Fermentation of resistant starches: influence of in vitro models on colon carcinogenesis

Resistant starch type 2 (RS2) and type 3 (RS3) containing preparations were digested using a batch (a) and a dynamic in vitro model (b). Furthermore, in vivo obtained indigestible fractions from ileostomy patients were used (c). Subsequently these samples were fermented with human feces with a batch and a dynamic in vitro method. The fermentation supernatants were used to treat CAC02 cells. Cytotoxicity, anti-genotoxicity against hydrogen peroxide (comet assay) and the effect on barrier function measured by trans-epithelial electrical resistance were determine. Dynamically fermented samples led to high cytotoxic activity, probably due to additional compounds added during in vitro fermentation. As a consequence only batch fermented samples were investigated further. Batch fermentation of RS resulted in an anti-genotoxic activity ranging from 9-30% decrease in DNA damage for all the samples, except for RS2-b. It is assumed that the changes in RS2 structures due to dynamic digestion resulted in a different fermentation profile not leading to any anti-genotoxic effect. Additionally, in vitro batch fermentation of RS caused an improvement in integrity across the intestinal barrier by approximately 22% for all the samples. We have demonstrated that batch in vitro fermentation of RS2 and RS3 preparations differently pre-digested are capable of inhibiting the initiation and promotion stage in colon carcinogenesis in vitro.

Potential anti-cancer effects of virgin olive oil phenols on colorectal carcinogenesis models in vitro

The traditional Mediterranean diet is thought to represent a healthy lifestyle; especially given the incidence of several cancers including colorectal cancer is lower in Mediterranean countries compared to Northern Europe. Olive oil, a central component of the Mediterranean diet, is believed to beneficially affect numerous biological processes. We used phenols extracted from virgin olive oil on a series of in vitro systems that model important stages of colon carcinogenesis. The effect the extract on DNA damage induced by hydrogen peroxide was measured in HT29 cells using single cell microgel-electrophoresis. A significant anti-genotoxic linear trend (p=0.011) was observed when HT29 cells were pre-incubated with olive oil phenols (0, 5, 10, 25, 50, 75, 100 microg/ml) for 24 hr, then challenged with hydrogen peroxide. The olive oil phenols (50, 100 microg/ml) significantly (p=0.004, p=0.002) improved barrier function of CACO2 cells after 48 hr as measured by trans-epithelial resistance. Significant inhibition of HT115 invasion (p<0.01) was observed at olive oil phenols concentrations of 25, 50, 75, 100 microg/ml using the matrigel invasion assay. No effect was observed on HT115 viability over the concentration range 0, 25, 50 75, 100 microg/ml after 24 hr, although 75 and 100 microg/ml olive oil phenols significantly inhibited HT115 cell attachment (p=0.011, p=0.006). Olive oil phenols had no significant effect on metastasis-related gene expression in HT115 cells. We have demonstrated that phenols extracted from virgin olive oil are capable of inhibiting several stages in colon carcinogenesis in vitro.

A central role for heme iron in colon carcinogenesis associated with red meat intake

Epidemiology shows that red and processed meat intake is associated with an increased risk of colorectal cancer. Heme iron, heterocyclic amines and endogenous N-nitroso compounds (NOC) are proposed to explain this effect, but their relative contribution is unknown. Our study aimed at determining, at nutritional doses, which is the main factor involved and proposing a mechanism of cancer promotion by red meat. The relative part of heme iron (1% in diet), heterocyclic amines (PhIP+MeIQx, 50+25 μg/kg in diet) and NOC (induced by NaNO2+NaNO3 0.17+0.23 g/l of drinking water) was determined by a factorial design and preneoplastic endpoints in chemically-induced rats and validated on tumors in Min mice. The molecular mechanisms (genotoxicity, cytotoxicity) were analyzed in vitro in normal and Apc- deficient cell lines and confirmed on colon mucosa. Heme iron increased the number of preneoplastic lesions but dietary heterocyclic amines and NOC had no effect on carcinogenesis in rats. Dietary hemoglobin increased tumor load in Min mice (control diet: 67±39 mm2; 2,5% hemoglobin diet: 114±47 mm2, p=0.004). In vitro, fecal water from rats given hemoglobin was rich in aldehydes and was cytotoxic to normal cells, but not to premalignant cells. The aldehydes 4-hydroxynonenal and 4-hydroxyhexenal were more toxic to normal versus mutated cells and were only genotoxic to normal cells. Genotoxicity was also observed in colon mucosa of mice given hemoglobin. These results highlight the role of heme iron in the promotion of colon cancer by red meat and suggest that heme iron could initiate carcinogenesis through lipid peroxidation.

