Elizabeth K. Lund

Antioxidants, reactive oxygen and nitrogen species, gene induction and mitochondrial function.

Redox-sensitive cell signalling Thiol groups and the regulation of gene expression Redox-sensitive signal transduction pathways Protein kinases Protein phosphatases Lipids and phospholipases Antioxidant (electrophile) response element Intracellular calcium signalling Transcription factors NF-?B AP-1 p53 Cellular responses to oxidative stress Cellular responses to change in redox state Proliferation Cell death Immune cell function Reactive oxygen and nitrogen species – good or bad? Reactive oxygen species and cell death Reactive oxygen species and inflammation Are specific reactive oxygen species and antioxidants involved in modulating cellular responses? Specific effects of dietary antioxidants in cell regulation Carotenoids Vitamin E Flavonoids Inducers of phase II enzymes Disease states affected Oxidants, antioxidants and mitochondria Introduction Mitochondrial generation of reactive oxygen and nitrogen species Mitochondria and apoptosis Mitochondria and antioxidant defences Key role of mitochondrial GSH in the defence against oxidative damage Mitochondrial oxidative damage Direct oxidative damage to the mitochondrial electron transport chain Nitric oxide and damage to mitochondria Effects of nutrients on mitochondria Caloric restriction and antioxidants Lipids Antioxidants Techniques and approaches Mitochondrial techniques cDNA microarray approaches Proteomics approaches Transgenic mice as tools in antioxidant research Gene knockout and over expression Transgenic reporter mice Conclusions Future research needs

Effect of oat gum on the physical properties of the gastrointestinal contents and on the uptake of D-galactose and cholesterol by rat small intestine in vitro

Recent reports indicate that oats have a relatively low glycaemic effect in comparison with other carbohydrate food, and that their consumption leads to a reduction in plasma-cholesterol levels in man. These properties may be due to a soluble non-starch polysaccharide in oats. The present study was undertaken to explore the physiological properties of this material. Three groups of male Wistar rats were meal-fed on a control diet free of soluble dietary fibre for 10 d before being given a 10 g meal of either the control diet, a diet containing oat gum (beta-glucan), or finely ground rolled oats. The contents of the stomach, small intestine and caecum were later recovered and the weight, water content and viscosity were measured. The small intestinal contents from oat-gum-fed or oat-fed rats had a higher wet: dry weight ratio than that of the controls, and a higher viscosity. In in vitro studies the rate of uptake of D-galactose by jejunal rings was reduced in the presence of oat gum. The estimated Michaelis-Menten constant for the carrier-mediated component in the presence of oat gum was higher than that for controls, but the maximum transport rates were similar. Cholesterol uptake by everted jejunal sacs was progressively inhibited by increasing concentrations of oat gum in the mucosal medium. It is concluded that increased viscosity of the contents of the small intestine may contribute to the low glycaemic index and hypocholesterolaemic effects of oats in man. Oats appear to be amongst the few palatable sources of viscous dietary fibre in the conventional Western diet.

Ulcerative colitis and irritable bowel patients exhibit distinct abnormalities of the gut microbiota.

BackgroundPrevious studies suggest a link between gut microbiota and the development of ulcerative colitis (UC) and irritable bowel syndrome (IBS). Our aim was to investigate any quantitative differences in faecal bacterial compositions in UC and IBS patients compared to healthy controls, and to identify individual bacterial species that contribute to these differences.MethodsFaecal microbiota of 13 UC patients, 11 IBS patients and 22 healthy volunteers were analysed by PCR-Denaturing Gradient Gel Electrophoresis (DGGE) using universal and Bacteroides specific primers. The data obtained were normalized using in-house developed statistical method and interrogated by multivariate approaches. The differentiated bands were excised and identified by sequencing the V3 region of the 16S rRNA genes.ResultsBand profiles revealed that number of predominant faecal bacteria were significantly different between UC, IBS and control group (p < 10-4). By assessing the mean band numbers in UC (37 ± 5) and IBS (39 ± 6), compared to the controls (45 ± 3), a significant decrease in bacterial species is suggested (p = 0.01). There were no significant differences between IBS and UC. Biodiversity of the bacterial species was significantly lower in UC (μ = 2.94, σ = 0.29) and IBS patients (μ = 2.90, σ = 0.38) than controls (μ = 3.25, σ = 0.16; p = 0.01). Moreover, similarity indices revealed greater biological variability of predominant bacteria in UC and IBS compared to the controls (median Dice coefficients 76.1% (IQR 70.9 - 83.1), 73.8% (IQR 67.0 - 77.5) and 82.9% (IQR 79.1 - 86.7) respectively). DNA sequencing of discriminating bands suggest that the presence of Bacteroides vulgatus, B. ovatus, B. uniformis, and Parabacteroides sp. in healthy volunteers distinguishes them from IBS and UC patients. DGGE profiles of Bacteroides species revealed a decrease of Bacteroides community in UC relative to IBS and controls.ConclusionMolecular profiling of faecal bacteria revealed abnormalities of intestinal microbiota in UC and IBS patients, while different patterns of Bacteroides species loss in particular, were associated with UC and IBS.

