Modulation of detoxification enzymes by watercress: in vitro and in vivo investigations in human peripheral blood cells.

BACKGROUND: Epidemiological studies indicate that consumption of cruciferous vegetables (CV) can reduce the risk of cancer. Supposed mechanisms are partly the inhibition of phase I and the induction of phase II enzymes. AIM: The aim of this study was to investigate in vitro and in vivo effects of watercress (WC), a member of the CV family, on chemopreventive parameters using human peripheral blood mononuclear cells (PBMC) as surrogate cells. We investigated the hypothesis that WC reduces cancer risk by inducing detoxification enzymes in a genotype-dependent manner. METHODS: In vitro gene expression and enzyme activity experiments used PBMC incubated with a crude extract from fresh watercress (WCE, 0.1-10 microL/mL with 8.2 g WC per 1 mL extract) or with one main key compound phenethyl isothiocyanate (PEITC, 1-10 microM). From an in vivo perspective, gene expression and glutathione S-transferase (GST) polymorphisms were determined in PBMC obtained from a human intervention study in which subjects consumed 85 g WC per day for 8 weeks. The influence of WC consumption on gene expression was determined for detoxification enzymes such as superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPX1), whilst the SOD and GPX activities in red blood cells were also analysed with respect to GST genotypes. RESULTS: In vitro exposure of PBMC to WCE or PEITC (24 h) increased gene expression for both detoxification enzymes GPX1 (5.5-fold, 1 microL/mL WCE, 3.7-fold 1 microM PEITC) and SOD2 (12.1-fold, 10 microL/mL WCE, 7.3-fold, 10 microM PEITC), and increased SOD2 activity (1.9-fold, 10 microL/mL WCE). The WC intervention had no significant effect on in vivo PBMC gene expression, as high individual variations were observed. However, a small but significant increase in GPX (p = 0.025) and SOD enzyme activity (p = 0.054) in red blood cells was observed in GSTM1*0, but not in GSTM1*1 individuals, whilst the GSTT1 genotype had no impact. CONCLUSION: The results indicate that WC is able to modulate the enzymes SOD and GPX in blood cells in vitro and in vivo, and suggest that the capacity of moderate intake of CV to induce detoxification is dependent in part on the GSTM1 genotype.

The database dbEST correctly predicts gene expression in colon cancer patients.

This study aims to test the predictive power of gene expression data derived from NIH's database dbEST, which collects gene expression results from a large number and variety of DNA array experiments. The motivation of this study is to make comparable experimental studies, which are usually performed only for one or a few tissues or organs, with a wide variety of other tissues. Confirmation of a good predictive power of dbEST would put a number of interesting and partially surprising recent findings, solely based on data mining, on a more solid basis than available so far. The expression of nine genes (eIF4E, DDX6, HAT1, USP28, HSP90(beta, PKM2, PLK1, COX2 and OPN) plus two calibration genes in paired normal and cancer colon tissues of eight individual patients was investigated by quantitative RT-PCR and compared with the predictions made by the data-base. GUS and beta-actin reveal only little variation among different patients, making them good internal calibration standards. In normal colon tissue, data mining correctly predicts the expression of all nine genes, which covers two orders of magnitude. In cancer, dbEST is somewhat less precise, but still valuable for the comparison with clinical results.

Carob fibre compounds modulate parameters of cell growth differently in human HT29 colon adenocarcinoma cells than in LT97 colon adenoma cells.

An extract of the Mediterranean carob (Ceratonia siliqua L.) pod (carob fibre extract), products formed after its fermentation by the gut flora and the major phenolic ingredient gallic acid (GA), were comparatively investigated for their influence on survival and growth parameters of colon adenocarcinoma HT29 cells and adenoma LT97 cells. Hydrogen peroxide (H2O2) formation in the cell culture media was quantified. After 1h 97+/-4 microM or 70+/-15 microM were found in HT29 medium and 6+/-1 microM or 3+/-3 microM in LT97 medium for carob fibre extract or GA, respectively. After 72 h carob fibre extract reduced survival of rapidly proliferating HT29 cells (by 76.4+/-12.9%) whereas metabolic activity and DNA-synthesis were only transiently impaired. Survival of slower growing LT97 cells was less decreased (by 21.5+/-12.9%), but there were marked effects on DNA-synthesis (reduction by 95.6+/-7%, 72 h). GA and fermented carob fibre did not have comparable effects. Thus, carob fibre extract resulted in H2O2 formation, which, however, could not explain impairment of cell growth. The differently modulated growth of human colon cell lines was more related to proliferation rates and impairment of DNA-synthesis than to H2O2 formation.

