Mechanisms of primary cancer prevention by butyrate and other products formed during gut flora-mediated fermentation of dietary fibre.

Dietary fibres are indigestible food ingredients that reach the colon and are then fermented by colonic bacteria, resulting mainly in the formation of short-chain fatty acids (SCFA) such as acetate, propionate, and butyrate. Those SCFA, especially butyrate, are recognised for their potential to act on secondary chemoprevention by slowing growth and activating apoptosis in colon cancer cells. Additionally, SCFA can also act on primary prevention by activation of different drug metabolising enzymes. This can reduce the burden of carcinogens and, therefore, decrease the number of mutations, reducing cancer risk. Activation of GSTs by butyrate has been studied on mRNA, protein, and enzyme activity level by real-time RT-PCR, cDNA microarrays, Western blotting, or photometrical approaches, respectively. Butyrate had differential effects in colon cells of different stages of cancer development. In HT29 tumour cells, e.g., mRNA GSTA4, GSTP1, GSTM2, and GSTT2 were induced. In LT97 adenoma cells, GSTM3, GSTT2, and MGST3 were induced, whereas GSTA2, GSTT2, and catalase (CAT) were elevated in primary colon cells. Colon cells of different stages of carcinogenesis differed in post-transcriptional regulatory mechanisms because butyrate increased protein levels of different GST isoforms and total GST enzyme activity in HT29 cells, whereas in LT97 cells, GST protein levels and activity were slightly reduced. Because butyrate increased histone acetylation and phosphorylation of ERK in HT29 cells, inhibition of histone deacetylases and the influence on MAPK signalling are possible mechanisms of GST activation by butyrate. Functional consequences of this activation include a reduction of DNA damage caused by carcinogens like hydrogen peroxide or 4-hydroxynonenal (HNE) in butyrate-treated colon cells. Treatment of colon cells with the supernatant from an in vitro fermentation of inulin increased GST activity and decreased HNE-induced DNA damage in HT29 cells. Additional animal and human studies are needed to define the exact role of dietary fibre and butyrate in inducing GST activity and reducing the risk of colon cancer.

Influence of inorganic and organic iron compounds on parameters of cell growth and survival in human colon cells.

Increased risk for development of colon cancer is associated with red meat intake and iron toxicity is discussed for one underlying mechanism. Anyhow, for iron itself only limited evidence is found. In this study, effects of different iron compounds on proliferation of HT29 carcinoma and LT97 adenoma human colon cells were investigated. After treatment of cells with inorganic (ferrous sulfate: FeSO4 and ferric nitrilotriacetate: FeNTA) and organic (hemoglobin and hemin) iron sources (24-72 h), number of cells and metabolic activity were measured. Under normal cell culture conditions, neither iron compound elevated cell growth in either cell line with the exception of FeNTA which induced LT97 cell growth significantly. Distinct inhibition of cell proliferation was measured for organic iron. Serum-free incubation of HT29 cells revealed growth promoting properties of iron under deficiency. Even though organic iron, especially hemin, was a potent growth factor, both substances showed also dose-dependent cytotoxic effects. In conclusion, these data emphasize that not iron itself, but merely organic iron may promote carcinogenic events. Since promotion of proliferation was only detectable under deficiency, cytotoxic properties of organic iron may be of more importance in colon carcinogenesis.

Apple polyphenols modulate expression of selected genes related to toxicological defence and stress response in human colon adenoma cells.

Apples contain significant amounts of flavonoids that are potentially cancer risk reducing by acting antioxidative or antiproliferative and by favorably modulating gene expression. The purpose of this study was to investigate whether polyphenols from apples modulate expression of genes related to colon cancer prevention in preneoplastic cells derived from colon adenoma (LT97). For this, LT97 cells were treated with effective concentrations of apple extracts (AEs). RNA was isolated and used for synthesis and labeling of cDNA that was hybridized to cDNA-arrays. Gene expression studies were performed using a commercial cDNA-array from Superarray that contains a limited number of genes (96 genes) related to drug metabolism, and a custom-made cDNA microarray that contains a higher number of genes (300 genes, including some genes from Superarray) related to mechanisms of carcinogenesis or chemoprevention. Real-time PCR and enzyme activity assays were additionally performed to confirm selected array results. Treatment of cells with AE resulted in 30 and 46 genes expressed over cut-off values (>or=1.5- or

Dietary Acid Load and Potassium Intake Associate with Blood Pressure and Hypertension Prevalence in a Representative Sample of the German Adult Population.