Review article: nutrition, obesity and colorectal cancer

Background The age‐adjusted incidence of colorectal cancer is higher in prosperous industrialized countries than elsewhere. Dietary factors may account for 75% of sporadic colorectal cancer in the west, but the mechanisms remain obscure.

The influence of dietary lipids on the composition and membrane fluidity of rat hepatocyte plasma membrane

Weanling male Wistar rats were fed for five weeks on standard rat chow (23 g fat/kg diet) or one of four synthetic diets with butterfat, coconut oil, corn oil, or fish oil as the main lipid source (100 g fat/kg diet). In all diets, 10% of the fat was provided as corn oil to prevent essential fatty acid deficiency. Significant differences were observed in the saturated, monounsaturated, and polyunsaturated fatty acid composition, and in the ratio of cholesterol to phospholipid, in the hepatocyte membranes. The fluidity of hepatocyte plasma membranes was assessed using the fluorescence recovery after photobleaching technique and steady-state fluorescence anisotropy of diphenylhexatriene. No significant differences were found in the fluidity of plasma membranes between animals on the different fat diets, despite diet-induced changes in their fatty acid composition. However, the proportion of lipid free to diffuse in the plasma membrane varied with diet, being significantly greater (P<0.05) in animals fed chow (63.7%), coconut oil (61.5%), and butterfat (57.6%) diets than in those fed the corn oil (47.3%) diet. Animals fed fish oil showed an intermediate (50.0%) proportion of lipid free to diffuse. The data support the hypothesis that dietary lipids can change both the chemical composition and lateral organization (lipid domain structure) of rat hepatocyte plasma membranes.

Health benefits of seafood; is it just the fatty acids?

There is a considerable body of literature suggesting a wide range of health benefits associated with diets high in seafood. However, the demand for seafood across the world now exceeds that available from capture fisheries. This has created a rapidly increasing market for aquaculture products, the nutrient composition of which is dependent on feed composition. The use of fishmeal in this food chain does little to counteract the environmental impact of fisheries and so the on-going development of alternative sources is to be welcomed. Nevertheless, an in-depth understanding as to which nutrients in seafood provide benefit is required to permit the production of foods of maximal health benefit to humans. This paper reviews our current knowledge of the beneficial nutrient composition of seafood, in particular omega-3 fatty acids, selenium, taurine, vitamins D and B12, in the context of the development of environmentally sustainable aquaculture.

L‐proline transport by brush border membrane vesicles prepared from human placenta.

1. Brush border microvillous plasma membranes were prepared from syncytiotrophoblast of human term placenta by a method of differential centrifugation. 2. Such plasma membranes form closed, osmotically active, right‐side‐out vesicles into which L‐proline (188 microM) is shown to be transported in a time‐dependent manner. There is no detectable metabolism of L‐proline within the vesicles during 30 min of incubation. 3. Transient accumulation of L‐proline to levels of up to three times its equilibrium value occurs in the presence of an inward gradient of sodium chloride. The proline and sodium are shown to have reached electrochemical equilibrium by 30 min, at which stage there is about 100 pmol L‐proline mg protein‐1. 4. This transient accumulation is abolished by the prior equilibration of the sodium chloride gradient, or by the replacement of sodium by inwardly directed gradients of other cations. Entry of the amino acid into the vesicles is also shown to be influenced by the permeability of the anion in the medium and by an imposed potassium diffusion potential. L‐Proline transport across the brush border membrane of human placental syncytiotrophoblast is thus a sodium‐dependent, electrogenic process. 5. Studies of the transport processes indicate saturation at higher concentrations of L‐proline with a ‘Km’ of about 1 mM; Vmax averaged about 2 nmol mg protein‐1 min‐1 varied considerably between preparations. 6. L‐Proline (188 microM) transport is inhibited competitively by the presence of many amino acids and by the dipeptide glycyl‐L‐proline. The Ki for inhibition by methyl AIB is 300 microM. 7. These findings are discussed in relation to the mechanism of transplacental amino acid transfer.

