoma cell line HT29 clone 19a as a target. The fluorescence and length of the tails of the comet images reflects the degree of DNA damage in single cells. The mean DNA damage, expressed as the ratio of tail intensity (fluorescence in the tail) to total intensity of the comet after incubation with faecal water from volunteers consuming diet 1 was about twice as high as for diet 2. The susceptibility of the cells incubated with faecal water to DNA damage caused by additional hydrogen peroxide treatment showed no significant differences between the two diets. Generation of oxidized pyrimidine and purine bases revealed no differences after pretreatment with both types of faecal water. The results indicate that diets high in fat and meat but low in dietary fibre increase the genotoxicity of faecal water to colonic cells and may contribute to an enhanced risk of colorectal cancer.
Abbreviations: BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; Endo III, Escherichia coli endonuclease III; FPG-protein, Escherichia coli formamidopyrimidine-DNA glycosylase; TI, tail intensity.
 |
Introduction
|
---|
Epidemiological studies have suggested that the incidence of colon cancer is influenced by the composition of the diet (16). In these studies, the risk of colorectal cancer was associated with a high intake of fat and meat and a low intake of dietary fibre and vegetables. However, the mechanisms which increase the risk of colorectal cancer are not clear. Several studies suggest that DNA damage in human colonic adenocarcinoma cells can be caused by specific food ingredients or their metabolic products (79). High meat consumption for instance leads to higher levels of amino compounds, nitrosamines, an altered gut flora and changed enzyme patterns (10,11). Factors can be generated which may be either cytotoxic or genotoxic themselves or increase the formation of genotoxic compounds in the gut lumen (12,13). Rafter et al. (14,15) demonstrated that the consumption of fat and meat results in an enhancement of erythrocyte lysis by faecal water of individuals who consumed these diets.
A suitable method to assess the genotoxic effects of faecal water is the single cell gel electrophoresis or Comet assay. The assay is a sensitive and rapid technique enabling the detection of DNA strand breaks and alkali-labile sites in individual cells (1620). It has already been used previously to measure genotoxicity of nutritional contaminants in human target tissues (12,21,22). The Comet assay has also been used to assess genotoxicity of faecal water in human colonic adenocarcinoma cell lines (8,9). In the present study, we have now compared the influence of the soluble components of faecal water on DNA damage in HT29 clone 19a cells after a diet rich in fat (50% of total energy intake), meat and sugar but poor in vegetables and free of wholemeal products (diet 1) to a second diet enriched in vegetables and wholemeal products but poor in fat (19% of total energy intake) and meat (diet 2).
The fragmentation of DNA of HT29 clone 19a cells has been shown not to underlie apoptosis-associated mechanisms (23), which is considered to be important in the measurement of genotoxic effects. Hydrogen peroxide (H2O2) is known to induce oxidative DNA damage (16,17). Therefore, HT29 clone 19a cells treated with faeces water were additionally exposed to H2O2 to assess whether this pretreatment alters the resistance of these cells to exogenous oxidative stress.
Oxidized pyrimidine and purine bases are an important type of damage to DNA and have been implicated in enhancing the processes of tumorigenesis (24). Escherichia coli endonuclease III (Endo III), an enzyme which specifically recognizes oxidized pyrimidine bases, and E.coli formamidopyrimidine-DNA glycosylase (FPG-protein), which recognizes 8-hydroxydesoxyguanosine and other purines, excise these oxidized bases from the DNA and induce nicks at the base sites (2527). These lesions result in a more extensive response in the Comet assay, which are a reflection of oxidative genetic damage. Steady-state levels of these lesions were measured after incubating the colon cells with the different faecal waters.
 |
Materials and methods
|
---|
Study design
The study was conducted with the approval of the Ethics Committee of the Robert Bosch Hospital with the informed consent of the participants. Seven healthy volunteers (four men and three women, median age 29, range 2757 years) were included in the study. The food quantities for each diet were calculated using standard formulae according to the individual energy requirements (28). The quantities were weighed to reach an identical composition of the diets per 10 MJ for each participant (Table I
). The composition of the diets was calculated using a standard nutrition table (29). To obtain identical meals for all participants, diets were prepared in the kitchen of the Robert Bosch Hospital (Stuttgart, Germany).