Diets rich in fruits and vegetables, like the Dietary Approaches to Stop Hypertension (DASH)-diet, are usually characterized by high potassium intake and reduced dietary acid load, and have been shown to reduce blood pressure (BP). However, the relevance of potential renal acid load (PRAL) for BP has not been compared with the relevance to BP of urinary biomarker (K-urine)- and dietary food frequency questionnaire (K-FFQ)-based estimates of potassium intake in a general adult population sample. For 6788 participants (aged 18-79 years) of the representative German Health-Interview and Examination Survey for Adults (DEGS1), associations of PRAL, K-urine, and K-FFQ with BP and hypertension prevalence were cross-sectionally examined in multivariable linear and logistic regression models. PRAL was significantly associated with higher systolic BP (p = 0.0002) and higher hypertension prevalence (Odds ratio [OR] high vs. low PRAL = 1.45, p = 0.0004) in models adjusted for age, sex, body mass index (BMI), estimated sodium intake, kidney function, relevant medication, and further important covariates. Higher estimates of K-FFQ and K-urine were related to lower systolic BP (p = 0.04 and p < 0.0001) and lower hypertension prevalence (OR = 0.82, p = 0.04 and OR = 0.77, p = 0.02) as well as a lower diastolic BP (p = 0.03 and p = 0.0003). Our results show, for the first time in a comparative analysis of a large representative population sample, significant relationships of BP and hypertension prevalence with questionnaire- and biomarker-based estimates of potassium intake and with an estimate of dietary acid load.

Hemoglobin and hemin induce DNA damage in human colon tumor cells HT29 clone 19A and in primary human colonocytes.

Epidemiological findings have indicated that red meat increases the likelihood of colorectal cancer. Aim of this study was to investigate whether hemoglobin, or its prosthetic group heme, in red meat, is a genotoxic risk factor for cancer. Human colon tumor cells (HT29 clone 19A) and primary colonocytes were incubated with hemoglobin/hemin and DNA damage was investigated using the comet assay. Cell number, membrane damage, and metabolic activity were measured as parameters of cytotoxicity in both cell types. Effects on cell growth were determined using HT29 clone 19A cells. HT29 clone 19A cells were also used to explore possible pro-oxidative effects of hydrogen peroxide (H2O2) and antigenotoxic effects of the radical scavenger dimethyl sulfoxide (DMSO). Additionally we determined in HT29 clone 19A cells intracellular iron levels after incubation with hemoglobin/hemin. We found that hemoglobin increased DNA damage in primary cells (> or =10 microM) and in HT29 clone 19A cells (> or =250 microM). Hemin was genotoxic in both cell types (500-1000 microM) with concomitant cytotoxicity, detected as membrane damage. In both cell types, hemoglobin and hemin (> or =100 microM) impaired metabolic activity. The growth of HT29 clone 19A cells was reduced by 50 microM hemoglobin and 10 microM hemin, indicating cytotoxicity at genotoxic concentrations. Hemoglobin or hemin did not enhance the genotoxic activity of H2O2 in HT29 clone 19A cells. On the contrary, DMSO reduced the genotoxicity of hemoglobin, which indicated that free radicals were scavenged by DMSO. Intracellular iron increased in hemoglobin/hemin treated HT29 clone 19A cells, reflecting a 40-50% iron uptake for each compound. In conclusion, our studies show that hemoglobin is genotoxic in human colon cells, and that this is associated with free radical mechanisms and with cytotoxicity, especially for hemin. Thus, hemoglobin/hemin, whether available from red meat or from bowel bleeding, may pose genotoxic and cytotoxic risks to human colon cells, both of which contribute to initiation and progression of colorectal carcinogenesis.

Sodium intake in Germany.

For many years, a high sodium intake has been discussed as a potential risk factor in the development of hypertension and, consequently, cardiovascular diseases. As part of the German Health Interview and Examination Survey for Adults (DEGS1), which was conducted between 2008 and 2011, sodium excretion in casual urine samples was used as a biomarker to measure sodium intake. DEGS1 observed that the median daily sodium intake of women (3.4 g) as well as men (4.0 g) exceeds the levels recommended by German and international organisations. Among other factors, men's higher sodium intake could be explained by their higher energy intake. In addition, DEGS1 demonstrates an association between women's sodium intake and age; however, no equivalent correlation was identified for men. Furthermore, although high socio-economic status is associated with lower sodium intake in men, no comparable correlation was observed among women.

