1 Food and Colon Carcinogenesis Laboratory, ISTAB, Bordeaux 1 University, 33405 Talence cedex, France,
2 Danone Vitapole, 15 avenue Galilée, 92350, le Plessis-Robinson, France,
3 Membrane Traffic and Epithelial Cells Signalisation Laboratory, U538 INSERM, Medicine Faculty, St-Antoine, Paris, France and
4 SFRI Laboratory, Berganton,33127 St-Jean d'Illac, France
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Abstract |
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Abbreviations: ACF, aberrant crypt foci;; CYP, cytochrome P450;; EROD, ethoxyresorufin deethylase;; HAA, heterocyclic aromatic amines;; IQ, 2-amino-3-methylimidazo[ 4,5-f]quinoline;; LAB, lactic acid bacteria;; MeIQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline;; MROD, methoxyresorufin deethylase;; 8-oxodGuo, 8-oxo-7,8-dihydro-2'desoxyguanosine;; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine;; SD, standard deviation.
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Introduction |
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Some of these agents are formed during food preparation, such as heterocyclic aromatic amines (HAA). These compounds were discovered by Sugimura et al. (3) and are produced during the cooking of food with high creatine, free amino acid and sugar content, such as cooked muscle meats and fish, arising from Maillard and Strecker degradation products. Among these compounds, the most frequent in the human diet are 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,4-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and more rarely, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) (47). The mutagenic potential of HAA is very high, particularly in in vitro bacterial tests such as the Ames test. Moreover, a number of in vivo studies have shown that HAA induce multi-site tumours in various animals, such as rodents and non-human primates (8). There is a growing interest in the use of HAA as carcinogenic compounds in experimental anticarcinogenesis studies, especially for induction of colorectal cancer in rats. Because carcinogenic compounds such as HAA are present in human alimentation, they can provide more pertinent information than typical chemicals, such as 1,2-dimethylhydrazine, used to induce colon cancer. Moreover, HAA induce the initiation step of the carcinogenesis process, whereas the majority of carcinogenic food compounds are implicated in cancer promotion. For this reason, a carcinogenesis model using HAA as carcinogens is of interest for the study of various cancer chemopreventive agents. These agents often act by inhibiting the initiation phase of chemical tumourigenesis.
Among diet factors that could contribute to a decrease of human colon cancer, probiotics are of interest. Indeed, in vitro and in vivo studies show that several lactic acid bacteria (LAB) present in fermented milk have antimutagenic and anticarcinogenic properties. Using the Ames test, Cassand et al. (9) and Abdelali et al. (10) have shown that Bifidobacterium animalis DN-173 010 has an inhibitory effect towards indirect mutagenic agents in vitro. Other in vitro studies, also using the Ames test, demonstrated antimutagenic effects of various fermenting LAB, especially bifidobacteria, towards IQ, MeIQ and PhIP (11,12). Several in vivo studies have demonstrated the protective effects of milk, fermented milk and various LAB towards chemically induced colon carcinogenesis in rats, particularly using 1,2-dimethylhydrazine or azoxymethanol, its metabolite, as carcinogenic agents and aberrant colon crypts as carcinogenesis markers (1318). Two short-term studies, using 1,2-dimethylhydrazine as a genotoxic agent in rats, showed that various LAB (Lactobacillus, Bifidobacterium and Streptococcus thermophilus) are able to decrease colon DNA damage screened using the comet assay (19,20). Only one study has investigated the effect of a LAB alone on HAA-induced rat colon carcinogenesis. Reddy and Rivenson (21) found 100% colon tumour inhibition in F344 male rats feeding diet supplemented with 0.5% lyophilized Bifidobacterium longum, using IQ at 125 p.p.m. for 58 weeks as a carcinogenic treatment. However, in this study, the number of colon tumours in the HAA control rats was very small. Lastly, in a dietary intervention study in humans where Lactobacillus acidophilus was given in healthy volunteers, there was a decrease in the fecal mutagenicity induced previously by a fried meat diet, rich in HAA (22).
