Grapefruit juices impair the bioaccessibility of ß-carotene from orange-fleshed sweet potato but not its intestinal uptake by Caco-2 cells.

Among various factors influencing ß-carotene (Bc) bioavailability, information on interactions between carotenoids or other micronutrients such as flavonoids during a meal that contains different plant-derived foods is quite limited. Because orange-fleshed sweet potato (OFSP) is an important Bc-rich staple food, a source of vitamin A in developing countries, this study focused on the effect of citrus fruit juice carotenoids and flavonoids on Bc bioaccessibility from OFSP. In vitro digestion coupled with the Caco-2 cell culture model was used to evaluate the bioaccessibility and cellular uptake of Bc from OFSP in the presence of pink grapefruit (pGF) or white grapefruit (wGF) juices. The addition of grapefruit juices significantly decreased the bioaccessibility, by up to 30%, but not the cellular uptake of Bc from boiled OFSP. Lycopene, but more probably naringin, present in grapefruit juices was suspected to be responsible for the inhibitory effect of the citrus juices on Bc bioaccessibility. This inhibition was apparently due in part to competition for incorporation between Bc and naringin into mixed micelles during in vitro digestion. In contrast, Bc uptake from dietary micelles was not impaired by naringin.

Staphylococcal enterotoxin A: Partial unfolding caused by high pressure or denaturing agents enhances superantigenicity.

The effect of transient exposure of Staphylococcus aureus enterotoxin A (SEA) to high pressure and/or denaturing agents was examined by assessing the toxin superantigenicity and immunoreactivity, and by monitoring pressure-induced changes in fluorescence emission spectra. Pressurization of SEA at 600 MPa and 45 degrees C in Tris-HCl buffer (20 mM, pH 7.4) resulted in a marked increase in both T-cell proliferation (superantigenicity) and immunoreactivity. In opposite, pressurization at 20 degrees C did not change significantly SEA superantigenicity and immunoreactivity, indicating some toxin baro-resistance. Exposure of SEA to 8 M urea at atmospheric pressure or at 600 MPa and 20 degrees C, also led to a marked increase of superantigenicity (but not of immunoreactivity). In contrast, exposure of SEA to sodium-dodecylsulfate (30 mM) led to an increase of immunoreactivity with some effect on superantigenicity after pressurization at 45 degrees C only. High pressure up to 600 MPa induced spectral changes which at 20 degrees C were fully reversible upon decompression. At 45 degrees C, however, a sharp break of the centre of spectral mass mainly due to tryptophan residues was observed at 300 MPa, and irreversible spectral changes mainly related to tyrosine residues subsisted after pressure release, indicating a marked protein conformational transition. Urea 8 M further increased SEA structural changes at 600 MPa and 20 degrees C. These results indicate that SEA, under a combination of high pressure and mild temperature, as well as in the presence of urea, partly unfolds to a structure of strongly increased T-cell proliferative ability.

Superoxide production: a procalcifying cell signalling event in osteoblastic differentiation of vascular smooth muscle cells exposed to calcification media.

Recent studies showed that hydrogen peroxide (H(2)O(2)) enhanced bone markers expression in vascular smooth muscle cells (VSMCs) implicated in osteoblastic differentiation. This study aimed at investigating the role of NAD(P)H oxidase in vascular calcification processes. A7r5 rat VSMCs were incubated with beta-glycerophosphate (10 mm) or uremic serum to induce a diffuse mineralization. H(2)O(2) production by VSMCs was determinated by chemiluminescence. NAD(P)H oxidase sub-unit (p22(phox)), Cbfa-1, ERK phosphorylation and bone alkaline phosphatase (ALP) expressions were measured by Western blotting. VSMCs exhibited higher production of H(2)O(2) and early expression of p22(phox) with beta-glycerophosphate or uremic serum within 24 h of treatment. beta-glycerophosphate-induced oxidative stress was associated with Cbfa-1 expression followed by ALP expression and activity, meanwhile the VSMCs expressing ALP diffusely calcified their extracellular matrix. Interestingly, diphenyleneiodonium partly prevented the osteoblastic differentiation. Results from this model strongly suggest a major implication of vascular NAD(P)H oxidase in vascular calcification supported by VSMCs osteoblastic differentiation.