Ferric iron is genotoxic in non-transformed and preneoplastic human colon cells.

Iron could be a relevant risk factor for carcinogenesis since it catalyses the formation of reactive oxygen species (ROS), which damage DNA. We previously demonstrated genotoxic effects by ferric iron using the human colon cancer cell line HT29. Here we investigated ferric iron in primary non-transformed colon cells and in a preneoplastic colon adenoma cell line (LT97), which both are suitable models to study effects of carcinogens during early stages of cell transformation. Genetic damage was determined using the Comet assay. Comet FISH (fluorescence in situ hybridization) was used to assess specific effects on TP53. Fe-NTA (0-1000 microM, 30 min, 37 degrees C) significantly induced single strand breaks in primary colon cells (500 microM Fe-NTA: Tail intensity [TI] 22.6%+/-5.0% versus RPMI control: TI 10.6%+/-3.9%, p<0.01) and in LT97 cells (1000 microM Fe-NTA: TI 26.8%+/-7.3% versus RPMI control: TI 11.1%+/-3.7%, p<0.01). With the Comet FISH protocol lower concentrations of Fe-NTA significantly increased DNA damage already at 100 and 250 microM Fe-NTA in primary colon and LT97 adenoma cells, respectively. This damage was detected as an enhanced migration of TP53 signals into the comet tail in both cell types, which indicates a high susceptibility of this tumor relevant gene towards Fe-NTA. In conclusion, Fe-NTA acts genotoxic in non-transformed and in preneoplastic human colon cells, in which it also enhances migration of TP53 at relatively low concentrations. Translated to the in vivo situation these results suggest that iron overload putatively contributes to a genotoxic risk during early stages of colorectal carcinogenesis on account of its genotoxic potential in non-tumorigenic human colon cells.

DEP-1 protein tyrosine phosphatase inhibits proliferation and migration of colon carcinoma cells and is upregulated by protective nutrients.

The transmembrane protein-tyrosine phosphatase (PTP) DEP-1 (density-enhanced phosphatase) is a candidate tumor suppressor in the colon epithelium. We have explored the function of DEP-1 in colon epithelial cells by inducible re-expression in a DEP-1-deficient human colon cancer cell line. Density-enhanced phosphatase-1 re-expression led to profound inhibition of cell proliferation and cell migration, and was associated with cytoskeletal rearrangements. These effects were dependent on the PTP activity of DEP-1 as they were not observed with cells expressing the catalytically inactive DEP-1 C1239S variant. shRNA-mediated suppression of DEP-1 in a colon epithelial cell line with high endogenous DEP-1 levels enhanced proliferation, further supporting the antiproliferative function of DEP-1. Nutrients, which are considered to be chemoprotective with respect to colon cancer development, including butyrate, green tea and apple polyphenols, had the capacity to elevate transcription of endogenous DEP-1 mRNA and expression of DEP-1 protein. Upregulation of DEP-1 expression, and in turn inhibition of cell growth and migration may present a previously unrecognized mechanism of chemoprevention by nutrients.

Ferric iron increases ROS formation, modulates cell growth and enhances genotoxic damage by 4-hydroxynonenal in human colon tumor cells.