Effects of cellular redox balance on induction of apoptosis by eicosapentaenoic acid in HT29 colorectal adenocarcinoma cells and rat colon in vivo

BACKGROUND AND AIMS Epidemiological evidence suggests n-3 polyunsaturated lipids may protect against colorectal neoplasia. Consumption of fish oil modulates crypt cytokinetics in humans, and crypt apoptosis in animal models. To explore these effects, we investigated involvement of caspase enzymes and cellular redox balance in the induction of apoptosis by eicosapentaenoic acid (EPA) in HT29 cells, and in rat colon in vivo. METHODS Survival of HT29 cells grown with EPA in the presence of caspase inhibitors, antioxidants, or buthionine sulphoximine, an inhibitor of glutathione neosynthesis, was determined. The effects of EPA enriched fish oil and glutathione depletion on apoptosis in rat colon were assessed using microdissected crypts. RESULTS Treatment of HT29 cells with EPA reduced viable cell number and activated caspase 3, prior to cell detachment. Antioxidants and caspase inhibitors blocked HT29 cell death whereas glutathione depletion increased it. Rats fed fish oil had higher crypt cell apoptosis than those fed corn oil, and glutathione depletion enhanced this effect. CONCLUSIONS Incorporation of EPA into colonic epithelial cell lipids increases apoptosis. The results of this study, using both an animal and cell line model, support the hypothesis that this effect is mediated via cellular redox tone, and is sensitive to glutathione metabolism. The data suggest a mechanism whereby polyunsaturated fatty acids may influence the susceptibility of colorectal crypt cells to induction or progression of neoplasia.

Effect of eicosapentaenoic acid on the proliferation and incidence of apoptosis in the colorectal cell line HT29

Fish oil has been shown to reduce the induction of colorectal cancer in animal models by a mechanism which may involve suppression of mitosis, increased apoptosis, or both. We used the human colonic adenocarcinoma cell line HT29 to explore the effects of the long-chain n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) on cell proliferation and death in vitro. Cells were cultured in media containing EPA at 5, 10, and 15 μg/mL. Cell number and thymidine incorporation were used to quantify proliferation, and cell cycle effects were studied using flow cytometry. Gel electrophoresis, annexin-V binding, and morphological criteria were used to characterize apoptosis. Adherent cells and freely floating detached cells were treated as two distinct populations. In the presence of EPA at 10 and 15 μg/mL there was a marked reduction in the growth rate of adherent HT29 colonies, owing to an increased detachment of adherent cells. After treatment with 10 or 15 μg/mL EPA the proportion of adherent cells in S-phase increased, indicating either a block in late S-phase or early G2. Floating cells showed evidence of extensive DNA cleavage, but the proportion of floating cells with sub G0 DNA content declined on treatment with 10 or 15 μg/mL EPA even though the number of floating cells increased. We conclude that EPA does not inhibit mitosis of adherent cells, but increases the rate at which they become detached from the substrate, probably at an early stage in the initiation of apoptosis. This mechanism may be analogous to “anoikis”, or induction of apoptosis in response to loss of cell contact, and may contribute to the anticarcinogenic effects of fish oil in vivo.

Non-Nutritive Bioactive Constituents of Plants: Dietary Sources and Health Benefits of Glucosinolates

A high intake of cruciferous vegetables is associated with a reduced risk of cancer, particularly lung and those of the gastrointestinal tract. This protective effect has been linked to the presence of glucosinolates in these vegetables. Certain metabolites of the glucosinolates, particularly the isothiocyanates and nitriles have been shown to both modify xenobiotic metabolising enzymes and induce cell cycle arrest and apoptosis. Different metabolites are more or less effective in mediating each response depending on the model system employed. It is likely that a combination of these responses explains the chemo-preventive characteristics of Brassicas and that a combination of different cruciferous vegetables will provide optimal protection.