Both direct and indirect effects account for the pro-inflammatory activity of enteropathogenic mycotoxins on the human intestinal epithelium: stimulation of interleukin-8 secretion, potentiation of interleukin-1beta effect and increase in the transepithelial passage of commensal bacteria.

Mycotoxins are fungal secondary metabolites responsible of food-mediated intoxication in animals and humans. Deoxynivalenol, ochratoxin A and patulin are the best known enteropathogenic mycotoxins able to alter intestinal functions resulting in malnutrition, diarrhea, vomiting and intestinal inflammation in vivo. Although their effects on intestinal barrier and transport activities have been extensively characterized, the mechanisms responsible for their pro-inflammatory effect are still poorly understood. Here we investigated if mycotoxin-induced intestinal inflammation results from a direct and/or indirect pro-inflammatory activity of these mycotoxins on human intestinal epithelial cells, using differentiated Caco-2 cells as model and interleukin 8 (IL-8) as an indicator of intestinal inflammation. Deoxynivalenol was the only mycotoxin able to directly increase IL-8 secretion (10- to 15-fold increase). We also investigated if these mycotoxins could indirectly stimulate IL-8 secretion through: (i) a modulation of the action of pro-inflammatory molecules such as the interleukin-1beta (IL-1beta), and/or (ii) an increase in the transepithelial passage of non-invasive commensal Escherichia coli. We found that deoxynivalenol, ochratoxin A and patulin all potentiated the effect of IL-1beta on IL-8 secretion (ranging from 35% to 138% increase) and increased the transepithelial passage of commensal bacteria (ranging from 12- to 1544-fold increase). In addition to potentially exacerbate established intestinal inflammation, these mycotoxins may thus participate in the induction of sepsis and intestinal inflammation in vivo. Taken together, our results suggest that the pro-inflammatory activity of enteropathogenic mycotoxins is mediated by both direct and indirect effects.

Selective transport of staphylococcal enterotoxin A through in vitro generated human M cells.

Staphylococcal enterotoxins are responsible for food poisoning and toxic shock syndrome due to their superantigen activity on T cells. Although their activity necessarily involves passage through the intestinal epithelium, little is known about this critical step. In the present study, we compared the in vitro transport of staphylococcal enterotoxin A through human intestinal absorptive and M cells. We found that the transport of the toxin through M cells was polarized and temperature-sensitive, in contrast with the less efficient transport of the toxin by absorptive cells. These data suggest the involvement of M cells in the intestinal absorption of staphylococcal enterotoxins.

Beta-cryptoxanthin from citrus juices: assessment of bioaccessibility using an in vitro digestion/Caco-2 cell culture model.

Beta-Cryptoxanthin (beta-CX), a provitaminic carotenoid of potential interest for health, is found principally in Citrus fruit in both free and esterified forms. Little is known about the intestinal absorption of beta-CX especially with regard to the esterified forms. The aim of this study was to evaluate the absorption of free and esterified beta-CX using simulated digestion coupled with the Caco-2 model. Bioaccessibility was investigated by measuring the transfer of carotenoids from different citrus juices into micelles using an in vitro digestion system. Then, carotenoid uptake was evaluated by adding carotenoid-rich micelles (from the in vitro digestion) or synthetic micelles (made from synthetic lipids and carotenoids purified from citrus juice) to human intestinal cells (Caco-2 TC7 clone). Our results showed that beta-cryptoxanthin esters (beta-CXE) were partially hydrolysed during the in vitro digestion. The bioaccessibility of free beta-CX measured was significantly higher (40 (SD 1.05) %) than that of beta-carotene (30 (SD 1.9) %) and beta-CXE (16 (SD 1.5) %). In the same way, the incorporation of free beta-CX (27 (SD 1.01) %) into synthetic micelles exceeded (P<0.05) that of beta-carotene (10 (SD 0.7) %) and beta-CXE (8.8 (SD 0.4) %). In the case of micelles from in vitro digestion, the uptake of beta-carotene, free beta-CX and beta-CXE forms by Caco-2 cells was 14.3 (SD 1.8), 3.9 (SD 1.3), and 0.7 (SD 0.08) % respectively. These results showed a preferential uptake by Caco-2 cells of beta-carotene and free beta-CX compared with the two esters of beta-CX.

Relative contribution of phytates, fibers, and tannins to low iron and zinc in vitro solubility in pearl millet (Pennisetum glaucum) flour and grain fractions.

