Chemopreventive effects of α-tocopherol and its long-chain metabolites α-13'-hydroxy- and α-13'-carboxychromanol in LT97 colon adenoma cells.

Anticancer effects of vitamin E (tocopherols) have been studied extensively. While in vitro and animal studies showed promising results regarding anticancer effects of tocopherols, human intervention studies failed to reproduce these results. In vivo, α-tocopherol (α-TOH) is metabolized to the long-chain metabolites (LCM) 13'-hydroxychromanol (α-13'-OH) and 13'-carboxychromanol (α-13'-COOH), which likely reach the large intestine. The LCM showed antiproliferative effects in different colon cancer cell lines, but the exact mechanism of action remains unclear. To further clarify the chemopreventive action of the LCM, premalignant LT97 colon adenoma cells were treated with α-TOH, α-13'-OH and α-13'-COOH to study their impact on growth, apoptosis, antigenotoxicity, and ROS-scavenging capacity as well as expression of selected genes involved in detoxification and the cell cycle. Growth inhibitory potential was observed for α-13'-OH (IC50: 37.4 µM) and α-13'-COOH (IC50: 5.8 µM) but not for α-TOH in the tested concentrations. Levels of caspase-3 activity and expression of genes regulating the cell cycle and detoxification remained unchanged. However, α-TOH, α-13'-OH and α-13'-COOH exhibited antigenotoxic and partly ROS-scavenging capacity. The results indicate that the LCM exert chemopreventive effects via ROS-scavenging capacity, the protection against DNA damage and the induction of cell death via caspase-independent mechanisms in premalignant colon cells.

Measurement of trace elements in murine liver tissue samples: Comparison between ICP-MS/MS and TXRF.

BACKGROUND: Trace elements exhibit essential functions in many physiological processes. Thus, for research focusing on trace element homeostasis and metabolism analytical methods allowing for multi-element analyses are fundamental. Small sample amounts may be a big challenge in trace element analyses especially if also other end points want to be addressed in the same sample. Therefore, the aim of the present study was to examine trace elements (iron, copper, zinc, and selenium) in murine liver tissue prepared by a RIPA buffer-based lyses method. METHODS AND RESULTS: After centrifugation, lysates and pellets were obtained and trace elements were analyzed with TXRF in liver lysates. The results were compared to that obtained by a standard microwave-assisted acidic digestion with subsequent ICP-MS/MS analysis of the same liver tissue, liver lysates, and remaining pellets. In addition, trace element concentrations, determined in murine serum with both methods, were compared. For serum samples, both TXRF and ICP-MS/MS provide similar and highly correlating results. Furthermore, in liver lysate samples prepared with RIPA buffer, comparable trace element concentrations were measured by TXRF as with the standard digestion technique and ICP-MS/MS. Only marginal amounts of trace elements were detected in the pellets. CONCLUSION: Taken together, the results obtained by the present study indicate that the RIPA buffer-based method is suitable for sample preparation for trace element analyses via TXRF, at least for the here investigated murine liver samples.

Effect of a regular consumption of traditional and roasted oat and barley flakes on blood lipids and glucose metabolism-A randomized crossover trial.

Background: Regular consumption of the soluble dietary fiber ß-glucan is associated with decreased total cholesterol (TC), low-density lipoprotein (LDL) cholesterol and blood glucose. Barley and oat flakes as natural sources of ß-glucan were roasted to improve sensory quality. The aim of this study was to investigate whether roasting of barley and oat flakes changes the physiological impact of the ß-glucan-rich flakes on glucose and lipid metabolism. Method: A five-armed randomized crossover trial design was used. The intervention study was conducted from May 2018 to May 2019 and included 32 healthy subjects with moderately increased LDL cholesterol (≥2.5 mmol/L). During the 3-week intervention periods, 80 g of roasted or traditional barley or oat flakes, or four slices of white toast bread per day were consumed for breakfast. At the start and the end of each intervention, fasting and postprandial blood was taken. The intervention periods were separated by 3-week wash-out periods. Results: During the interventions with the cereal flakes, TC and LDL cholesterol concentrations were significantly reduced compared to baseline values by mean differences of 0.27-0.33 mmol/L and 0.21-0.30 mmol/L, respectively (p < 0.05), while high-density lipoprotein (HDL) cholesterol was only reduced after the intervention with barley flakes (p < 0.05). After the intervention period with toast, TC and HDL cholesterol increased (p < 0.05). The fasting levels of triglycerides, fasting blood glucose and insulin did not change in any group. The effects of traditional and roasted varieties on blood lipids did not differ between the groups. Conclusion: The regular consumption of traditional or roasted barley and oat flakes contributes to the management of cardiovascular diseases by improving TC and LDL cholesterol. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT03648112, identifier NCT03648112.