Iron is a relevant risk factor for colorectal cancer due to its genotoxic properties. Here we hypothesised that iron-overload causes other toxic effects, which contribute to carcinogenesis. For this, we investigated formation of reactive oxygen species (ROS), DNA repair, cell growth and glutathione (GSH) in human colon tumor cells (HT29 clone 19A) treated with ferric nitrilotriacetate (Fe-NTA, 0-2000 microM). Intracellular formation of ROS was analysed with the peroxide-labile fluorescent dye carboxy-dichlorodihydrofluorescine-diacetate. DNA repair, reflected as the persistency of DNA damage induced by selected genotoxins, was determined with the Comet assay. Cell growth and GSH were measured by fluorimetrical analysis. Key findings were that ROS formation increased with time (1000 microM Fe-NTA, p < 0.001). DNA damage was largely repaired after 120 min, but was not affected by 10 microM Fe-NTA. In contrast, 10 microM Fe-NTA significantly increased DNA damage induced by 4-hydroxynonenal. Doses of 25 microM Fe-NTA increased cell growth (p < 0.05), whereas high concentrations (2000 microM) resulted in growth arrest (p < 0.05), that was accompanied by increased GSH levels (p < 0.01). In conclusion, high concentrations of Fe-NTA caused cellular effects, which reflect a stress response, and resulted in formation of ROS. Carcinogenic risks from ferric iron could be derived also from lower concentrations, which enhance tumor cell growth and cause progenotoxic effects.

The main catechin of green tea, (-)-epigallocatechin-3-gallate (EGCG), reduces bleomycin-induced DNA damage in human leucocytes.

Interest in the beneficial effects of green tea has led to investigations on activities by the main catechin (-)-epigallocatechin-3-gallate (EGCG). This antioxidative compound could contribute to cancer chemoprevention by acting antigenotoxic. To further explore this hypothesis we investigated antigenotoxic potentials of low EGCG concentrations in human peripheral leucocytes. Leucocytes isolated from whole blood were (1) stimulated with phytohaemagglutinin, (2) damaged with genotoxic bleomycin, and (3) post-incubated to allow DNA repair. After each phase DNA integrity was measured with the comet assay. EGCG (2, 20, 100 microM) was added either during phases 1, 2 or 3 or during the whole process (1-3), to delineate mechanisms of antigenotoxicity reflecting induction of detoxification (phase 1), scavenging of radicals (phase 2), stimulation of repair (phase 3), respectively. Bleomycin induced breaks and endonuclease III specific damage, but EGCG did not affect damage or repair of these lesions when added during phases 1, 2 or 3. However, the application of EGCG during phases 1 and 2 significantly reduced both bleomycin-induced breaks and endonuclease III sensitive sites. EGCG added during all phases impaired persistence of damage. Our studies show that the continuous presence of EGCG can reduce radical-induced DNA damage in primary leucocytes, possibly due to a combination of different mechanisms. Together the findings support the hypotheses that EGCG acts protective in human cells.

Assessment of DNA damage and its modulation by dietary and genetic factors in smokers using the Comet assay: a biomarker model.

Methods are needed to assess exposure to genotoxins in humans and to improve understanding of dietary cancer prevention. The Comet assay was used to detect smoking-related exposures and dietary modulations in target tissues. Buccal scrapings, blood and faeces were collected from 38 healthy male volunteers (smokers and non-smokers) during a dietary intervention study with bread supplemented with prebiotics+/-antioxidants. GSTM1-genotype was determined with PCR. Buccal and peripheral lymphocytes were analysed for DNA damage using the Comet assay. Genotoxicity of faecal water (FW) was assayed in human colon HT29 clone 19A cells. 'Tail intensity' (TI) was used as a quantitative indicator of DNA damage in the Comet assay. Intervention with bread reduced DNA damage in lymphocytes of smokers (8.3+/-1.7% TI versus 10.2+/-4.1% TI, n=19), but not of non-smokers (8.6+/-2.8% TI versus 8.3+/-2.7% TI, n=15). Faecal water genotoxicity was reduced only in non-smokers (9.4+/-2.9% TI versus 18.9+/-13.1% TI, n=15) but not in smokers (15.5+/-10.7% TI versus 20.4+/-14.1% TI, n=13). The Comet assay was efficient in the detection of both smoking-related exposure (buccal cells) and efficacy of dietary intervention (faecal samples). Smokers and non-smokers profited differently from the intervention with prebiotic bread+/-antioxidants. Stratification of data by genotype enhanced specificity/sensitivity of the intervention effects and contributed important information on the role of susceptibility.

Genetic damage and repair in human rectal cells for biomonitoring: sex differences, effects of alcohol exposure, and susceptibilities in comparison to peripheral blood lymphocytes.