For the first 12 days of the study the volunteers received a diet rich in fat (50% of total energy intake), meat and sugar but poor in vegetables and free of wholemeal products (diet 1). This diet was followed by 1 week of `normal' nutrition to eliminate influences of diet 1, as a change in the effects of nutrition was shown in other studies even after 47 days (8,14). The nutrition in this week corresponded to the standard menu served at the hospital. During the second period of the study the volunteers received a diet enriched in vegetables and wholemeal products but poor in fat (19% of total energy intake) and meat (diet 2) over a second period of 12 days.
At the end of each 12 day period, faeces was collected on the morning of day 13. To enforce quantitative collection of the contents of the colon, the volunteers ingested 23 l of saline (154 mM NaCl), which was collected quantitatively in four fractions. The first and second faeces samples had normal consistencies, while the last two fractions consisted mainly of clear liquid only. Therefore, the first two samples were weighed, combined, homogenized, frozen, lyophilized and stored at 80°C until further processing.
Faecal water preparation
Double-distilled water was added to each lyophilized faeces sample to reconstitute its original water content before lyophilization. All faecal samples were coded so that the origin of the samples was unknown during further processing and analysis. The samples were homogenized (Ultra Turrax, model TP 1810; Janke & Kunkel KG, Staufen im Breisgau, Germany) and centrifuged at 36 000 g for 2 h at 4°C. The supernatant (i.e. `faecal water') was carefully decanted and used for incubation of HT29 cells and measurement of its ability to cause DNA strand breaks as detected by the Comet assay.
Comet assay
The Comet assay was performed according to McKelvey-Martin et al. (16) and Pool-Zobel et al. (27) with minor modifications. Microscope slides with one rough side were covered with 30 µl of normal melting point agarose (Sea Kern HGT Agarose; FMC BioProducts, Rockland, ME). After the agarose solidified, 85 µl of normal melting point agarose was applied on top of the first layer together with a coverslip. All slides were stored in a humidified box at 4°C and used within 48 h.
The human colon cell line HT29 was established by J.Fogh (Memorial Sloan Kettering Cancer Centre, New York, NY) in 1964 and its subclone 19a was terminally differentiated with 5 mM sodium butyrate (characterized by C.Augeron and C.Laboisse) (30). The HT29 clone 19a cells used in this assay were a kind gift of Laboisse and were cultured as monolayers in Dulbecco's modified Eagle's medium (DMEM, containing 10% fetal calf serum, 20 mM glutamine and 1% penicillin/streptomycin; Gibco BRL Life Technologies, Eggenstein, Germany) for 7 days at 37°C with 5% atmospheric CO2. For use in the genotoxicity assay with faecal water samples, cells were detached after 5 min incubation with 0.25% trypsinEDTA at 37°C, centrifuged for 5 min at 200 g and resuspended in DMEM at a concentration of 2x106 cells/ml.