In vitro digestions were performed on pearl millet flours with decreased phytate contents and on two dephytinized or nondephytinized pearl millet grain fractions, a decorticated fraction, and a bran fraction with low and high fiber and tannin contents, respectively. Insoluble residues of these digestions were then incubated with buffer or enzymatic solutions (xylanases and/or phytases), and the quantities of indigestible iron and zinc released by these different treatments were determined. In decorticated pearl millet grain, iron was chelated by phytates and by insoluble fibers, whereas zinc was almost exclusively chelated by phytates. In the bran of pearl millet grain, a high proportion of iron was chelated by iron-binding phenolic compounds, while the rest of iron as well as the majority of zinc were chelated in complexes between phytates and fibers. The low effect of phytase action on iron and zinc solubility of bran of pearl millet grain shows that, in the case of high fiber and tannin contents, the chelating effect of these compounds was higher than that of phytates.

Ethanol and polyphenolic free wine matrix stimulate the differentiation of human intestinal Caco-2 cells. Influence of their association with a procyanidin-rich grape seed extract.

The effect of daily contact with ethanol on Caco-2 cell differentiation was investigated. Pure ethanol (1%) and a polyphenolic free wine matrix (polyphenol-free wine containing 1% ethanol) associated or not with a procyanidin-rich grape seed extract (GSE) were added to Caco-2 cells from confluency for 2 h a day after successive incubation in salivary, gastric, and pancreatic media. Treatment with 1% ethanol did not appear to be cytotoxic to cells, but it also stimulated Caco-2 cell differentiation, particularly in the first days following confluency, and this effect was more marked when associated with polyphenolic free wine matrix constituents. This activation resulted in an increase in microvillar density, organization, and elongation (+70%) and was associated with strong stimulation of sucrase-isomaltase (+780%) and a concomitant regular increase in cell protein content (+50-88%). While the presence of GSE in alcoholic solutions did not modify the morphological pattern observed in cells subjected to ethanol and polyphenolic free wine matrix alone, it had a clear reducing effect on their microvillus elongation (-30%). However, these stimulating effects of ethanol on morphological differentiation were attenuated from day 10 postconfluency, which could suggest cell cytoprotection against ethanol. These are the first results in support of the notion that moderate concentration of ethanol may stimulate the differentiation of Caco-2 cells, particularly when integrated with a polyphenolic free wine matrix.

Iron and zinc in vitro availability in pearl millet flours (Pennisetum glaucum) with varying phytate, tannin, and fiber contents.

Simulations of gastro-intestinal digestion, used to estimate in vitro iron and zinc availability, were performed on two kinds of samples: (i) samples with decreased phytate contents from whole pearl millet flour and (ii) nondephytinized or dephytinized samples from two pearl millet grain fractions, a decorticated fraction with low fiber and tannin contents and a bran fraction with high fiber and tannin contents. Iron and zinc in vitro availabilities of whole pearl millet flour were significantly improved by phytate degradation, even if the IP6 were not all degraded. Total dephytinization of decorticated fraction led to a marked increase in iron and zinc in vitro availabilities, but that of bran fraction had no effect on either iron or zinc in vitro availability. Even if phytates are involved in reducing in vitro iron and zinc availability in pearl millet flour, fibers and tannins play an important role by chelating a high proportion of iron and zinc in grain hulls.

Dietary wine phenolics catechin, quercetin, and resveratrol efficiently protect hypercholesterolemic hamsters against aortic fatty streak accumulation.

The effects of the phenolic compounds catechin (Cat), quercetin (Qer), and resveratrol (Res) present in red wine on early atherosclerosis were studied in hamsters. Hamsters (n = 32) were divided into 4 groups of 8 and fed an atherogenic diet for 12 weeks. They received by force-feeding 7.14 mL/(kg of body wt.day) Cat, Qer, or Res in water [2.856 mg/(kg of body wt.day) for Cat and 0.1428 mg/(kg of body wt.dday) for Qer and Res], mimicking a moderate consumption of alcohol-free red wine (equivalent to that supplied by the consumption of about two glasses of red wine per meal for a 70 kg human), or water as control. Plasma cholesterol concentration was lower in groups that consumed phenolics than in controls. The increase in plasma apolipoprotein (Apo) A1 concentration was mainly due to Cat (26%) and Qer (22%) and to a lesser extent, but nonsignificantly, Res (19%). Apo-B was not affected. Plasma antioxidant capacity was not improved, and there was no sparing effect on plasma vitamins A and E. Plasma iron and copper concentrations were not modified nor were liver super oxide dismutase and catalase activities. A sparing effect of Qer on liver glutathione peroxidase activity appeared, whereas Cat and Res exhibited a smaller effect. Aortic fatty streak area was significantly reduced in the groups receiving Cat (84%) or Qer (80%) or Res (76%) in comparison with the controls. These findings demonstrate that catechin, quercetin, and resveratrol at nutritional doses prevent the development of atherosclerosis through several indirect mechanisms.

