Modulation of chemical stability and in vitro bioaccessibility of beta-carotene loaded in kappa-carrageenan oil-in-gel emulsions.

In the present paper, ionotropically structured κ-carrageenan based oil-in-gel (o/g) emulsions were tested as potential carrier systems for the delivery of ß-carotene. In situ ionic gelation was induced by Na+, K+ or Ca2+ added at the level of 0.2-0.6% (w/w). All o/g emulsions exerted a true gel like behaviour with storage modulus (G') being reduced according to the order: K+>Ca2+>Na+. Ionic gelation induced a moderate increase in the microscopically assessed lipid droplets radii. O/g emulsions containing monovalent ions exerted the highest ß-carotene retention throughout isothermal storage particularly at high (37 and 55°C) temperatures. Notwithstanding, increasing ionic strength resulted in acceleration of ß-carotene degradation rates for all cation species. ß-Carotene bioaccessibility was significantly lower in Ca2+o/g emulsions due to the formation of complexes between the biopolymer matrix containing ß-carotene and bile salts. A good correlation between ß-carotene bioaccessibility, physical and colloidal aspects of the micellar digesta fractions was observed.

Proteomic response of inflammatory stimulated intestinal epithelial cells to in vitro digested plums and cabbages rich in carotenoids and polyphenols.

Due to their anti-oxidant and anti-inflammatory potential, polyphenol and carotenoid-rich plant foods have been suggested as promising phytochemicals in the prevention of or as adjuvants regarding inflammatory bowel diseases (IBD). In the present study, we investigated whether plum (Italian Plum, Prunus cocomilla), or cabbage (Kale, Brassica oleracea var. sabellica), selected for their high phytochemical content, are able to reduce inflammation in cellular models of the intestinal epithelium, employing proteomic methods. For this purpose, plum/cabbage (carotenoid content: 1.9 mg per 100 g resp. 13 mg per 100 g; polyphenol content: 83 mg per 100 g resp. 27 mg per 100 g) were gastro-intestinally digested, and aliquots exposed (18 h) to either a monoculture (Caco-2) or a triple culture (Caco-2/HT-29-MTX (90 : 10, v/v) with THP-1 like macrophages), stimulated (with LPS, TNF-α, and IL-1ß) to induce inflammation. Cells (Caco-2, Caco-2/HT-29-MTX, and THP-1) were then harvested separately, and proteomic analyses of total cell extracts were carried out by 2D-DIGE. In the monoculture, 68 protein-spots were significantly (p < 0.05, expression ratio >1.5) differentially regulated due to the Kale and Italian plum digesta, and in the co-culture 206 protein-spots, compared to digesta without plum/cabbage. These belonged to 27 (monoculture) and 76 (coculture) uniquely identified proteins, suggesting the coculture to be a more sensitive model. Proteins included antioxidant enzymes such as catalase, superoxide dismutase and glutathione-S-transferases. Only 3 proteins were differentially regulated in the THP-1 cells, perhaps as these were only indirectly exposed. The results show promise regarding some aspects related to IBD complications, however, employing phytochemical-rich food items should be further investigated in in vivo trials.

Inflammation related responses of intestinal cells to plum and cabbage digesta with differential carotenoid and polyphenol profiles following simulated gastrointestinal digestion.

SCOPE: Plums/cabbages represent fruits/vegetables rich in carotenoids and polyphenols, and have been associated with anti-inflammatory properties. METHODS AND RESULTS: We tested four plum (Italian Plum, Plum 620, Ersinger, and Cherry Plum) and cabbage varieties (Duchy, Kalorama, Kale, Scots Kale) with contrasting carotenoid/polyphenol content for their capability to alter inflammation/oxidative stress following simulated gastrointestinal digestion. Digesta were exposed to Caco-2(TC-7) and to a triple-culture(Caco-2/HT-29-MTX (90:10 v/v) including THP-1 like macrophages), stimulated to induce inflammation (10 µg/mL LPS, 100 ng/mL TNF-α, 25 ng/mL IL-1-ß for 24 h, the last 18 h with digesta). Endpoints investigated included IL-6, IL-8, PGE-2, NO (all ELISA), NF-κB, MAPK, IL-6, IL-8, iNOS, Nrf2, COX-2 (real-time-PCR) and Nrf2 (immunostaining). IL-6 secretion was reduced in THP-1 cells by Scots Kale and Kalorama (up to 22%, p<0.05), and IL-8 secretion in the coculture (up to 35% in plums, p<0.05). This was accompanied by decreased NF-kB expressions in THP-1 cells (up to 30%, p<0.05). Nrf2 translocation to the nucleus was partly reduced by plums and cabbages (up to 40% (p<0.05). CONCLUSIONS: Some varieties, especially in the triple-culture, reduced inflammation, though this was unrelated to concentrations of carotenoids/polyphenols. The potential of phytochemical-rich fruits and vegetables to ameliorate gastrointestinal inflammation should be further investigated.

Effects of silver nanoparticles and ions on a co-culture model for the gastrointestinal epithelium.