INTRODUCTION: Cells other than lymphocytes may be preferable as surrogate biomarkers during exposure monitoring. In nutritional toxicology, cells from colorectal tissues are particularly relevant for studying associations between food and cancer. Thus, we have previously shown that colonic cells of males have higher levels of DNA damage than females, which (among other factors) could be due to a higher consumption of alcoholic beverages by males. To test this hypothesis, we have performed a first exploratory study to compare DNA damage in rectal cells from biopsies of male patients with alcohol abuse and of male and female controls. Peripheral blood lymphocytes were additionally monitored to assess systemic exposure loads. METHODS: Cells were isolated and subjected to microgelelectrophoresis +/- endonuclease III to measure DNA breaks and oxidized pyrimidine bases ("comet-assay"). Cell aliquots were treated with H(2)O(2) for 5min in suspension culture and processed immediately or after 60min to determine induced damage and its persistence. RESULTS: Pooled data from subjects of all groups revealed that oxidative DNA damage in rectal cells directly correlated to damage in lymphocytes. Female controls had lower levels of DNA damage than male controls, confirming the previous studies. An unexpected result was that male alcohol abusers had significantly less genetic damage than male controls. Also, repair was detected in lymphocytes of male alcohol abusers and female controls, but not in male controls. CONCLUSION: This is the first time the comet-assay has been used to detect genotoxicity in human rectal cells as a biomonitoring tool. Our pilot study confirms earlier reports on sex differences and indicates a good correlation between damage in rectal cells and damage in lymphocytes and implies that alcohol exposure enhances endogenous defence.

The 10 basic requirements for a scientific paper reporting antioxidant, antimutagenic or anticarcinogenic potential of test substances in in vitro experiments and animal studies in vivo.

There is increasing evidence that chemicals/test substances cannot only have adverse effects, but that there are many substances that can (also) have a beneficial effect on health. As this journal regularly publishes papers in this area and has every intention in continuing to do so in the near future, it has become essential that studies reported in this journal reflect an adequate level of scientific scrutiny. Therefore a set of essential characteristics of studies has been defined. These basic requirements are default properties rather than non-negotiables: deviations are possible and useful, provided they can be justified on scientific grounds. The 10 basic requirements for a scientific paper reporting antioxidant, antimutagenic or anticarcinogenic potential of test substances in in vitro experiments and animal studies in vivo concern the following areas: (1) Hypothesis-driven study design; (2) The nature of the test substance; (3) Valid and invalid test systems; (4) The selection of dose levels and gender; (5) Reversal of the effects induced by oxidants, carcinogens and mutagens; (6) Route of administration; (7) Number and validity of test variables; (8) Repeatability and reproducibility; (9) Statistics; and (10) Quality Assurance.

Putative colon cancer risk factors damage global DNA and TP53 in primary human colon cells isolated from surgical samples.

This study describes a novel in vitro method in genetic toxicology that is based on detection of chemical-induced DNA damage connected with altered migration of TP53 in primary human colonocytes. Techniques were developed to isolate high numbers of human epithelial colon cells from surgical tissues. High quantities of viable cells were obtained per donor. The primary cells were treated with the endogenous risk factors trans-2-hexenal, and hydrogen peroxide. Global DNA damage and repair were measured by single-cell gel electrophoresis (Comet assay). We compared responses of primary colon cells to HT29clone19A, a differentiated human colon tumour cell line, for which the karyotype was analysed with 24-colour FISH. Both compounds were genotoxic in both cell types and most of the induced DNA damage was repaired after 30 min. Specific migration of TP53 was determined by fluorescence in situ hybridization (Comet FISH). Using primary colon cells, we quantified the migration of TP53 signals into the comet tails. In these cells TP53 was more sensitive than global DNA for genotoxicity induced by trans-2-hexenal and H(2)O(2). HT29clone19A cells cannot be used for Comet FISH because of their aberrant karyotype. The approach described allows us to obtain more knowledge of putative risk factors in colon carcinogenesis.

Multitude multicolor chromosome banding (mMCB) - a comprehensive one-step multicolor FISH banding method.