Potential Role of ROS in Butyrate- and Dietary Fiber-Mediated Growth Inhibition and Modulation of Cell Cycle-, Apoptosis- and Antioxidant-Relevant Proteins in LT97 Colon Adenoma and HT29 Colon Carcinoma Cells.

The aim of the present study was to examine whether reactive oxygen species (ROS) contribute to chemopreventive effects of fermentation supernatants (FS) of different dietary fibers (Synergy1®, oat-, barley-, yeast ß-glucan, Curdlan) and butyrate as a fermentation metabolite. LT97 and HT29 cells were treated with butyrate and FS alone or with N-acetyl-cysteine (NAC) and their impact on ROS formation, cell growth, and protein expression (Cyclin D2, p21, PARP, Bid, GPx2) was investigated. Butyrate and FS significantly decreased cell growth. ROS levels were significantly increased, particularly in LT97 cells, while co-treatment with NAC decreased ROS formation and growth inhibitory effects in both cell lines. After treatment with butyrate and FS, Cyclin D2 expression was reduced in LT97 cells and p21 expression was increased in both cell lines. Levels of full-length PARP and Bid were decreased, while levels of cleaved PARP were enhanced. GPx2 expression was significantly reduced by fiber FS in HT29 cells. A notable effect of NAC on butyrate- and FS-modulated protein expression was observed exclusively for PARP and Bid in HT29 cells. From the present results, a contribution of ROS to growth inhibitory and apoptotic effects of butyrate and FS on LT97 and HT29 cells cannot be excluded.

Use of the ß-Glucan-Producing Lactic Acid Bacteria Strains Levilactobacillus brevis and Pediococcus claussenii for Sourdough Fermentation-Chemical Characterization and Chemopreventive Potential of In Situ-Enriched Wheat and Rye Sourdoughs and Breads.

The aim of the present study was to examine ß-glucan production and the potential prebiotic and chemopreventive effects of wheat and rye sourdoughs and breads generated with wild-type and non-ß-glucan-forming isogenic mutant strains of Levilactobacillus brevis and Pediococcus claussenii. Sourdough and bread samples were subjected to in vitro digestion and fermentation. Fermentation supernatants (FS) and pellets (FP) were analyzed (pH values, short-chain fatty acids (SCFA), ammonia, bacterial taxa) and the effects of FS on LT97 colon adenoma cell growth, viability, caspase-2 and -3 activity, genotoxic and antigenotoxic effects and on gene and protein expression of p21, cyclin D2, catalase and superoxide dismutase 2 (SOD2) were examined. Concentrations of SCFA were increased and concentrations of ammonia were partly reduced in the FS. The relative abundance of Bifidobacteriaceae was increased in all FPs. Treatment with FS reduced the growth and viability of LT97 cells and significantly increased caspase-2 and -3 activities without exhibiting genotoxic or antigenotoxic effects. The p21 mRNA and protein levels were increased while that of cyclin D2 was reduced. Catalase and SOD2 mRNA and protein expression were marginally induced. The presented results indicate a comparable chemopreventive potential of wheat and rye sourdoughs and breads without an additional effect of the formed ß-glucan.

Olive Oil Extracts and Oleic Acid Attenuate the LPS-Induced Inflammatory Response in Murine RAW264.7 Macrophages but Induce the Release of Prostaglandin E2.