In this context, the aim of the present study was to evaluate the effects of some LAB on HAA-induced colon carcinogenesis in rats. A mixture of three HAAIQ, MeIQ and PhIPwas used because they are present in the Western-type diet and are colon carcinogens. Moreover, the carcinogenic potential of this HAA mixture has been evaluated previously in the rat colon (E.Tavan, C.Barbé, C.Cayuela, J.M.Antoine and P.Cassand, in press).
Therefore, the HAA carcinogenesis model was used in this study in order to test the prevention potential of fermented milk, food products in human alimentation. Two different LAB strains were used. These strains, B.animalis DN-173 010 and S.thermophilus DN-001 158, were chosen after in vitro antimutagenic screening assays performed previously in our laboratory, using the Ames mutatest with IQ as genotoxic agent (E.Tavan, C.Cayuela, J.M.Antoine and P.Cassand, in press).
The aims of this study were, firstly, to evaluate the effects of fermented milk on aberrant crypt foci (ACF) induction, using HAA as initiating agents of colon carcinogenesis. This is the first time that dairy products have been assayed towards with HAA in an ACF carcinogenesis model. A second goal of this study was to investigate the level of action of fermented milk using early markers of the carcinogenesis process, especially markers of genotoxicity: 8-oxodeoxyguanosine (8-oxodGuo) level, 3D test and comet assay. To our knowledge, 8-oxodGuo has never been characterized in rat colon after a HAA treatment, nor used in anticarcinogenic studies using HAA. The 3D test and comet assay have also never been used in long-term in vivo studies with HAA. The most widely used in vivo method to investigate protective effects against HAA is the 32P post-labelling method; however, this marker is not simply correlated to ACF induction, and only detects specific types of adducts.
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Materials and methods |
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Rats were randomly distributed into four groups of 15 animals. Different diets were used in each group during the 15 experimental weeks: a standard powdered diet for rodent maintenance (9609 Harlan, Gannat, France) was supplemented with 20% tap water (group 1), with 30% non-fermented skimmed milk (group 2), with 30% B.animalis DN-173 010-fermented milk (group 3), with 30% S.thermophilus DN-001 158-fermented milk (group 4). Supplements were added in amounts calculated to provide isocaloric diets to the different groups.
After 1 week of acclimatization, rats were fed HAA for 7 weeks. The IQ, MeIQ and PhIP were purchased from Toronto Research Chemicals (Ontario, Canada). Chronic exposure induction was chosen in order to better mimic a human diet, with a cumulative dose of 250 mg of a IQ, MeIQ and PhIP mixture (ratio 1:1:1, weight basis) per kg body weight. The daily dose of HAA per rat was dissolved in 115 µl of distilled water, 15% dimethylsulfoxide (DMSO), 0.2% 0.1 N HCl, then mixed with the food.
Rat feces were recovered the day before the HAA supplementation period, after 3 weeks and at the end of the 7 week induction, in order to follow HAA excretion. After the 7 HAA treatment weeks, seven rats of each group were killed for measurement of DNA lesions and evaluation of metabolic activity in the liver and colon. The other rats were kept on their specific diets and killed 8 weeks later for numeration of ACF (Figure 1).
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Feces mutagenicity biomonitoring
Feces were recovered at days 0, 24 and 49 of the treatment period. Feces samples were homogenized in 5 ml of distilled water, filtered on a 0.8 µm GS Millipore membrane, and then polar mutagenic compounds were extracted with a normal phase Disposable Spe Column (Mallinckrodt Baker B.V., Holland) eluted with 5 ml acetone. Acetone was evaporated under vacuum using a Speed-Vac and the residue was diluted in 100 µl of DMSO. The mutagenic potential of 30 µl of this feces extract dissolved in DMSO was evaluated using the Ames test after 1 h of incubation, using Salmonella typhimurium TA98 and a rat hepatic metabolism system (S9mix obtained from Aroclor-induced SpragueDawley rats) (23). Negative controls were performed with DMSO alone. Each feces sample was tested on triplicate plates and the experiment was repeated twice.