The cell suspension (900 µl) was incubated at 37°C for 30 min with 100 µl faecal water in triplicate (Figure 1
). After incubation, 4x100 µl were taken from each sample and the cells were centrifuged for 8 min at 100 g. Cell membrane integrity, which correlates closely with cell viability (31) was determined in aliquots by trypan blue exclusion. After centrifugation, supernatants were removed and cells were resuspended in 75 µl of 0.7% low melting point agarose (Sea Plaque GTG Agarose; FMC BioProducts) dissolved in phosphate-buffered saline (pH 7.4, containing 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4·2H2O) and applied to the prepared slides. The agarose was allowed to solidify on ice and 75 µl of low melting point agarose was finally added on top of the cells. In an additional treatment, 50 µl of H2O2 (75 µmol/l) were applied on top of another slide of each sample for 5 min on ice and then washed three times in 0.9% saline solution for 5 s. All slides were then transferred into a lysis solution for 1 h at 4°C (pH 8.0, containing 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1% sodium N-laurylsarcosine, 1% Triton X-100 and 10% dimethylsulphoxide). After lysis, the cells on the third slide of each sample were washed three times with Endo III enzyme reaction buffer [40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml bovine serum albumin (BSA), pH 8.0] for 5 min. Endo III (50 µl, 1 µg/ml) was applied to the cells and incubated at 37°C for 45 min. On the fourth slide of each sample, cells were washed three times with FPG-protein reaction buffer (0.02 mM TrisHCl, 0.1 M NaCl, 1 mM EDTA, 0.5 mg/ml BSA, pH 7.5) for 5 min. FPG-protein (50 µl, 1 µg/ml) was applied to the cells and incubated at 37°C for 30 min. The controls were treated accordingly with cells that had been incubated with 100 µl of saline (0.9% NaCl solution) instead of faecal water.
All slides were then transferred to an electrophoresis chamber and placed in ice-cold electrophoresis buffer (1 mM Na2EDTA and 300 mM NaOH, pH 13.5) for 20 min to allow the DNA to unwind before applying a current of 300 mA at 25 V for 20 min. After electrophoresis, slides were washed with neutralization buffer (0.4 M Tris, pH 7.5), stained with 100 µl ethidium bromide (10 µg/ml) and stored at 4°C for not longer than 60 h before analysis. All preparative steps with the isolated HT29 clone 19a cells were performed under red light.
Slides were analysed at 500x magnification using a fluorescence microscope (Leica, Solms, Germany). The tail intensity (TI, percentage of fluorescence in the tail of the total comet) of 50 randomly selected cells on each slide was measured using an image analyser (Perceptive Instruments, Halstead, UK).
Statistical analysis
The results are expressed as mean TI as a percentage (±SD). The Wilcoxon matched pairs test was used for data comparison of the two diets. The MannWhitney U-test was used for data comparison between the diets and the control. Significant differences were considered at P < 0.05 and P < 0.01. The data were analysed using the STATISTICA for WindowsTM statistical software (release 5.1; StatSoft, Tulsa, OK).
 |
Results
|
---|
The total dry weight of faeces after subjects consumed diet 1 (49.9 ± 17.4 g) was significantly lower than that being excreted by subjects consuming diet 2 (69.3 ± 25.6 g).
Incubation of the HT29 clone 19a cells with faecal water obtained from volunteers having consumed diets 1 or 2 showed no detectable toxicity in the trypan blue exclusion assay in comparison with the toxicity of physiological saline. Cell viability exceeded 90% in all experiments (data not shown).
The mean TI of HT29 cell DNA after incubation with faecal water obtained after consumption of diet 1 was significantly higher (~2-fold) than the TI of the HT29 cell DNA treated with faecal water of volunteers who consumed diet 2 (Figure 2
).

View larger version (0K):
[in this window]
[in a new window]
|
Fig. 2. Effect of faecal water from volunteers consuming a diet rich in fat, meat and sugar but poor in vegetables and free of wholemeal products (diet 1, black shading) and a diet enriched in vegetables and wholemeal products but poor in fat and meat (diet 2, grey shading) and effect of the control (0.9% NaCl, no shading) on the tail intensity of DNA in HT29 clone 19a cells subjected to the Comet assay. Mean values of tail intensity ± SD, expressed as a percentage of total intensity; *P < 0.02 versus diet 2 with faecal water incubation, n = 7; P < 0.01 versus diets 1 and 2 with faecal water incubation, n = 7 for diet 1 and diet 2, n = 14 for the control.
|
|
The TI of HT29 cell DNA incubated with faecal water from diet 1 or with faecal water from diet 2 was considerably higher than that of HT29 cell DNA incubated with saline only as a control (Figure 2
).