Olive oil contains high amounts of oleic acid (OA). Although OA has been described to inhibit inflammatory processes, the effects of olive oil on cellular mechanisms remain poorly understood. Therefore, we compared the effects of major fatty acids (FA) from olive oil with those of olive oil extracts (OOE) on inflammatory mediators and alterations in the cellular phospholipid composition in murine macrophages. Upon treatment with different OOE, FA compositions of lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophages were analyzed using gas chromatography. Olive oil extracts and OA significantly reduced the LPS-induced expression of inducible nitric oxide synthase (iNos), cyclooxygenase (Cox2), and interleukin-6 mRNA. In addition, a significant decrease in Cox2 and iNos protein expression was observed. The formation of nitric oxide was significantly reduced, while the formation of prostaglandin (PG) E2 from arachidonic acid significantly increased after treatment with OOE or OA. The latter was associated with a shift in the phospholipid FA composition from arachidonic acid to OA, resulting in an elevated availability of arachidonic acid. Together, OOE and OA mediate anti-inflammatory effects in vitro but increase the release of arachidonic acid and hereinafter PGE2, likely due to elongation of OA and competitive incorporation of fatty acids into membrane phospholipids.

Thermal Processing has no Impact on Chemopreventive Effects of Oat and Barley Kernels in LT97 Colon Adenoma Cells.

The unique dietary fiber composition with high contents of ß-glucan contributes to the health-promoting properties of oat and barley and may mediate a reduction of colon cancer risk. In the present study, chemopreventive effects of oat and barley (beta®barley) kernels were investigated. In order to address the impact of thermal processing on these effects, kernels were roasted (150-180 °C, approx. 20 min), digested and fermented using an In Vitro human digestion model. Concentrations of short-chain fatty acids (SCFA) and ammonia were determined in fermentation supernatants (FS). Growth inhibition, apoptosis, DNA integrity and gene expression of catalase were analyzed in LT97 colon adenoma cells. Concentrations of SCFA, particularly butyrate, were higher in oat/barley FS (2.2-fold, on average), while ammonia levels were significantly lower (0.7-fold, on average) than in the fermentation control. Treatment of LT97 cells with FS of oat/barley kernels led to a significant time- and dose-dependent growth reduction, a significant increase in caspase-3 activity and enhanced levels of catalase mRNA, without exhibiting genotoxic effects. In general, the results indicate a chemopreventive potential of In Vitro fermented oat and waxy winter barley mediated mainly by growth inhibitory and apoptotic effects, which are preserved after thermal processing.

Chemopreventive effects of raw and roasted oat flakes after in vitro fermentation with human faecal microbiota.

The aim of the present study was to analyse chemopreventive effects of oat flakes under consideration of processing. Thin and thick flakes were roasted and subjected to an in vitro digestion and fermentation. Fermentation supernatants (FS) were characterised and chemopreventive effects were analysed in LT97 colon adenoma cells. Compared to the fermentation control, pH values were decreased (from pH 6.3 to pH 5.0) and concentrations of SCFA, in particular butyrate, were increased in oat FS (2.6-fold, on average). Ammonia levels were not altered. Oat FS significantly decreased cell growth time- and dose-dependently. Caspase 3 activity was significantly increased (9.7-fold, on average). Oat FS slightly increased the mRNA expression of CAT (2.0-fold), SOD2 (1.7-fold) and GSTP1 (2.8-fold), on average, while GPX1 mRNA (0.3-fold) was decreased. The results indicate a chemopreventive potential of in vitro digested oat flakes regarding colon cancer development mediated mostly by growth inhibition and apoptosis, unaffected by roasting.

In Vitro Digested Nut Oils Attenuate the Lipopolysaccharide-Induced Inflammatory Response in Macrophages.