Quantification of 8-oxodGuo
After killing the animals, livers and colons were removed. Colons were rinsed with 0.9% NaCl at 4°C and the epithelium was recovered. Liver and colonic epithelium were homogenized with potter and Ultra-Turrax, respectively, in 8 vol. of 800 mM guanineHCl, 30 mM EDTA (pH 8), 30 mM TrisHCl (pH 8), 5% Tween 20, 0.5% Triton X-100 buffer, provided by Quiagen S.A. (Courtaboeuf, France). Samples were kept at 80°C until DNA extraction, by anion-exchange chromatography, with the Midi Kit from Quiagen S.A. For extraction, 50 mg of liver and 75 mg of colon tissues were used. About 3 µg of DNA/mg of liver and 6 µg of DNA/mg of colon tissue were recovered. DNA was enzymatically digested with 10 U of Nuclease P1 (Sigma Chemical Co., St Quentin, France) for 1.5 h at 37°C in 300 mM sodium acetate, 1 mM zinc sulfate buffer (pH 5.3) and then with 2 U of phosphatase alkaline (Sigma) for 30 min at 37°C in 500 mM TrisHCl, 1 mM EDTA buffer (pH 8). Digestion was stopped with 1/2 vol. of chloroform and samples were centrifuged at 10 500 g for 10 min, then the aqueous phase was recovered for DNA analysis (24,25). The 8-oxodGuo was carried out by high performance liquid chromatography (HPLC) (System Gold 118, Beckman) coupled to electrochemical detection (405, Kontron). Separation of 8-oxodGuo from 2'-deoxyribonucleosides was performed on an Ultrasphere C18 pre-column (ODS, 45x4.6 mmx5 µm, Interchrom) and an Uptisphere column (ODB, 250x4.6 mmx5 µm, Interchrom). The column was eluted in isocratic mode using a mobile phase composed of 10% methanol, 10 mM citric acid, 25 mM sodium acetate, 30 mM sodium hydroxide and 10 mM acetic acid. The elution flow rate was set at 0.8 ml/min. The detection sensitivity of the electrochemical detector was 0.5 nA/V for an oxidation potential of 650 mV. The 8-oxodGuo quantification was done in accordance with a calibration curve obtained previously with pure standard. For the usual expression in number of 8-oxodGuo residues per 105 deoxyguanosine, this was also quantified by coupling a UV detector (System Gold 166, Beckman), at the output of the HPLC column, with a wavelength of 260 nm. This quantification was done for three rats per group.
DNA damage quantification by `3D Test'
This method was used for three rats per group and performed as described by Salles et al. (26). Briefly, damaged DNA was incubated with repair and replication enzymes, which permit a previously biotine-marked base to be incorporated each time that an excision-base repair has occurred. Two antibodies were then used, one directed against biotine conjugated to peroxidase, and one with a luminescent substrate directed against peroxidase. The light emitted was then measured with a luminometer. The signal, in relative light units (RLU) was thus directly proportional to the repair rate and so to the DNA damage rate. After liver and colon DNA extraction, the `3D Test' was performed using the SFRI kit (St-Jean d'Illac, France), with 100 ng of extracted DNA/well.
DNA damage quantification using the comet assay
After sacrifice, colons of four rats per group were removed, cut into small pieces and washed at 37°C in Merchant solution (0.14 M NaCl, 1.47 mM K2HPO4, 2.7 mM KCl, 8.1 mM Na2HPO4). Then, the tissue pieces were incubated at 37°C for 10 min in Merchant solution supplemented with 0.53 mM Na2EDTA (pH 7.4). The supernatant was removed and colonic epithelium was further incubated in a solution of 0.05% collagenase IV (Sigma) in MEM buffer (Sigma) under shaking, for 30 min at 37°C. After filtration of the supernatant on 100 µm mesh, the resulting cell suspensions were centrifuged at 900 g for 5 min and then resuspended in 1 ml PBS (Gibco). The cells were counted and their viability simultaneously evaluated by the Trypan blue exclusion method (at least 80% of the cells must be viable for the comet assay).