Phenolics from commercialized grape extracts prevent early atherosclerotic lesions in hamsters by mechanisms other than antioxidant effect.

The aim of this study was to evaluate the antiatherosclerotic effect of commercially available phenolic-rich extracts from grape seeds (ExGrape seeds, EGS; grape seed extract, GSE) and marc (ExGrape total, EGT) in cholesterol-fed hamsters and to investigate possible operating mechanisms. These extracts fed at a moderate dose mimicking two glasses of red wine per meal reduced plasma cholesterol (-11% on average) but did not affect plasma antioxidant capacity of hamsters. The extracts prevented the development of aortic atherosclerosis by 68% (EGS), 63% (EGT), and 34% (GSE). Elsewhere, in an ex vivo experiment using rat aortic rings, EGS (7 microg/mL) induced 77% endothelium-dependent relaxation, whereas EGT and GSE (30 microg/mL) induced 84 and 72%, respectively. These results suggests that phenolic extracts from grape seeds and marc are beneficial in inhibiting atherosclerosis by indirect mechanism(s).

Grape seed extract affects proliferation and differentiation of human intestinal Caco-2 cells.

The effect of daily contact of a grape seed extract (GSE) on Caco-2 cell proliferation and differentiation was investigated. GSE at 400 mg/L was added to Caco-2 cells for 2 h a day after successive incubation in saliva, gastric, and pancreatic media. When applied at the beginning of the cell culture, GSE triggered inhibition of cell growth associated with a possible cytotoxic reaction. On the other hand, when the treatment was applied to confluent cells, treated cells displayed a higher protein content than control cells and a more developed brush border, with taller and denser microvilli. These observations were accompanied by stimulation of alkaline phosphatase activity, especially at day 5 postconfluency, with a 2.2-fold increase in comparison with the control. On the other hand, aminopeptidase N activity was inhibited throughout the differentiation period in GSE-treated cells to reach 28.8% of control cell activity on day 30. GSE did not affect either sucrase-isomaltase activity or cytoplasmic lactate dehydrogenase (LDH) activity, which otherwise appeared to be a good cellular marker. GSE treatment of Caco-2 cells thus inhibited their proliferation from seeding onward and stimulated both proliferation and differentiation after confluency.

Hydroxycinnamic acids do not prevent aortic atherosclerosis in hypercholesterolemic golden Syrian hamsters.

The protective effect of hydroxycinnamic acids, i.e. caffeic acid (CA) and sinapic acid (SA) present in wine, and chlorogenic acid (CHA) present in apple, compared to a red wine phenolic extract (RWPE) was investigated in hamsters fed an atherogenic diet for 12 weeks. Five groups of 8 hamsters fed such a diet received by force-feeding RWPE, CA or SA in water, mimicking a moderate consumption of alcohol-free red wine. Controls received water and CHA force-feeding was extrapolated from apple consumption. Plasma cholesterol concentration was lower in group that received RWPE (-22%) and hydroxycinnamic acids had no effect. Plasma apolipoprotein Apo-A1 concentration was not affected; consumption of RWPE only decreased Apo-B concentration (-46%). Liver superoxide dismutase activity was 33% lower and glutathione peroxidase activity was 67% greater in the group receiving RWPE compared to controls; there was no effect when CA, SA or CHA were given. All the phenolic compounds significantly increased plasma antioxidant capacity (about 28% on average) compared with controls. Aortic fatty streak area was significantly reduced in the group receiving RWPE (-30%) in comparison with controls and hydroxycinnamic acids. Our findings demonstrate that chronic ingestion of the nonalcoholic components of red wine, mainly polyphenols, prevent the development of atherosclerosis in hamster and that wine hydroxycinnamic acids are not the phenolic compounds involved in such a beneficial effect.