Nut consumption is known for its health benefits, in particular in inflammatory diseases. A possible mechanism for these effects could be their beneficial fatty acid composition. Nuts mainly contain mono- and polyunsaturated fatty acids, which have anti-inflammatory properties. However, studies investigating the effects of nut extracts on inflammatory processes on the molecular level are rare. We therefore prepared oily nut extracts after in vitro digestion and saponification of the fat-soluble constituents. Besides chromatographic analysis, cell culture experiments were performed using murine macrophages (RAW264.7) to study the capacity of different nut extracts (hazelnut, almond, walnut, macadamia, and pistachio) to modulate inflammatory processes. Oleic acid was the main fatty acid in hazelnut, almond, macadamia, and pistachio extracts. Both oily nut extracts and pure oleic acid significantly reduced the LPS-induced expression of iNos, Cox2, Tnfα, Il1ß, and Il6 mRNAs. iNos protein expression was down-regulated followed by reduced nitric oxide formation. Thus, nut extracts at concentrations achievable in the digestive tract inhibit the expression and formation of inflammatory mediators in macrophages. Hence, a beneficial contribution of nut consumption to inflammatory diseases can be assumed. We are convinced that these results provide new insights on the molecular mechanisms involved in the health-beneficial effects of nuts.

Chemopreventive Potential of In Vitro Fermented Raw and Roasted Hazelnuts in LT97 Colon Adenoma Cells.

BACKGROUND/AIM: Due to their unique composition of health-promoting compounds, the consumption of hazelnuts may contribute to the prevention of colon cancer. MATERIALS AND METHODS: Since hazelnuts are often consumed roasted, the impact of different roasting conditions (RC1=140.6°C/25 min, RC2=155.1°C/20 min and RC3=180.4°C/21 min) on chemopreventive effects of in vitro fermented hazelnuts was analyzed in LT97 colon adenoma cells. RESULTS: FS (2.5%) of raw and roasted hazelnuts reduced H2O2-induced DNA damage while 5% FS significantly induced gene expression of SOD2 (3.0-fold) and GSTP1 (2.1-fold). GPx1 mRNA levels were significantly decreased (0.6-fold) by FS (2.5%). The growth of LT97 cells was significantly reduced by hazelnut FS in a time- and dose-dependent manner. Hazelnut FS (5%) increased the numbers of early apoptotic cells (9.6% on average) and caspase-3 activities (6.4-fold on average). CONCLUSION: These results indicate a chemopreventive potential of in vitro fermented hazelnuts which is largely unaffected by the roasting process.

Influence of roasting on the chemopreventive potential of in vitro fermented almonds in LT97 colon adenoma cells.

The consumption of almonds may contribute to the prevention of colon cancer due to their unique composition of health promoting compounds. Since almonds are often consumed roasted, the impact of different roasting (R) conditions (R1 = 139.2 °C/25 min, R2 = 161.5 °C/20 min and R3 = 170.8 °C/15 min) on chemopreventive effects of in vitro-fermented almonds was analysed in LT97 colon adenoma cells. Fermentation supernatants (FS) of raw and roasted almonds had no genotoxic effects. FS obtained from raw or mildly roasted almonds (R1) significantly increased mRNA levels of CAT (4.6-fold), SOD2 (5.6-fold) and GSTP1 (3.9-fold) but not of GPx1. FS of almonds significantly reduced the growth of LT97 cells in a time- and dose-dependent manner. Treatment with 5% almonds FS increased the number of early apoptotic cells (17.4%, on average) and caspase-3 activity (4.9-fold, on average). The results indicate a chemopreventive potential of in vitro-fermented almonds which is largely independent of the roasting process.

Chemopreventive Potential of Raw and Roasted Pistachios Regarding Colon Carcinogenesis.

Pistachios are rich in health-promoting bioactive compounds such as B vitamins, γ-tocopherol, polyphenols and dietary fiber, which could contribute to the reduction of colon cancer risk in terms of chemoprevention (Fischer, S.; Glei, M. Health-Potential of Nuts. Ernaehrungs Umsch. Int. 2013, 60, 206-215.). Since pistachios are often consumed roasted, the present study aims at investigating the influence of different roasting conditions (RC) on potential chemopreventive effects of pistachios in colon adenoma cells such as growth and apoptosis, genotoxic- and anti-genotoxic effects and modulation of gene expression of detoxifying enzymes (CAT, SOD2, GPx1, and GSTP1). Fermentation supernatants (FS) were obtained from raw and roasted (RC1 = 141 °C/25 min, RC2 = 160 °C/15 min and RC3 = 185 °C/21 min) pistachios after in vitro fermentation. FS of pistachios significantly reduced LT97 cell growth in a time- and dose-dependent manner. Compared to the blank control, pistachio FS (2.5%) led to a significant average reduction of H2O2-induced DNA damage (1.5-fold). Levels of CAT mRNA were significantly increased (1.3-fold, on average for 5% FS). Pistachio FS (5%) significantly increased the number of early apoptotic cells (up to 2.1-fold) and levels of caspase-3 activities (up to 6.9-fold). The present results confirm a chemopreventive potential of pistachios, which is mediated by growth inhibition, induction of apoptosis and anti-genotoxic effects, as well as induction of CAT. These effects remain mostly unaffected by roasting.