When classifying the TI of HT29 cell DNA incubated with faecal water into five groups of different ranks of DNA damage intensity (Figure 3
), there was a pronounced higher proportion of cells with TI of >35% induced by faecal water from the diet 1 group than from the diet 2 group. Cells incubated with saline had only a few comets with a TI >6% (Figure 3
).

View larger version (0K):
[in this window]
[in a new window]
|
Fig. 3. Classification of 50 HT29 clone 19a cells, which were incubated for 30 min with faecal water from volunteers consuming a diet rich in fat, meat and sugar but poor in vegetables and free of wholemeal products (diet 1), a diet enriched in vegetables and wholemeal products but poor in fat and meat (diet 2) and with 0.9% NaCl (control), into five classes of tail intensity (TI), representing increased DNA damage; n = 7 for diets 1 and 2, n = 14 for the control.
|
|
No significant differences were found between the two diets when HT29 cells were additionally treated with H2O2 (Table II
). However, H2O2 treatment of HT29 cells which were preincubated with faecal water of volunteers who had consumed diets 1 and 2 were more sensitive to the genotoxic action of H2O2 than cells preincubated with physiological saline (Table II
).
View this table:
[in this window]
[in a new window]
|
Table II. Effect of faecal water from volunteers consuming a diet rich in fat, meat and sugar but poor in vegetables and free of wholemeal products (diet 1) and a diet enriched in vegetables and wholemeal products but poor in fat and meat (diet 2) and effect of 0.9% NaCl (control) on mean DNA damage in HT29 clone 19a cells before and after H2O2 (75 µmol/l) treatment and treatment with E.coli endonuclease (Endo III) (50 µl, 1 µg/ml) and E.coli formamidopyrimidine-DNA glycosylase (FPG-protein) (50 µl, 1 µg/ml)a
|
|
Additional E.coli Endo III treatment of DNA from HT29 cells preincubated with faecal water induced no significant differences in TI between the two diets (Table II
). Also, no differences were observed between the two diets after E.coli FPG-protein treatment. However, the TI after FPG-protein treatment were significantly higher than after treatment with Endo III (Table II
). Finally, Endo III as well as FPG-protein lesions were more pronounced after preincubation with faecal water from both diets than after preincubation with physiological saline, but revealed no differences between the two diets (Table II
).
 |
Discussion
|
---|
In the present study, the influence of faecal water on DNA damage in a human colon tumor cell line was investigated in samples obtained after two different dietary conditions. The Comet assay is a useful method to measure and quantify DNA damage (strand breaks) in individual cells (1618). According to the higher mobility of smaller DNA fragments during electrophoresis, the intensity of the tail increases in proportion to the extent of DNA damage (16).
The damage to DNA nearly doubled on treatment with faecal water from volunteers consuming a diet rich in fat, meat and sugar but poor in vegetables and free of wholemeal products (diet 1) compared with faecal samples after the diet enriched in vegetables and wholemeal products but poor in fat and meat (diet 2). One reason for the greater damage to DNA resulting from diet 1 might be a lower concentration of compounds possessing the ability to complex metal ions, such as dietary fibre (Table I
) or phytate. Phytate especially binds the transition metal Fe2+/3+, which catalyses the Fenton reaction (32). Thus, Fe2+/3+ could be responsible for increased formation of hydroxyl radicals after the release of superoxide by cells, produced by the respiratory chain. In previous investigations by our group (33), enhanced formation of hydroxyl radicals was found in faeces from subjects consuming diet 1 in comparison with those on diet 2. The significantly higher Fe2+/3+ concentration (+42%) in the faeces from diet 1 might have contributed to the ~13-fold increased production of hydroxyl radicals (33). These radicals can react directly with DNA resulting in oxidized bases and ultimately mutations after replication (34). Another reason for enhanced DNA fragmentation by extracts from faeces derived from diet 1 may be the lower intake of Ca2+ and Mg2+ ions (Table I
). These ions are thought to protect cells from the toxic effects of free fatty or bile acids by formation of insoluble complexes with these acids (7,9,35). Consumption of diet 1 also resulted in a lower intake of carotene and vitamin C (Table I
). Both substances have antioxidant properties and are hypothesized to decrease cancer risk by preventing cell damage by trapping free radicals or quenching singlet oxygen (3638). Nitrosamines, which are present in meats processed by nitrite curing and formed endogenously in the stomach lumen at acidic pH from amine and nitrite precursors, could also contribute to the higher genotoxicity of the faeces from diet 1 (13). Furthermore, a change in dietary fibre and fat consumption has been shown to alter both the composition and the metabolic processes of the gut flora (39,40). Changes in the intestinal flora might proceed in parallel with altered short chain acid production or bile acid metabolism (41,42) after elevated dietary fibre ingestion. The role of these compounds has to be clarified by further investigations of their impact on DNA fragmentation.