Hydrogen peroxide generation in caco-2 cell culture medium by addition of phenolic compounds: effect of ascorbic acid.

Phenolic compounds have recently attracted special attention due to their beneficial health effects; their intestinal absorption and bioavailability need, therefore, to be investigated and Caco-2 cell culture model appeared as a promising tool. We have shown herein that the addition of a grape seed extract (GSE) to Dulbecco's modified Eagle's medium (DMEM) used for Caco-2 cell culture leads to a substantial loss of catechin, epicatechin and B2 and B3 dimers from GSE in the medium after 24 h and to a production of hydrogen peroxide (H2O2). When 1420 microM ascorbic acid is added to the DMEM, such H2O2 production was prevented. This hydrogen peroxide generation substantially involves inorganic salts from the DMEM. We recommend that ascorbic acid be added to circumvent such a risk.

Red wine phenolic compounds reduce plasma lipids and apolipoprotein B and prevent early aortic atherosclerosis in hypercholesterolemic golden Syrian hamsters (Mesocricetus auratus).

The effects of a red wine phenolic extract (PE) on plasma lipoproteins and early atherosclerosis were studied in hamsters. Hamsters (n = 32) were divided into 4 groups of 8 and fed an atherogenic diet for 8 wk. They received by force- feeding 7.14 mL/(kg. d) PE in 2.6 mol/L ethanol (E + PE) or PE in water (W + PE), mimicking a moderate consumption of red wine or alcohol-free red wine [30.4 mg/(kg. d)], or 2.6 mol/L ethanol (E-PE) or water (W-PE) as their respective controls. Plasma cholesterol and triglyceride concentrations were lower in groups that consumed PE. The decrease in plasma apolipoprotein (Apo) B concentration was due mainly to PE and was significantly lower in Group E + PE than in Group E-PE (-7.5%) and in Group W + PE than in Group W-PE (-40%). Apo-A1 was not affected. PE significantly increased plasma antioxidant capacity by 9% in Group E + PE and 18% in Group W + PE compared with their respective controls. Liver glutathione peroxidase activity was 67% greater in the group receiving PE in water compared with the group given water; there was no effect when PE was given in ethanol relative to its control. Aortic fatty streak area (AFSA) was significantly reduced in the groups receiving PE in ethanol (-32%) or PE in water (-29%) in comparison with their respective controls. Ethanol significantly reduced AFSA by 60% (Group E-PE vs. Group W-PE) or 62% (Group E + PE vs. Group W + PE). These data suggest that ethanol is a complementary component of phenolics in the benefits of red wine for hamsters and that chronic ingestion of PE in ethanol prevents the development of atherosclerosis through several mechanisms. With moderate consumption of red wine, ethanol can improve the effects of phenolic compounds. However, alcohol-free red wine appears to be a very good alternative to red wine.

Assessment of selenium bioavailability from high-selenium spirulina subfractions in selenium-deficient rats.

It was previously found that the bioavailability of Se from Se-rich spirulina (SeSp) was lower than that from selenite or selenomethionine when fed to Se-deficient rats. The present study examined the bioavailability of Se from SeSp subfractions: a pellet (P) issuing from the centrifugation of a suspension of broken SeSp and a retentate (R) resulting from ultrafiltration of the supernatant through a 30 kDa exclusion membrane. Animals were fed a torula yeast based diet with no Se (deficients) or supplemented with 75 microg of Se/kg of diet as sodium selenite (controls) for 42 days. Se-deficient rats were then repleted for 56 days with Se (75 microg/kg of diet) supplied as sodium selenite, SeSp, P, or R. During this period, controls continued to receive sodium selenite. Speciation of Se in subfractions showed that the majority was present in the form of high molecular weight compounds; free selenomethionine was only a minor constituent. Gross absorption of Se from sodium selenite, P, and R was not different and was higher than from SeSp. Only retentate allowed full replenishment of Se concentration in liver and kidney (as did sodium selenite) and glutathione peroxidase (GSHPx) activity in liver, kidney, plasma, and erythrocytes. The bioavailabilities of Se in retentate, as assessed by slope ratio analysis using selenite as a reference Se, were 89 and 112% in the tissue Se content and 106-133% in the GSHPx activities. SeSp and P exhibited a gross bioavailability of <100%. These results indicate that Se in retentate is highly bioavailable and represents an interesting source of Se for food supplementation.