Chemopreventive potential of in vitro fermented nuts in LT97 colon adenoma and primary epithelial colon cells.

Due to their beneficial nutritional profile the consumption of nuts contributes to a healthy diet and might reduce colon cancer risk. To get closer insights into potential mechanisms, the chemopreventive potential of different in vitro fermented nut varieties regarding the modulation of genes involved in detoxification (CAT, SOD2, GSTP1, GPx1) and cell cycle (p21, cyclin D2) as well as proliferation and apoptosis was examined in LT97 colon adenoma and primary epithelial colon cells. Fermentation supernatants (FS) of nuts significantly induced mRNA expression of CAT (up to 4.0-fold), SOD2 (up to 2.5-fold), and GSTP1 (up to 2.3-fold), while GPx1 expression was significantly reduced by all nut FS (0.8 fold on average). Levels of p21 mRNA were significantly enhanced (up to 2.6-fold), whereas all nut FS significantly decreased cyclin D2 expression (0.4-fold on average). In primary epithelial cells, expression of CAT (up to 3.5-fold), GSTP1 (up to 3.0-fold), and GPx1 (up to 3.9-fold) was increased, whereas p21 and cyclin D2 levels were not influenced. Nut FS significantly inhibited growth of LT97 cells and increased levels of early apoptotic cells (8.4% on average) and caspase 3 activity (4.6-fold on average), whereas caspase 3 activity was not modulated in primary colon cells. The differential modulation of genes involved in detoxification and cell cycle together with an inhibition of proliferation and induction of apoptosis in adenoma cells might contribute to chemopreventive effects of nuts regarding colon cancer.

B-vitamins, carotenoids and α-/γ-tocopherol in raw and roasted nuts.

The concentrations of B-vitamins, carotenoids and tocopherols in nuts may differ between species and might be influenced by roasting. Thiamine, riboflavin, pyridoxine, lutein, zeaxanthin, ß-carotene and α-/γ-tocopherol were determined in different varieties of raw and roasted nuts using HPLC (fluorescence/UV-vis detection). The analysis revealed remarkable concentrations of thiamine and pyridoxine in pistachios (57%, 79% of the recommended daily intake/100g (RDI), respectively) and riboflavin in almonds (119% of the RDI). Pistachios were rich in lutein/zeaxanthin and contained highest ß-carotene levels among nuts. Almonds and hazelnuts were abundant in α-tocopherol (>4-fold the RDI for tocopherol equivalents) while pistachios and walnuts were rich in γ-tocopherol. Roasting had a diminishing effect on thiamine, carotenoids and tocopherols especially in almonds and walnuts. Nuts could make a valuable contribution to a healthy diet in regard to B-vitamins, lutein/zeaxanthin and tocopherols. A reduction in micronutrient content by roasting is reliant on the nut variety and specific micronutrient.

Foetal cord blood contains higher portions of n-3 and n-6 long-chain PUFA but lower portions of trans C18:1 isomers than maternal blood.