Multicolor chromosome banding (MCB) using one single chromosome-specific MCB probe set per experiment was previously reported as powerful tool in molecular cytogenetics for the characterization of all kinds of human marker chromosomes. However, a quick analysis of karyotypes with highly complex chromosomal changes was hampered by the problem that up to 24 MCB experiments were necessary for a comprehensive karyotype description. To overcome that limitation the 138 available region-specific microdissection-derived libraries for all human chromosomes were combined to one single probe set, called multitude MCB (mMCB). A typical fluorescence banding pattern along the human karyotype is produced, which can be evaluated either by transforming these profiles into chromosome region-specific pseudo-colors or more reliably by studying the fluorescence profiles. The mMCB probe set has been applied on chromosomes of normal male and female probands, two primary myelodysplastic syndromes and two solid tumor cell lines. Additionally, a cell line of Gorilla gorilla (GGO) studied previously by single chromosome-specific MCB was reevaluated by the mMCB method. All results were in concordance with those obtained in parallel or by other cytogenetic and molecular cytogenetic approaches indicating that mMCB is a powerful multicolor FISH banding tool for fast characterization of complex karyotypes.

Experimental evidences on the potential of prebiotic fructans to reduce the risk of colon cancer.

Inulin is extracted from the chicory root. It is a set of fructans with its monomers linked by means of beta(2-1) bonds. This linkage cannot be hydrolysed by either pancreatic or by brush border digestive enzymes in the upper intestinal tract of humans. As such the carbohydrates arrive in the colon, where they are fermented by bifidobacteria and other lactic acid producing bacteria, thus enhancing their relative populations in the gut. Recent research in experimental animal models revealed that inulin has significant anticarcinogenic properties. It acts chemopreventively by reducing the incidence of azoxymethane (AOM) - induced aberrant crypt foci and tumours in the colon. These effects may be due to the stimulation of bifidobacteria, which themselves have been shown to act as antigenotoxic in the colon and to reduce AOM-induced tumours. Also fermentation products, including the short-chain fatty acid butyrate, could contribute to the protective effects. In this case a mechanism may be the induction of apoptosis of already transformed cells. The experimental evidence from animal studies and from studies elucidating potential mechanisms strongly supports the possibility that inulin will contribute to reducing risks for colon cancer in humans. In order to obtain more insight into this possibility, human dietary intervention studies relating biomarkers of reduced risk to inulin consumption are needed.

Methods of in vitro toxicology.

In vitro methods are common and widely used for screening and ranking chemicals, and have also been taken into account sporadically for risk assessment purposes in the case of food additives. However, the range of food-associated compounds amenable to in vitro toxicology is considered much broader, comprising not only natural ingredients, including those from food preparation, but also compounds formed endogenously after exposure, permissible/authorised chemicals including additives, residues, supplements, chemicals from processing and packaging and contaminants. A major promise of in vitro systems is to obtain mechanism-derived information that is considered pivotal for adequate risk assessment. This paper critically reviews the entire process of risk assessment by in vitro toxicology, encompassing ongoing and future developments, with major emphasis on cytotoxicity, cellular responses, toxicokinetics, modelling, metabolism, cancer-related endpoints, developmental toxicity, prediction of allergenicity, and finally, development and application of biomarkers. It describes in depth the use of in vitro methods in strategies for characterising and predicting hazards to the human. Major weaknesses and strengths of these assay systems are addressed, together with some key issues concerning major research priorities to improve hazard identification and characterisation of food-associated chemicals.

Contribution of apoptosis to responses in the comet assay.

Apoptosis, a physiological process of selected cell deletion, leads to DNA fragmentation in typical segments of 180 base pairs. DNA strand breaks are also an effect induced by genotoxic compounds. The aim of this study was to compare these two types of damaging potentials by a known genotoxic substance and an apoptosis-inducing agent in HT-29 colon adenocarcinoma cells. The cells were incubated for 24h with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent DNA damage-inducing agent, staurosporine, an inhibitor of protein kinase C and apoptosis-inducing agent, and hydrogen peroxide, a source of reactive oxygen species. Apoptosis was measured with the Annexin V affinity assay which detects the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the cytoplasmic membrane, an early event in the apoptotic process. DNA damage as an end point of genotoxicity was detected by single cell microgel electrophoresis, also called "comet assay". The results show that apoptosis does not necessarily need to correlate or coincide with DNA damage observed with genotoxic substances in the comet assay. The representative apoptosis-inducing agent (staurosporine) did not induce strand breaks in the tested concentrations (0.5 and 1.0microM); genotoxic doses of the strand break inducing agent MNNG did not induce apoptosis. Therefore, the comet assay can be used as a specific test for detecting genotoxicity, and the results are not necessarily confounded by concomittant processes leading to apoptosis.