The alkaline comet assay was performed as described by Lebailly et al. (27) for the detection of single- and double-strand breaks. A suspension of 2x105 cells was prepared and 75 µl of the suspension were rapidly spread on frosted microscope slides pre-coated with agarose. Slides were then put into a tank fill with a lysis solution (2.5 M NaCl, 10 mM Tris, 0.1 M Na2EDTA, pH 10, 10% DMSO, 1% Triton X-100 both freshly added) for at least 1 h at room temperature. The slides were removed from the lysis solution and incubated in a fresh electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH 13). Electrophoresis was carried out for 20 min at 20 V, 900 mA. After electrophoresis, slides were gently washed in neutralization buffer (0.4 M Tris, pH 7.5), and stained with 60 µl ethidium bromide solution (20 µg /ml). Slides were observed with a fluorescence microscope (515 nm excitation and between 515 and 561 nm emission), and 250x magnification. Two slides were performed per rat, at least 50 randomly selected cells were visually analysed per slide and damage was classified into four categories for a qualitative evaluation: undamaged cells, slightly damaged cells, damaged cells and highly damaged cells.
Liver and colon microsomes preparation
Liver and colon epithelium were removed, minced and homogenized in 3 vol. of ice-cold 0.15 M KCl solution. The post-mitochondrial fraction, obtained by centrifugation of the homogenate at 9000 g for 20 min, was centrifuged for 1 h at 105 000 g. Final microsomal preparations were resuspended in 100 mM potassium phosphate buffer with 20% glycerol (pH 7.4). Protein levels in the microsomal preparations were determined as described previously (28).
Evaluation of hepatic CYP1A1 and 1A2 by determination of enzymatic activity
The ethoxyresorufin deethylase (EROD) and methoxyresorufin deethylase (MROD) activities were assayed in liver microsomes using the methods reported previously (2931). The resorufine metabolism by cytochromes P450 1A1 and 1A2 leads to the formation of ethoxyresorufine and methoxyresorufine, respectively. A final volume of 1 ml of 50 mM phosphate, 5 mM MgCl2 buffer (pH 7.4), 1 mg of microsomal proteins and 0.15 mM 7-ethoxyresorufine or 7-methoxyresorufine was used for incubation. The reaction, occurring at 25°C for 3 min, was started by addition of 7.5 mM NADPH and stopped by 1 ml of 5% trichloracetic acid. After centrifugation at 2000 g for 5 min, 500 µl of supernatant was mixed with 2.5 ml of 0.5 M phosphate buffer (pH 7.4) and the absorbance was measured by fluorimetry at 530 nm (excitation wavelength) and 585 nm (emission wavelength). The EROD and MROD activities were expressed in nanomoles of resorufine formed per minute per mg of proteins, and were measured in three rats per group.
Evaluation of liver and colon CYP1A1 and 1A2 by immuno-quantification
Concurrently with the evaluation of the enzymatic activity of CYP1A1 and 1A2, the immuno-quantification of these two cytochrome P450 (CYP450) isoforms were assayed both in liver and colon tissues for the same rats. Proteins from hepatic and colonic microsomes were separated first by sodium dodecyl sulfate denaturing polyacrylamide gel electrophoresis (32). Western blots were then performed using a method described previously (33,34): proteins were transferred from the gel to a nitrocellulose membrane which was incubated with 1/1000 diluted complex IgG goat conjugated to anti-CYP1A primary antibody (Dachi Pure Chemicals) and then a second time with 1/5000 diluted peroxidase-conjugated rabbit anti-goat IgG secondary antibody complex (Sigma). After these treatments, the membrane was washed and incubated with Super Signal® Substrate working solution (Pierce), and exposed to a standard autoradiographic film which was then developed and fixed. The optical density of bands corresponding to CYP1A1 and 1A2 were determined by scanning with a densitometer (MultiScan HG-Bio 1D Vilber Loumat).
Identification and quantification of ACF
Immediately after being killed, the rat colons were removed and prepared according to Bird (35). ACF were distinguished from surrounding non-involved crypts by their slit-like opening, increased size, staining and pericryptal zone. They were scored blindly by a single observer, in eight rats per group.
Statistical analysis
The level of significance was tested using one-way analysis of variance (ANOVA) followed by unpaired Student's test, except for ACF numeration where the post-test used was the Tukey test, the minimum level of significance accepted being P < 0.05 in both cases.