Dietary exposure to selected perfluoroalkyl acids (PFAAs) in four European regions.

The dietary exposure to selected PFAAs was estimated in four selected European states (Belgium, the Czech Republic, Italy and Norway) representing Western, Southern, Eastern and Northern Europe. The harmonised sampling programme designed in the European Union project PERFOOD was targeted at identifying seven selected PFAAs, including perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), in food items that are most important both in terms of consumption and based on known high contamination patterns. The estimated average dietary exposure for adults (18-64 years) and children (3-9 years) is generally below or close to 1 ng kg⁻¹ BW day⁻¹ for all seven PFAAs. Considering the high consumption of food groups that contribute most to the exposure does not result in estimates exceeding 4 ng kg⁻¹ BW day⁻¹. Thus, based on the TDIs proposed by EFSA for PFOS (150 ng kg⁻¹ BW day⁻¹) and PFOA (1500 ng kg⁻¹ BW day⁻¹), no concern can be identified. There are distinct dietary exposure patterns from region to region as a result of different food consumption and contamination patterns. Foods of plant origin (e.g. fruit and vegetables) are most important for the dietary exposure to PFHxA, PFOA and PFHxS, while the consumption of foods of animal origin (particularly fish and seafood) mostly contributes to the dietary exposure to PFDA and PFUnDA. For the dietary exposure to PFNA and PFOS, food of animal and plant origin contributes with equal importance. In conclusion, region-to-region differences as well as the relative importance of food of different origin for each PFAA should be paid more attention in further research.

Occurrence of selected perfluorinated alkyl acids in lunch meals served at school canteens in Italy and their relevance for children's intake.

Ready-to-eat servings may be more contaminated with perfluorinated alkyl acids (PFAAs) than the corresponding unprocessed foods due to the presence of PFAAs in and transfer from food contact materials (FCM) and cookware. Therefore, the presence of selected PFAAs in meals served weekly at lunch time in six Italian school canteens was assessed. Five towns were selected representing different areas with local water and food supply. Daily lunch menus were sampled and pooled to form a composite. Analyses were carried out on the weekly composite from each canteen. UPLC-MS/MS quantification limits were in the 6.0-12 pg g⁻¹ range for the selected PFAAs (PFHxA, PFOA, PFNA, PFDA, PFUnDA, PFHxS, branched and non-branched PFOS). Non-branched PFOS was quantified in four out of six composites, with levels ranging from 14 to 25 pg g⁻¹, while PFOA and PFDA were determined in two out of six in the range 6.5-8.2 pg g⁻¹. Theoretical estimates and analytical results in the same order of magnitude indicate a negligible contribution from food processing and serving to meal contamination. When composite analytical data are transposed into dietary estimates, it is shown that Italian school-age children have intakes in the range of 0.3-1.1 and 0.5-1.4 ng kg⁻¹ bw day⁻¹ for PFOA and PFOS respectively, well below the corresponding tolerable daily intakes (TDIs).

Perfluorinated alkylated substances in vegetables collected in four European countries; occurrence and human exposure estimations.

The human diet is recognised as one possible major exposure route to the overall perfluorinated alkylated substances (PFAS) burden of the human population, resulting directly from contamination of dietary food items, as well as migration of PFAS from food packaging or cookware. Most European countries carry out national monitoring programs (food basket studies) to monitor contamination with pollutants. Usually, for PFASs, non-coordinated approaches are used in Europe, since food basket studies are mainly carried out by national authorities following national requirements and questions, making comparisons between different countries difficult. A harmonised sampling campaign collecting similar food items in a uniform procedure enabling direct comparison between different regions in Europe was designed. We selected four countries (Belgium, Czech Republic, Italy and Norway), representing the four regions of Europe: West, East, South and North. In spring 2010 and 2011, 20 different types of vegetables were sampled in Belgium, Czech Republic, Italy and Norway. Perfluorinated carboxylic acids (PFCAs) were the main group of detected PFASs, with perfluorinated octanoic acid (PFOA) as the most abundant PFCA (with exception of samples from Czech Republic), followed by perfluorinated hexanoic acid and perfluorinated nonanoic acid. Dietary intake estimates for PFOA show only low human exposure due to vegetable consumption for adults and children, mostly governed by high intake of potatoes.

Gene expression profiles in human peripheral blood mononuclear cells as biomarkers for nutritional in vitro and in vivo investigations.