Biomarkers in disease and health.

Biomarkers have considerable potential in aiding the understanding of the relationship between diet and disease or health. However, to assess the role, relevance and importance of biomarkers on a case by case basis it is essential to understand and prioritise the principal diet and health issues. In the majority of cases, dietary compounds are only weakly biologically active in the short term, have multiple targets and can be both beneficial and deleterious. This poses particular problems in determining the net effect of types of foods on health. In principle, a biomarker should be able to contribute to this debate by allowing the measurement of exposure and by acting as an indicator either of a deleterious or of an enhanced health effect prior to the final outcome. In this review, the examples chosen - cancer (stomach, colon/rectal, breast); coronary heart disease and osteoporosis - reflect three major diet-related disease issues. In each case the onset of the disease has a genetic determinant which may be exacerbated or delayed by diet. Perhaps the most important factor is that in each case the disease, once manifest, is difficult to influence in a positive way by diet alone. This then suggests that the emphasis for biomarker studies should focus on predictive biomarkers which can be used to help in the development of dietary strategies which will minimise the risk and be of greater benefit.

Protective role of probiotics and prebiotics in colon cancer.

Ingestion of viable probiotics or prebiotics is associated with anticarcinogenic effects, one mechanism of which is the detoxification of genotoxins in the gut. This mechanism was shown experimentally in animals with use of the rat colon carcinogen 1,2-dimethylhydrazine and by determining endpoints that range from tumorigenesis to induction of DNA damage. Because of the complexity of cancer initiation, cancer progression, and the exposure of cancer in the gut, many types of interactions may be envisaged. Notably, some of our newer studies showed that short-lived metabolite mixtures isolated from milk that was fermented with strains of Lactobacillus bulgaricus and Streptococcus thermophilus are more effective in deactivating etiologic risk factors of colon carcinogenesis than are cellular components of microorganisms. Ingestion of prebiotics results in a different spectrum of fermentation products, including the production of high concentrations of short-chain fatty acids. Gut flora, especially after the ingestion of resistant starch, induces the chemopreventive enzyme glutathione transferase pi in the colon of the rat. Together, these factors lead to a reduced load of genotoxic agents in the gut and to an increased production of agents that deactivate toxic components. Butyrate is one such protective agent and is associated with lowering cancer risk. It was recently shown that buytrate may inhibit the genotoxic activity of nitrosamides and hydrogen peroxide in human colon cells. In humans, the ingestion of probiotics leads to the excretion of urine with low concentrations of components that are genotoxic in human colon cells and high concentrations of components that induce oxidized DNA bases.

Butyrate induces glutathione S-transferase in human colon cells and protects from genetic damage by 4-hydroxy-2-nonenal.

Butyrate, one of the major products of gut fermentation, is known to inhibit proliferation, induce apoptosis and differentiation, and increase phase II enzyme activities in tumor cells, whereas little information is available on protective effects in less-transformed colon cells. The aim of this study was to investigate whether the chemoprotective mechanism of glutathione S-transferase (GST) induction by butyrate could also play a role in earlier stages of colon carcinogenesis and whether chemoresistance of cells toward the endogenous genotoxic risk factor 4-hydroxy-2-nonenal (HNE) could be a consequence of butyrate treatment. As cell models, we used the human tumor cell lines HT29 and HT29 clone 19A, a differentiated subclone with properties resembling primary colon cells. We determined the expression of GSTP1 protein (enzyme-linked immunosorbent assay), the major GST in HT29, GSTP1 mRNA (Northern blotting), GST activity, intracellular glutathione, and total protein. The genotoxic impact of HNE (100-200 microM) was compared in butyrate-treated and nontreated cells using single-cell microgel electrophoresis. Our results show that GSTP1 mRNA, GSTP1 protein, GST activity, and total protein were increased (1.2- to 2.5-fold) and glutathione levels were maintained after 24-72 h of incubation with 4 mM butyrate. Moreover, a marked reduction of HNE-induced genotoxicity was caused by preincubation with butyrate. Butyrate also induced the phosphorylation of extracellular signal-regulated kinases (ERK1/2, Western blotting) after 5-30 min, which indicates a regulation of GST expression by this signal pathway. Most effects were greater in HT29 parent cells than in clone cells. In conclusion, butyrate enhances expression of GST and other proteins in both cell lines, which leads to an enhanced chemoprotection, reducing the impact of HNE genotoxicity. Thus butyrate could play a role in early and later stages of cancer prevention by reducing exposure to relevant risk factors.