Carcinogenesis of the upper gastrointestinal tract induced by N-methyl-N '-nitro-nitrosoguanidine and reflux of duodenal contents in the rat

Purpose: To investigate the combined effects of reflux of duodenal contents through the pylorus and treatment with N-methyl-N'-nitro-nitrosoguanidine ( MNNG) on the development of lesions in the glandular stomach, at the gastrojejunal anastomosis and in the forestomach of rats. Methods: Eighty Male Wistar rats were divided into 4 groups: G1: MNNG + Reflux, G2: Reflux, G3: MNNG and G4: Gastrostomy. MNNG was given in the drinking water ( 100 mg/ml) for 12 weeks and then two groups ( G1 and G2) were submitted to a gastrojejunal anastomosis followed by section of the afferent loop and suture of both stumps to allow reflux of duodenal contents through the pylorus. The animals were sacrificed 18 and 36 weeks after surgery. The lesions obtained in the antral mucosa, at the gastrojejunal anastomosis and in the forestomach were analysed histologically. Results: Duodenal reflux induced proliferative lesions at both glandular and squamous mucosa of the stomach. In the antrum, adenomatous hyperplasia (AH) was observed in 20% and 50% of the animals at the 18(th) and 36(th) weeks respectively. Aditionally 85% of the animals presented AH at the gastrojejunal anastomosis and 60% developed squamous hyperplasia at the squamous portion of the stomach. MNNG treatment plus duodenal reflux enhanced the development of malignant tumors at both glandular and squamous mucosa, since there were 30% of antral adenocarcinomas and 45% of squamous carcinomas at the 18th week and the frequency of these malignant tumors rose to 50% in the antrum and 65% in the squamous mucosa at the 36th week. Conclusion: The reflux of duodenal contents through the pylorus enhanced the development of proliferative lesions, benign and malignant, in the glandular stomach and in the forestomach of rats.

No mutations found in exons of TP53, H-RAS and K-RAS genes in liver of male Wistar rats submitted to a medium-term chemical carcinogenesis assay

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of swim training on liver carcinogenesis in male Wistar rats fed a low-fat or high-fat diet

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

No mutations found in exon 2 of gene p16CDKN2A during rat tongue carcinogenesis induced by 4-nitroquinoline-1-oxide

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Lack of chemopreventive effects of ginger on colon carcinogenesis induced by 1,2-dimethylhydrazine in rats

Ginger (Zingiber officinale Roscoe) has been proposed as a promising candidate for cancer prevention. Its modifying potential on the process of colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) was investigated in male Wistar rats using the aberrant crypt foci (ACF) assay. Five groups were studied: Groups 1-3 were given four s.c. injections of DMH (40 mg/kg b.w.) twice a week, during two weeks, whereas Groups 4 and 5 received similar injections of EDTA solution (DMH vehicle). After DMH-initiation, the animals were fed a ginger extract mixed in the basal diet at 0.5% (Group 2) and 1.0% (Groups 3 and 4) for 10 weeks. All rats were killed after 12 weeks and the colons were analyzed for ACF formation and crypt multiplicity. The rates of cell proliferation and apoptosis were also evaluated in epithelial colonic crypt cells. Dietary consumption of ginger at both dose levels did not induce any toxicity in the rats, but ginger meal at 1% decreased significantly serum cholesterol levels (p < 0.038). Treatment with ginger did not suppress ACF formation or the number of crypts per ACF in the DMH-treated group. Dietary ginger did not significantly change the proliferative or apoptosis indexes of the colonic crypt cells induced by DMH. Thus, the present results did not confirm a chemopreventive activity of ginger on colon carcinogenesis as analyzed by the ACF bioassay and by the growth kinetics of the colonic mucosa. (c) 2005 Elsevier Ltd. All rights reserved.

DNA damage and aberrant crypt foci as putative biomarkers to evaluate the chemopreventive effect of annatto (Bixa orellana L.) in rat colon carcinogenesis