The significantly higher DNA damage in HT29 cells after incubation with faecal water of both diets in comparison with incubation with physiological saline implies that faecal water itself is genotoxic. This genotoxic effect has to be seen in contrast to the high percentage of cells with an integral cell membrane (>90%), which can be assumed to be viable. Loss of cell integrity, which was almost unaffected by faecal water in comparison with saline, seems therefore not to parallel the damage caused to DNA. In comparable studies, cell viability or cell survival after incubation with faecal water differs strongly from a very low cell survival of 20 ± 22% (8) to a cell viability of ~90% (9).
The standard deviation of DNA damage induced by faecal water samples after consumption of the same diet in the present study was quite low (14.0% for diet 1 and 6.9% for diet 2, as shown in Figure 2
). This is in contrast to other studies, where the volunteers did not consume identical diets and the results of faecal water-induced genotoxicity showed a high standard deviation. Glinghammar et al. (8) showed, in a crossover study with 12 volunteers, a standard deviation of DNA damage in CACO-2 cells after incubation with faecal water from a dairy product-rich diet of 93.4% and after a dairy product-free diet of 90.8%. Venturi et al. (9) demonstrated a standard deviation of DNA damage in CACO-2 cells of 113% for faecal water from a group of 12 Swedish people and 86.4% for a group of 23 English people. The low variations in the current study might result from identical diets according to the individual energy requirement for each participant. Alternatively, the unstable compounds causing the high variations of the quoted studies may no longer be present in the freeze dried samples used in this study. In any case, this point may play an important role in the comparison of the genotoxicity of faecal waters in further investigations.
In addition to the influence of faecal water on DNA damage in colonic cells, we also measured DNA fragmentation induced by H2O2 treatment of HT29 cells preincubated with faecal water of volunteers on diets 1 and 2, respectively. H2O2 is a common intermediate in a variety of oxidative stress situations (16,17) and has been demonstrated to induce DNA damage (20,27,43). H2O2 treatment of cells preincubated with faecal water from diet 2 showed a significant increase in strand breaks in comparison with cells incubated with faecal water from diet 2 not treated with H2O2. On the other hand, H2O2 treatment of cells preincubated with faecal water from volunteers who consumed diet 1 did not show a significant increase in DNA fragmentation in comparison with cells incubated with faecal water from diet 1. A possible explanation for these findings may be a saturation of strand breaks induced by genotoxic agents in the faecal water from diet 1, where the additional treatment with 75 µmol H2O2 did not result in an additional increase in DNA breakage.
To quantify the oxidative nucleic acid modifications in cellular DNA after incubation with faecal water resulting in single-strand breaks, we treated isolated cell DNA with E.coli Endo III and FPG-protein. Endo III and FPG-protein are specific for the cleavage of oxidized pyrimidine (Endo III) and purine (FPG-protein) bases, nicking the DNA at sites of oxidative damage (25,27). These enzymes play a fundamental role in the DNA repair system and are useful tools for revealing oxidized bases in the Comet assay. In both diets, the results show a higher level of DNA damage in cells after FPG-protein treatment than after treatment with Endo III. A reason for this may be differences in the activities of the two enzymes or a higher number of unsaturated bonds in purine bases, resulting in the formation of more stable intermediates after reaction with reactive oxygen species. Endo III treatment of cells preincubated with faecal water of volunteers who consumed diet 2 resulted in a significant increase in oxidized pyrimidine bases in comparison with cells incubated with faecal water from diet 2. On the other hand, oxidized pyrimidines were not increased with faecal water of volunteers who consumed diet 1, which may be due to the higher basic genotoxicity of diet 1.