Neurotensin-and EGF-induced metabolic activation of colon carcinoma cells is diminished by dietary flavonoid cyanidin but not by its glycosides.

Dietary polyphenols, including anthocyanidins and their glycosides anthocyanins, are suggested to be involved in the protective effects of fruits and vegetables against cancer. Very few data are available concerning the effects of anthocyanidins/anthocyanins on cellular processes induced by growth factors such as neurotensin and epidermal growth factor (EGF), which are implicated in the pathophysiology of colon cancer. Here, we show that neurotensin and EGF caused an increase in the extracellular acidification rate, which could reflect the activity of cellular metabolism, in the human carcinoma cell line HT29 clone 19A. Neurotensin and EGF also caused a strong rise in the intracellular Ca2+ concentration, induced phosphorylation of extracellular signal-regulated kinases (ERK1 and ERK2), and stimulated growth of human carcinoma cells. Cyanidin (10 microM), but not its glycosides cyanin and idaein, was able to inhibit the neurotensin- and EGF-induced increased rate of extracellular acidification. In contrast to N-ethyl-N-isopropyl amiloride, an inhibitor of Na+/H+ exchange, cyanidin did not alter the rate of intracellular pH recovery of cells loaded by NH3/NH4+, indicating that cyanidin inhibits cellular metabolism, rather than directly altering Na+/H+ exchange. Cyanidin, but not cyanin and idaein, was able to inhibit an increase in intracellular Ca2+ concentration induced by neurotensin. Neurotensin- and EGF-induced phosphorylation of ERKs was not affected by cyanidin, cyanin, and idaein at < or = 100 microM. Only cyanidin (100 microM), but not cyanin and idaein, was able to inhibit cellular growth induced by EGF. Thus these findings suggest that a dietary polyphenol cyanidin, but not its glycosides, is a potent inhibitor of mitogen-induced metabolic activity, increase in free intracellular Ca2+, and cellular growth of cultured colon carcinoma cells.

Effect of lactobacilli, bifidobacteria and inulin on the formation of aberrant crypt foci in rats.

BACKGROUND: Our studies were aimed at investigating the effect of lactic acid producing bacteria (LAB) or inulin, a natural source of non-digestible oligosaccharides derived from chicory, on the induction by carcinogens of aberrant crypt foci (ACF) in the colon, which are considered to be early precursor lesions of neoplasia. METHODS: Strains of Bifidobacterium longum, Lactobacillus casei and Lactobacillus acidophilus were administered to rats fed a purified high starch diet, under a variety of treatment protocols including daily gavage, via the drinking water and in the diet. The rats were treated with methyl-N-nitrosourea, 1,2-dimethylhydrazine, or azoxymethane (AOM) to induce ACF. RESULTS: In general, no consistent significant changes in ACF numbers were detected in these experiments. In one study, the basal diet of the rats was changed to one containing a higher level of fat (corn oil). Under these conditions, a significant decrease in AOM-induced colonic ACF was seen in rats given L. acidophilus or inulin. In a concurrent group of animals fed a low fat diet, no significant decrease in ACF was observed. CONCLUSIONS: The results indicate that the type of diet fed can influence the detection of protective effects of LAB and oligosaccharides and that against the background of a diet with a level of fat typical of a Western diet, evidence for a protective effect of L. acidophilus and inulin towards colon cancer was obtained