Chemoprevention opens new perspectives in the prevention of cancer and other degenerative diseases. Use of target-organ biological models at the histological and genetic levels can markedly facilitate the identification of such potential chemopreventive agents. Colon cancer is one of the highest incidence rates throughout the world and some evidences have indicated carotenoids as possible agents that decrease the risk of colorectal cancer. In the present study, we evaluate the activity of annatto (Bixa orellaria L.), a natural food colorant rich in carotenoid, on the formation of aberrant crypt foci (ACF) induced by dimethy1hydrazine (DMH) in rat colon. Further, we investigate, the effect of annatto on DMH-induced DNA damage, by the comet assay. Male Wistar rats were given s.c. injections of DMH (40 mg/kg body wt.) twice a week for 2 weeks to induce ACE They also received experimental diets containing annatto at 20, 200 or 1000 ppm for five 5 weeks before (pre-treatment), or 10 weeks after (post-treatment) DMH treatment. In both protocols the rats were sacrificed on week 15th. For the comet assay, the animals were fed with the same experimental diets for 2 weeks. Four hours before the sacrifice, the animals received an s.c. injection of DMH (40 mg/kg body wt.). Under such conditions, dietary administration of 1000 ppm annatto neither induce DNA damage in blood and colon cells nor aberrant crypt foci in rat distal colon. Conversely, annatto was successful in inhibiting the number of crypts/colon (animal), but not in the incidence of DMH-induced ACF, mainly when administered after DMH. However, no antigenotoxic effect was observed in colon cells. These findings suggest possible chemopreventive effects of annatto through their modulation of the cryptal cell proliferation but not at the initiation stage of colon carcinogenesis. (c) 2005 Elsevier B.V. All rights reserved.

Natural killer activity in a medium-term multi-organ bioassay for carcinogenesis

Natural killer (NK) cell activity was evaluated after the initiation and promotion steps in a medium-term multi-organ bioassay for carcinogenesis. NK cell activity was assessed in vitro by Cr-51 release assay at the 4th and 30th weeks of the experiment. Male Wistar rats were sequentially initiated with N-diethylnitrosamine (DEN i.p.), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN drinking water), N-methyl-N-nitrosourea (MNU i.p.), dihydroxy-di-N-propylnitrosamine (DHPN drinking water) and N,N'-dimethylhydrazine (DMH s.c.) at subcarcinogenic doses for 4 weeks (DMBDD initiation). One group was evaluated at the 4th week and the other was maintained without any further treatment until the 30th week. Two initiated groups were exposed through the diet to 2-acetylaminofluorene (2-AAF) or phenobarbital (PB), from the 6th until the 30th week, Five additional groups were studied to evaluate the effects of each initiator on NK activity. All groups submitted to initiation only, initiation plus promotion, or promotion only, developed significantly more preneoplastic lesions than the untreated control group. The main target organs for tumor development in the initiated animals n ere the liver and the colon, irrespective of treatment with 2-AAF or PB. NK cell activity was not affected bal exposure to genotoxic carcinogens after initiation, at the 4th week. Treatments only with PB or 2-AAF did not change NK cell activity, However, decreased NK cell activity was registered in the group only initiated with DMBDD and in the group given DMBDD+2-AAF. This late depression of NK cell activity at the 30th week could be related to the production of suppressing molecules by the tumor cells.

Chemically induced immunotoxicity in a medium-term multiorgan bioassay for carcinogenesis with Wistar rats

A variety of chemicals can adversely affect the immune system and influence tumor development. The modifying potential of chemical carcinogens on the lymphoid organs and cytokine production of rats submitted to a medium-term initiation-promotion bioassay for carcinogenesis was investigated. Male Wistar rats were sequentially initiated with N-nitrosodiethylamine (DEN), N-methyl-N-nitrosourea (MNU), N-butyl-N-(4hydroxybutyl)nitrosamine (BBN), dihydroxy-di-n-propylnitrosamine (DHPN), and 1,2-dimethylhydrazine (DMH) during 4 weeks. Two initiated groups received phenobarbital (PB) or 2-acetyl amino fluorene (2-AAF) for 25 weeks and two noninitiated groups received only PB or 2-AAF. A nontreated group was used as control. Lymphohematopoietic organs, liver, kidneys, lung, intestines, and Zymbal's gland were removed for histological analysis. Interleukin (IL)-2, IL-12, interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), IL-10, and transforming growth factor betal (TGF-beta1) levels were determined by ELISA in spleen cell culture supernatants. At the fourth week, exposure to the initiating carcinogens resulted in cell depletion of the thymus, spleen and bone marrow, and impairment of IL-2, IL-12, and IFN-gamma production. However, at the 30th week, no important alterations were observed both in lymphoid organs and cytokine production in the different groups. The results indicate that the initiating carcinogens used in the present protocol exert toxic effects on the lymphoid organs and affect the production of cytokines at the initiation step of carcinogenesis. This early and reversible depression of the immune surveillance may contribute to the survival of initiated cells facilitating the development of future neoplasia. (C) 2003 Elsevier B.V. All rights reserved.