HT29 cells showed a marked increase in DNA damage when cells of the controls were incubated with both enzymes. This indicates a high number of oxidixed pyrimidine and purine bases and represents unrepaired oxidative damage of DNA bases in the HT29 clone 19a cells. In primary human colon cells from biopsies similar increases were observed (22), whereas other human colon adenocarcinoma cells, like CACO-2 cells, did not show increased DNA damage under comparable conditions (9).
The present findings indicate that a diet rich in fat, meat and sugar but poor in vegetables and free of wholemeal products may significantly increase the genotoxicity of faecal water to colonic cells and therefore may increase the risk of colorectal cancer development.
 |
Acknowledgments
|
---|
The authors would like to thank Jürgen Erhardt for the collection of the faeces and Daniela Oberreuther for preparation of the HT29 clone 19a cells. Parts of this work were supported by the Deutsche Krebshilfe e. V.
 |
Notes
|
---|
3 To whom correspondence should be addressed Email: bodech{at}uni-hohenheim.de 
 |
References
|
---|
-
McKeown-Eyssen,G.E. and Bright-See,E. (1984) Dietary factors in colon cancer: international relationships. Nutr. Cancer, 6, 160170.[Medline]
-
Hill,M.J. (1997) Nutrition and human cancer. Ann. N.Y. Acad. Sci., 833, 6878.[Abstract]
-
Willett,W. (1989) The search for the causes of breast and colon cancer. Nature, 338, 389394.[ISI][Medline]
-
Weisburger,J.H. (1991) Carcinogens in our food and cancer prevention. Adv. Exp. Med. Biol., 289, 137151.[Medline]
-
Miller,A.B., Berrino,F., Hill,M., Pietinen,P., Riboli,E. and Wahrendorf,J. (1994) Diet in the aetiology of cancer: a review. Eur. J. Cancer, 30A, 207220.
-
World Cancer Research Fund and American Institute for Cancer Research (1997) Food, Nutrition and Prevention of Cancer: a Global Perspective. American Institute for Cancer Research, Washington, DC.
-
Allinger,U.G., Johansson,G.K., Gustafsson,J.A. and Rafter,J.J. (1989) Shift from a mixed to a lactovegetarian diet: influence on acidic lipids in fecal watera potential risk factor for colon cancer. Am. J. Clin. Nutr., 50, 992996.[Abstract]
-
Glinghammar,B., Venturi,M., Rowland,I.R. and Rafter,J.J. (1997) Shift from a dairy product-rich to a dairy product-free diet: influence on cytotoxicity and genotoxicity of fecal waterpotential risk factors for colon cancer. Am. J. Clin. Nutr., 66, 12771282.[Abstract]
-
Venturi,M., Hambly,R.J., Glinghammar,B., Rafter,J.J. and Rowland,I.R. (1997) Genotoxic activity in human faecal water and the role of bile acids: a study using the alkaline comet assay. Carcinogenesis, 18, 23532359.[Abstract]
-
Silvester,K.R., Bingham,S.A., Pollock,J.R., Cummings,J.H. and O'Neill,I.K. (1997) Effect of meat and resistant starch on fecal excretion of apparent N-nitroso compounds and ammonia from the human large bowel. Nutr. Cancer, 29, 1323.[ISI][Medline]
-
Hill,M.J. (1981) Diet and the human intestinal bacterial flora. Cancer Res., 41, 37783780.[Abstract]
-
Pool-Zobel,B.L., Lotzmann,N., Knoll,M., Kuchenmeister,F., Lambertz,R., Leucht,U., Schröder,H.G. and Schmezer,P. (1994) Detection of genotoxic effects in human gastric and nasal mucosa cells isolated from biopsy samples. Environ. Mol. Mutagen., 24, 2345.[ISI][Medline]
-
Mirvish,S.S. (1995) Role of N-nitroso compounds (NOC) and N-nitrosation in etiology of gastric, esophageal, nasopharyngeal and bladder cancer and contribution to cancer of known exposures to NOC. Cancer Lett., 93, 1748.[ISI][Medline]
-
Rafter,J.J., Child,P., Anderson,A.M., Alder,R., Eng,V. and Bruce,W.R. (1987) Cellular toxicity of fecal water depends on diet. Am. J. Clin. Nutr., 45, 559563.[Abstract]
-
Rafter,J., Geltner,U. and Bruce,R. (1987) Cellular toxicity of human faecal waterpossible role in aetiology of colon cancer. Scand. J. Gastroenterol., 129 (suppl.), 245250.