BACKGROUND/OBJECTIVE: An adequate supply of long-chain polyunsaturated fatty acids (LC PUFA) promotes foetal health and development, whereas generally, trans fatty acids (tFA) are considered to negatively interfere with LC PUFA metabolism. Nevertheless, to date, limited data concerning separate trans C18:1, such as t9 and t11, are available for maternal and foetal blood. Therefore, in this study the portions of individual trans C18:1, LC n-6, and n-3 PUFA in lipids of maternal and foetal plasma and erythrocyte membranes of German mother and child pairs (n=40) were analysed. RESULTS: Portions of linoleic acid and α-linolenic acid as LC precursors were lower (~0.4-fold); whereas the metabolites arachidonic acid (AA, n-6) and docosahexaenoic acid (DHA, n-3) were significantly higher (~2-fold) in foetal than in maternal plasma and erythrocytes. The main tFA in maternal and foetal blood were elaidic acid (C18:1t9; t9) and vaccenic acid (C18:1t11; t11). Portions of t9, t10, t11, and t12 in foetal blood lipids were lower (~0.5-fold) compared with maternal blood. In foetal lipids, t9 was higher than t11. The t9 correlated negatively with eicosapentaenoic acid (n-3) and AA in maternal and foetal lipids; whereas t11 correlated negatively only with foetal total LC n-6 (plasma and erythrocytes) and n-3 PUFA (erythrocytes). No correlation between maternal tFA and foetal PUFA was observed. CONCLUSIONS: 'Biomagnification' of LC n-6 and n-3 PUFA AA and DHA in foetal blood was confirmed, whereas single trans isomers were lower compared with maternal blood. Nevertheless, tFA intake, especially from industrial sources, should be as low as possible.

Loss of FADS2 function severely impairs the use of HeLa cells as an in vitro model for host response studies involving fatty acid effects.

SCOPE: Established epithelial cell lines equipped with pattern recognition receptors such as the Toll-like receptor (TLR)-2 are common tools for immune response studies on invading pathogens, e.g. the obligate intracellular species of Chlamydia. Moreover, such models are widely used to elucidate fatty acid-mediated immune effects. In several transformed cell lines, however, unusual loss of metabolic functions was described. The cell lines A549 and HeLa are poorly characterized in this respect. Therefore, we comparatively assessed the metabolic capacity of A549 and HeLa prior to proposed application as in vitro model for fatty acid effects on chlamydial infection. METHODOLOGY/PRINCIPAL FINDINGS: We incubated both cell lines either with substrates (C18:2n-6 or C18:3n-3) or products (C18:3n-6, C18:4n-3) of fatty acid desaturase-2 (FADS2), and analysed the fatty acid profiles after 24 h and 72 h by gas chromatography. Based on these data, we suspected that the complete discontinuation of normal biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) in HeLa was due to loss of FADS2 function. Consequently, prostaglandin E2 (PGE2) formation was less inducible by TLR2 stimulation in HeLa, likely as a result of not only insufficient supply of precursors but also weak cyclooxygenase-2 (COX-2) response. In accordance, Chlamydia infection rates were consistently lower in HeLa than in A549. Sequence analysis revealed no alteration within the FADS2 gene in HeLa. The FADS2 expression level, however, was significantly lower and, in contrast to A549, not regulated by C18:2n-6. A549 exhibited regular fatty acid metabolism and enzyme functionality. CONCLUSIONS/SIGNIFICANCE: Our data show that HeLa cells considerably differ from A549 at several stages of fatty acid metabolism. The poor metabolic potential of HeLa, mainly concerning FADS2 upstream of COX-2 function, calls into question whether these cells represent a good model to unveil fatty acid or downstream eicosanoid effects in the course of intracellular bacterial infection.

Analysis of DNA damage and repair by comet fluorescence in situ hybridization (Comet-FISH).

A useful tool in the detection of overall and region-specific DNA damage is the Comet-FISH technique. This method combines two well-established methods, the Comet assay (single cell gel electrophoresis), which makes it possible to detect and quantify DNA damage at the single cell level, and FISH (fluorescence in situ hybridization), a technique that allows the specific detection of selected DNA sequences. The influence of specific substances such as water pollutants or food ingredients on individual cells can be measured with the alkaline version of the Comet assay, which involves the embedding of cells in agarose on microscopic slides, lysis of cells, and separation of DNA via electrophoresis. In damaged cells a "comet tail" is formed by fractured DNA migrating from the nucleus (head of the comet) in the electric field.The damaged DNA (DNA strand breaks) correlates with the percentage of DNA in the tail. In combination with the FISH method, DNA damage or repair capacity in single cells can be measured using labelled probes, which hybridize to specific DNA sequences of interest. This protocol exemplarily provides a description of the Comet-FISH technique for the detection of DNA damage using hydrogen peroxide as a genotoxic model substance.