Changes in minor salivary glands during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis

We investigated the changes of minor salivary glands during 4NQO-induced rat tongue carcinogenesis. Histopathological examinations of serous and mucous tongue salivary glands of 30 male Wistar rats were performed after 4, 12 and 20 weeks of 50 ppm 4NQO chronicle administration in drinking water. Ten rats were used as control. Morphometric results were expressed as volume density (Vv %) of each of the components. Histopathological and morphometric changes in the salivary glands were evident only at 20 weeks following 4NQO administration and they included a significant (P < 0.05) decreased in the mean Vv of the serous acini compared with the control group accompanied by abnormal acini (Vv=14 %). Neither mucous acini nor ducts demonstrated significant changes. In conclusion, minor salivary glands are involved in the progression of 4NQO-induced carcinoma.

Lymphoproliferative response and T lymphocyte subsets in a medium-term multi-organ bioassay for carcinogenesis in Wistar rats

The lymphoproliferative response and T lymphocyte subsets were evaluated at different stages of carcinogenesis in male Wistar, rats sequentially initiated with N-diethylnitrosamine (DEN), N-butyl-N-4(hydroxybutyl)nitrosamine (BBN), N-methyl-N-nitrosourea (MNU), dihydroxy-di-N-propylnitrosamine (DHPN) and N,N'-dimethylhydrazine (DMH) (DMBDD initiation). One group was evaluated at the 4th week and other initiated group at the 30th week. Two initiated groups were also exposed through diet to 7-acetylaminofluorene (2-AAF) or phenobarbital (PB), from the 6th until the 30th week. Two groups received only 2-AAF or PB until the 30th week. Five groups were studied to evaluate the effects of each initiator. The lymphoproliferative response was induced in vitro by concanavalin A and the percentage of T lymphocyte subsets was determined by flow cytometry, All groups submitted to initiation only, initiation plus promotion, or promotion only, developed significantly more preneoplastic: lesions than the untreated control group. The main target organs for tumor development were the liver, colon, urinary bladder, kidneys and Zymbal glands, mainly in the group treated with DMBDD + 2-AAF, There were no alterations of the lymphoproliferative response and of the T lymphocyte subsets percentage in the DMBDD-treated group at the 4th and 30th weeks. At the 30th week, the T lymphocyte subsets percentage was also not affected in the initiated groups after treatments with 2-AAF or PB. The lymphoproliferative response, however, was decreased in the DMBDD + 2-AAF group and in the groups treated only with 2-AAF or PB, the present results indicate that the initiating chemicals used in the DMBDD initiation protocol do not exert any influence on the immune system. The alteration of lymphoproliferative response induced at the advanced stage of carcinogenesis without alteration of T lymphocyte subsets may indicate that the influence of 2-AAF and PB on the immune system is functional and not toxic. (C) 2000 Elsevier B.V. Ireland Ltd. All rights reserved.

Protective action of propolis on the rat colon carcinogenesis

Propolis is a honeybee product with several biological and therapeutical properties. Its effect on the process of colon carcinogenesis and DNA damage were evaluated in the male Wistar rats using the aberrant crypt foci (ACF) assay and the comet assay, respectively. For both tests, animals were treated with the colon carcinogen 1,2 dimethylhydrazine (DMH, 40 mg/kg, s.c.) for 2 weeks (two injections/week) in order to induce both DNA damage and ACF. The animals were divided into groups that received propolis (ethanolic extract) at three different doses (10, 30, and 90 mg/kg b.w., by gavage), either simultaneously or after DMH treatment. For the comet assay, peripheral blood samples were collected 4 h after the last DMH treatment. All animals were sacrificed at the 5th week for evaluation of ACF. The results show that only the intermediate dose (30 mg/kg) of propolis, administered after DMH initiation, is significantly associated to a smaller number of aberrant crypts in the distal colon. No effect on DNA damage in peripheral blood cells, however, was verified by the comet assay. These data suggest that propolis has a protective influence on the process of colon carcinogenesis, suppressing the development of preneoplastic lesions, and probably exerts no protection against the initiation of carcinogenesis.

Chemopreventive property of dietary ginger in rat urinary bladder chemical carcinogenesis

The modifying potential of ginger on the development of preneoplasia and tumors in the male Wistar rat urinary bladder was investigated in a 36-week-long initiation-promotion assay for chemical carcinogenesis. Groups G1 to G3 were given 0.05% N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in drinking water for 5 weeks and a 3% uracil meal for the subsequent 3 weeks. Groups G4 and G5 were treated with 3% uracil only for the same period. After these steps, groups G2, and G3 and G4 were fed for 26 weeks a ginger extract mixed at 0.5 and 1.0% in a basal diet, respectively. Thirty six weeks after the beginning of the experiment all rats were killed. The multiplicity of urothelial lesions (hyperplasia and neoplasia) was significantly lower (P = 0.013) in group G3 than in groups G1 and G2. The results suggest that 1.0% ginger meal exerts a protective effect on the post-initiation stage of rat chemically-induced urothelial carcinogenesis.