-
McKelvey-Martin,V.J., Green,M.H., Schmezer,P., Pool-Zobel,B.L., De Meo,M.P. and Collins,A. (1993) The single cell gel electrophoresis assay (comet assay): a European review. Mutat. Res., 288, 4763.[ISI][Medline]
-
Fairbairn,D.W., Olive,P.L. and O'Neill,K.L. (1995) The comet assay: a comprehensive review. Mutat. Res., 339, 3759.[ISI][Medline]
-
Olive,P.L., Wlodek,D., Durand,R.E. and Banath,J.P. (1992) Factors influencing DNA migration from individual cells subjected to gel electrophoresis. Exp. Cell Res., 198, 259267.[ISI][Medline]
-
Tice,R.R. and Strauss,G.H. (1995) The single cell gel electrophoresis/comet assay: a potential tool for detecting radiation-induced DNA damage in humans. Stem Cells, 13 (suppl. 1), 207214.
-
Holz,O., Jorres,R., Kastner,A., Krause,T. and Magnussen,H. (1995) Reproducibility of basal and induced DNA single-strand breaks detected by the single-cell gel electrophoresis assay in human peripheral mononuclear leukocytes. Int. Arch. Occup. Environ. Health, 67, 305310.[ISI][Medline]
-
Hartmann,A. and Speit,G. (1997) The contribution of cytotoxicity to DNA-effects in the single cell gel test (comet assay). Toxicol. Lett., 90, 183188.[ISI][Medline]
-
Pool-Zobel,B.L. and Leucht,U. (1997) Induction of DNA damage by risk factors of colon cancer in human colon cells derived from biopsies. Mutat. Res., 375, 105115.[ISI][Medline]
-
Battu,S., Rigaud,M. and Beneytout,J.L. (1998) Resistance to apoptosis and cyclooxygenase-2 expression in a human adenocarcinoma cell line HT29 CL. 19A. Anticancer Res., 18, 35793583.[ISI][Medline]
-
Cerutti,P.A. (1974) Excision repair of DNA base damage. Life Sci., 15, 15671575.[ISI][Medline]
-
Collins,A.R., Duthie,S.J. and Dobson,V.L. (1993) Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis, 14, 17331735.[Abstract]
-
Pflaum,M., Will,O. and Epe,B. (1997) Determination of steady-state levels of oxidative DNA base modifications in mammalian cells by means of repair endonucleases. Carcinogenesis, 18, 22252231.[Abstract]
-
Pool-Zobel,B.L., Bub,A., Müller,H., Wollowski,I. and Rechkemmer,G. (1997) Consumption of vegetables reduces genetic damage in humans: first results of a human intervention trial with carotenoid-rich foods. Carcinogenesis, 18, 18471850.[Abstract]
-
Rutenfranz,J. and Wenzel,H.G. (1980) Energiehaushalt. In Cremer,H.D., Hötzel,D. and Kühnau,J. (eds) Ernährungslehre und Diätetik, Band 1: Biochemie und Physiologie der Ernährung. Thieme Verlag, Stuttgart, Germany.