Detection of DNA damage by comet fluorescence in situ hybridization.

Comet fluorescence in situ hybridization (Comet-FISH) is a useful method to detect overall and region-specific DNA damage in individual cells. Two well-established methods are combined, the Comet assay (single cell gel electrophoresis) and fluorescence in situ hybridization (FISH). The Comet assay is the method of choice for the detection of DNA damage. With the alkaline version the influence of specific substances such as water pollutants or ingredients of food on individual cells can be easily measured. The Comet assay involves the embedding of cells in agarose on microscopic slides, lysis of cells, and separation of DNA via electrophoresis. Damaged DNA migrates from the nucleus (head of the comet) forming a tail. The percentage of DNA in the tail correlates with the degree of DNA strand breaks (DNA damage). The combination of FISH with the Comet assay uses labeled probes which hybridize specifically to selected DNA sequences. This allows the detection of specific DNA damage or repair capacity in single cells. Here we present exemplarily the Comet-FISH method by detection of DNA damage using hydrogen peroxide as a genotoxic model substrate.

Vaccenic acid-mediated reduction in cytokine production is independent of c9,t11-CLA in human peripheral blood mononuclear cells.

The ruminant trans fatty acid vaccenic acid (tVA) favorably alters markers of inflammation. However, it is not yet clear whether these effects are attributed to its endogenous partial conversion to c9,t11-CLA, which is known to possess anti-inflammatory properties. We compared the cytokine reducing potential of tVA to c9,t11-CLA in human T-helper (Th) cells as a main source of cytokine production during inflammation. Secondly, we assessed whether a bioconversion of tVA to c9,t11-CLA via stearoyl-CoA desaturase (SCD) encoded activity takes place in peripheral blood mononuclear cells (PBMC) in order to relate the outcomes of intracellular cytokine measurement to the degree of conversion. TVA reduced the percentage of both IL-2 and TNF-α expressing Th cells significantly, but to a lesser extent compared to c9,t11-CLA, as determined by flow cytometry after alloreactive stimulation of PBMC. Pre-treatment with the selective PPARγ antagonist T0070907 largely re-established the IL-2 and TNF-α positive Th cell population in both tVA and c9,t11-CLA treated cultures. Interestingly, while the portion of tVA dose-dependently increased within the cellular lipid fraction, the initially marginal amount of c9,t11-CLA remained unaltered. However, SCD mRNA although abundantly expressed in PBMC was not regulated by tVA. Conclusively, these results suggest that the cytokine reducing effect of tVA in human T cells is independent of c9,t11-CLA, since no bioconversion occurred. Moreover, the data provide evidence that tVA mechanistically acts in a manner similar to c9,t11-CLA.

Chemopreventive effects of in vitro digested and fermented bread in human colon cells.

PURPOSE: Bread as a staple food product represents an important source for dietary fibre consumption. Effects of wheat bread, wholemeal wheat bread and wholemeal rye bread on mechanisms which could have impact on chemoprevention were analysed in colon cells after in vitro fermentation. METHODS: Effects of fermented bread samples on gene expression, glutathione S-transferase activity and glutathione content, differentiation, growth and apoptosis were investigated using the human colon adenoma cell line LT97. Additionally, apoptosis was studied in normal and tumour colon tissue by determination of caspase activities. RESULTS: The expression of 76 genes (biotransformation, differentiation, apoptosis) was significantly upregulated (1.5-fold) in LT97 cells. The fermented bread samples were able to significantly increase glutathione S-transferase activity (1.8-fold) and glutathione content (1.4-fold) of the cells. Alkaline phosphatase activity as a marker of differentiation was also significantly enhanced (1.7-fold). The fermented bread samples significantly inhibited LT97 cell growth and increased the level of apoptotic cells (1.8-fold). Only marginal effects on apoptosis in tumour compared to normal tissue were observed. CONCLUSIONS: This is the first study which presents chemopreventive effects of different breads after in vitro fermentation. In spite of differences in composition, the results were comparable between the bread types. Nevertheless, they indicate a potential involvement of this staple food product regarding the prevention of colon cancer.