Identification of chemopreventive substances may be achieved by measuring biological endpoints in human cells in vitro. Since generally only tumour cells are available for such investigations, our aim was to test the applicability of peripheral blood mononuclear cells (PBMC) as an in vitro primary cell model since they mimic the human in vivo situation and are relatively easily available. Cell culture conditions were refined, and the basal variation of gene expression related to drug metabolism and stress response was determined. Results were compared with profiles of an established human colon cell line (HT29) as standard. For biomarker development of nutritional effects, PBMC and HT29 cells were treated with potentially chemopreventive substances (chrysin and butyrate), and gene expression was determined. Key results were that relevant stress response genes, such as glutathione S-transferase T2 (GSTT2) and GSTM2, were modulated by butyrate in PBMC as in HT29 cells, but the blood cells were less sensitive and responded with high individual differences. We conclude that these cells may serve as a surrogate tissue in dietary investigations and the identified differentially expressed genes have the potential to become marker genes for population studies on biological effects.

New insight into the influence of carob extract and gallic acid on hemin induced modulation of HT29 cell growth parameters.

Red meat intake is associated with an increased risk of developing cancer. This is possibly related to the heme content of red meat. Plant derived polyphenols might protect from cancer development via their antioxidant activities. In this study, the impact of an aqueous extract of carob (CE) on hemin-modulated proliferation was investigated. CE, gallic acid (GA) and a known iron chelator (deferoxamine: DFO) significantly reduced the number of human colon cancer HT29 cells. CE and GA were more effective under serum-free conditions than in normal cell culture medium. These effects were abolished by addition of 1 microM hemin at low concentrations of CE and GA. At higher concentrations of CE and GA, both substances reduced cell number despite hemin supplementation. Effects of CE, GA and DFO on cell number could not be linked to iron chelation even though CE and DFO were capable of chelating iron. Furthermore, the effects of high CE concentration point to antioxidative effects other than iron chelation. However, a connection to a reduction of colorectal cancer risk due to consumption of meat with high heme content by CE could not be drawn, since the effective concentrations are beyond the physiologically relevant concentrations.

Does an extract of carob (Ceratonia siliqua L.) have chemopreventive potential related to oxidative stress and drug metabolism in human colon cells?

Phenolic ingredients of an aqueous carob extract are well characterized and consist of mainly gallic acid (GA). In order to assess possible chemopreventive mechanisms of carob, which can be used as a cacao substitute, effects on expression of genes related to stress response and drug metabolism were studied using human colon cell lines of different transformation state (LT97 and HT29). Stress-related genes, namely catalase (CAT) and superoxide dismutase (SOD2), were induced by carob extract and GA in LT97 adenoma, but not in HT29 carcinoma cells. Although corresponding protein products and enzyme activities were not elevated, pretreatment with carob extract and GA for 24 h reduced DNA damage in cells challenged with hydrogen peroxide (H(2)O(2)). In conclusion, carob extract and its major phenolic ingredient GA modulate gene expression and protect colon adenoma cells from genotoxic impact of H(2)O(2). Upregulation of stress-response genes could not be related to functional consequences.

Impact of apple polyphenols on GSTT2 gene expression, subsequent protection of DNA and modulation of proliferation using LT97 human colon adenoma cells.

Apple extract (AE) enhances expression of glutathione S-transferases (e.g., GSTT2) in human colon cells (LT97). Therefore, aim of the present study was to identify functional consequences of GSTT2 induction by AE and to determine the relation of AE effects to isolated compounds. Polyphenol composition of AE was analyzed. LT97 cells were treated with AE or synthetic polyphenol mixture (SPM) under conditions that induced GSTT2, and challenged with GSTT2-2 substrate cumene hydroperoxide (CumOOH) to determine DNA damage using comet assay. Modulation of GSTT2 expression (real-time PCR) was reassessed, and the influence on cell proliferation and pro-oxidative potential of AE and SPM were assessed to understand additional mechanisms. Induction of GSTT2 by AE was accompanied by protection of LT97 cells from CumOOH-induced genotoxicity. Although SPM was unable to reflect AE-specific bioactivity related to GSTT2 modulation and anti-genotoxicity, inhibition of LT97 cell proliferation by SPM was comparable. Storage of AE caused changes in phenolic composition along with loss of activity regarding GSTT2 induction and amplified growth inhibition. At the applied concentrations, no H(2)O(2) formation was detectable with any of the substances. AE can protect against oxidatively induced DNA damage. Nevertheless, chemopreventive effects of AE strongly depend on the specific composition, which is modified by storage.