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Results |
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Discussion |
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Concomitantly with the inhibition of ACF initiation, we observed: (i) that hepatic CYP 1A2 metabolic activity is decreased in liver of rats fed fermented milk (groups 3 and 4), (ii) that feces mutagenicity increases during the HAA induction period only for the rats which had no dairy products incorporated in their diet (group 1) and (iii) that there was less damage in colon cells of rats fed milk or fermented milk (groups 2, 3 and 4). Those last results are not identical for each biomarker used: 8-oxodGuo oxidative damage decreased in colon cells of rats fed non-fermented milk (group 2) or fermented milk (groups 3 and 4). The 8-oxodGuo is a coding lesion inducing stable and transmissible mutations and has been shown to be involved in HAA-induced carcinogenesis (38,39; E.Tavan, C.Barbé, C.Cayuela, J.M.Antoine and P.Cassand, submitted), whereas DNA damage background in liver is not modified in our study. Thus, the present results may confirm that oxidative damage to DNA induced by HAA could be one of the mechanisms involved in mutagenesis in liver and colonic cells. This is in accordance with the study of Schut and Snyderwine (40), which showed that HAADNA adduct amount alone is not correlated with the tumour incidence. On the other hand, the `3D Test' and the comet assay, which measured breaks in DNA strands linked to genomic reparation processes, indicated less colon cell DNA damage only for rats fed fermented milk (groups 3 and 4). However, with the `3D Test', the heterocyclic amines do not affect the background level of DNA damage. In that case, the protective effects observed may be unrelated to the effects of heterocyclic amines. In contrast, using the comet assay, the dairy products show some protective effects.
The results obtained with the intermediate markers show that milk and fermented milks may act in the first steps of colon carcinogenesis. It is likely that LAB in fermented milk are linked to the different HAA, preventing their digestive absorption, causing the observed decrease in metabolic enzymatic activity in the liver. Indeed, several studies have shown the capacity LAB to bind chemicals, such as HAA, in vitro and in vivo (4143). There was, however, no effect of dairy products on the level of hepatic DNA lesions, even though this level has been shown to be increased after a HAA induction (38). In fact, the decrease in metabolic activity of CYP 1A2 should be not sufficient to involve hepatic DNA lesion reduction.
Moreover, in our study, a mixture of three different HAA, representing different structural types, was used. Therefore, the binding capacities LAB cannot be the same. This idea is supported by the study of Rajendran and Ohta (42), which showed that IQ is not effectively bound to the LAB they used, and IQ presents organ specificity towards liver while MeIQ and PhIP have a colon tropism. Bolognani et al. (41) pointed out that the binding capacities of LAB are not always correlated to major changes in absorption and distribution of the carcinogens in the body. Therefore, in the colon, the effects of dairy products on aberrant crypt formation would take place after HAA metabolism. Different mechanisms are possible: (i) alteration of physicochemical conditions in the colon such as pH (16) and of the metabolic activities of intestinal microflora like ß-glucuronidase bacterial activity (13,16,44), (ii) production of anti-mutagenic compounds by the dairy products, in the colon, or the production of components that are able to enhance the host's immune response (19,20,45,46) and (iii) modification of the intestinal microflora which may change the short-chain fatty acid profile (47).
The present study also shows that non-fermented milk has almost the same effect as fermented milk. These results are in accordance with a previous study, which showed that skimmed milk alone is able to decrease the incidence and number of ACF in 1,2-dimethylhydrazine treated rats (13). These findings support the idea that dairy products affect carcinogenesis in different ways, fermentation by LAB enhancing the protective mechanisms. Lankaputhra and Shah (11) and Sreekumar and Hosono (12), in vitro, found some antimutagenic compounds in milk (palmitic acid, a proteic fraction), and fermentation with LAB increases production of these compounds.
There is no difference between the two LAB strains used in this study, in the protective effect of fermented milk against HAA-induced carcinogenesis, whether in the number of aberrant crypts, or the intermediate biomarkers. This suggests that modulation of the various colonic parameters mentioned above is equivalent for both LAB. To confirm this, it would be interesting to measure the numbers of viable bifidobacteria in feces of rats fed the various diets.
In conclusion, the intermediate biomarkers used in this study showed that dairy products decreased aberrant colonic crypt formation, which may act before the early steps of the carcinogenesis process, the initiation stage. Therefore, dairy products may be an interesting food to help prevent colon tumourigenesis induced by human food carcinogens. This action seems to be slightly more efficient when milk has been fermented with LAB than with milk alone.
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Notes |
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Acknowledgments |
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References |
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