-
Souci,S.W., Fachmann,W. and Kraut,H. (1994) Die Zusammensetzung der Lebensmittel. Wissenschaftliche Verlagsgesellschaft, Stuttgart, Germany.
-
Rousset,M. (1986) The human colon carcinoma cell lines HT-29 and Caco-2: two in vitro models for the study of intestinal differentiation. Biochimie, 68, 10351040.[ISI][Medline]
-
Elia,M.C., Storer,R.D., Harmon,L.S., Kraynak,A.R., McKelvey,T.W., Hertzog,P.R., Keenan,K.P., DeLuca,J.G. and Nichols,W.W. (1993) Cytotoxicity as measured by trypan blue as a potentially confounding variable in the in vitro alkaline elution/rat hepatocyte assay. Mutat. Res., 291, 193205.[ISI][Medline]
-
Babbs,C.F. (1990) Free radicals and the etiology of colon cancer. Free Radic. Biol. Med., 8, 191200.[ISI][Medline]
-
Erhardt,J.G., Lim,S.S., Bode,J.C. and Bode,C. (1997) A diet rich in fat and poor in dietary fiber increases the in vitro formation of reactive oxygen species in human feces. J. Nutr., 127, 706709.[Abstract/Free Full Text]
-
Nagata,C., Kodama,M., Ioki,Y. and Kimura,T. (1982) Free radicals produced from chemical carcinogens and their significance in carcinogenesis. In Floyd,R.A. (ed.) Free Radicals and Cancer. Marcel Dekker, New York, NY.
-
Newmark,H.L., Wargovich,M.J. and Bruce,W.R. (1984) Colon cancer and dietary fat, phosphate and calcium: a hypothesis. J. Natl Cancer Inst., 72, 13231325.[ISI][Medline]
-
Sandstrom,B., Astrup,A.V., Dyerberg,J., Holmer,G., Poulsen,H.E., Stender,S., Kondrup,J. and Gudmand-Hoyer,E. (1994) The effect on health of dietary antioxidants and antioxidant supplements. Ugeskr. Laeger, 156, 76757679.[Medline]
-
Lee,I.M. (1999) Antioxidant vitamins in the prevention of cancer. Proc. Assoc. Am. Physicians, 111, 1015.[ISI][Medline]
-
Hennekens,C.H. (1994) Antioxidant vitamins and cancer. Am. J. Med., 97, 2S4S.[Medline]
-
Bourquin,L.D., Titgemeyer,E.C., Fahey,G.C.J. and Garleb,K.A. (1993) Fermentation of dietary fibre by human colonic bacteria: disappearance of, short-chain fatty acid production from and potential water-holding capacity of various substrates. Scand. J. Gastroenterol., 28, 249255.[ISI][Medline]
-
Silvi,S., Rumney,C.J., Cresci,A. and Rowland,I.R. (1999) Resistant starch modifies gut microflora and microbial metabolism in human flora-associated rats inoculated with faeces from Italian and UK donors. J. Appl. Microbiol., 86, 521530.[ISI][Medline]
-
Cummings,J.H. and Macfarlane,G.T. (1997) Role of intestinal bacteria in nutrient metabolism. J. Parenter. Enteral. Nutr., 21, 357365.[Abstract]
-
Fadden,K., Hill,M.J. and Owen,R.W. (1997) Effect of fibre on bile acid metabolism by human faecal bacteria in batch and continuous culture. Eur. J. Cancer Prev., 6, 175194.[ISI][Medline]
-
Cantoni,O., Sestili,P., Guidarelli,A., Palomba,L., Brambilla,L. and Cattabeni,F. (1996) Cytotoxic impact of DNA single vs double strand breaks in oxidatively injured cells. Arch. Toxicol., 18 (suppl.), 223235.
Received June 7, 1999;
revised August 12, 1999;
accepted August 18, 1999.