BACKGROUND: The increased incorporation of silver nanoparticles (Ag NPs) into consumer products makes the characterization of potential risk for humans and other organisms essential. The oral route is an important uptake route for NPs, therefore the study of the gastrointestinal tract in respect to NP uptake and toxicity is very timely. The aim of the present study was to evaluate the effects of Ag NPs and ions on a Caco-2/TC7:HT29-MTX intestinal co-culture model with mucus secretion, which constitutes an important protective barrier to exogenous agents in vivo and may strongly influence particle uptake. METHODS: The presence of the mucus layer was confirmed with staining techniques (alcian blue and toluidine blue). Mono and co-cultures of Caco-2/TC7 and HT29-MTX cells were exposed to Ag NPs (Ag 20 and 200 nm) and AgNO3 and viability (alamar blue), ROS induction (DCFH-DA assay) and IL-8 release (ELISA) were measured. The particle agglomeration in the media was evaluated with DLS and the ion release with ultrafiltration and ICP-MS. The effects of the Ag NPs and AgNO3 on cells in co-culture were studied at a proteome level with two-dimensional difference in gel electrophoresis (2D-DIGE) followed by Matrix Assisted Laser Desorption Ionization - Time Of Flight/ Time Of Flight (MALDI-TOF/TOF) mass spectrometry (MS). Intracellular localization was assessed with NanoSIMS and TEM. RESULTS: The presence of mucus layer led to protection against ROS and decrease in IL-8 release. Both Ag 20 and 200 nm NPs were taken up by the cells and Ag NPs 20 nm were mainly localized in organelles with high sulfur content. A dose- and size-dependent increase in IL-8 release was observed with a lack of cytotoxicity and oxidative stress. Sixty one differentially abundant proteins were identified involved in cytoskeleton arrangement and cell cycle, oxidative stress, apoptosis, metabolism/detoxification and stress. CONCLUSIONS: The presence of mucus layer had an impact on modulating the induced toxicity of NPs. NP-specific effects were observed for uptake, pro-inflammatory response and changes at the proteome level. The low level of overlap between differentially abundant proteins observed in both Ag NPs and AgNO3 treated co-culture suggests size-dependent responses that cannot only be attributed to soluble Ag.

Carotenoid and polyphenol bioaccessibility and cellular uptake from plum and cabbage varieties.

Plum and cabbage are rich in carotenoids and polyphenols. However, their bioactivity depends on their release and intestinal uptake. Four varieties of Brassicaceae (Duchy, Scots Kale, Kale, Kalorama) and Prunus (Cherry Plum, Plum 620, Ersinger, Italian Plum) were studied; bioaccessibility following in vitro digestion, cellular uptake (Caco-2 vs. co-culture cell model: Caco-2:HT-29-MTX (90:10%) and colonic fermentation were determined for carotenoids/polyphenols; the influence of certain kitchen preparations was likewise studied. Carotenoids were non-significantly influenced by the latter, while for polyphenols, boiling and steaming significantly reduced total phenolics (p<0.05). Carotenoid bioaccessibility did not differ significantly between Prunus vs. Brassicaceae varieties, but xanthophyll was higher than carotene bioaccessibility (p<0.01). Polyphenol bioaccessibility was low (<10%), possibly compromised by the cream containing test meal. Total carotenoid cellular uptake varied between varieties (0.3-4.1%), being higher for carotenes (4.1%) than for xanthophylls (1.6%, p<0.01), and were higher for the co-culture cell model compared to Caco-2 cells (p<0.01). Total carotenoid recovery in the colonic fraction varied from 4% to 25%. Lower bioaccessibility of carotenes thus appeared to be somewhat counterbalanced by higher cellular uptake. The potential positive role of the mucus layer for cellular uptake and the fate of the colonic digesta deserve further attention in the future.

Carotenoids, polyphenols and micronutrient profiles of Brassica oleraceae and plum varieties and their contribution to measures of total antioxidant capacity.

The consumption of phytochemicals such as carotenoids and polyphenols within whole fruits and vegetables has been associated with decreased incidence of various inflammation and oxidative stress related chronic diseases, which may be due to direct antioxidant effects, or indirect mechanisms such as affecting signal transduction/gene expression. Within the present study, we investigated the antioxidant composition of two major groups of vegetables and fruits, Brassica oleraceae and prunus spp., and estimated their contribution to antioxidant capacity. For this purpose, 17 plum and 27 Brassica varieties were collected in Luxembourg, and analysed for their individual polyphenol and carotenoid profile, vitamin C, dietary fibre, and minerals/trace elements, and their correlation with markers of antioxidant capacity (FRAP, ABTS, Folin-Ciocalteu). Total carotenoid and polyphenol content varied considerably between the different Brassica and plum varieties, with highest concentrations in the variety Kale (13.3 ± 0.58 mg/100g wet weight) and Cherry plum (1.96 ± 0.28 mg/100g) for carotenoids; and Kale (27.0 ± 0.91 mg/100g) and Kirks plum (185 ± 14 mg/100g) for polyphenols. In developed multiple linear-regression-models for Brassica, flavonoids, anthocyanins, lutein and vitamin C were found to be the best predictors of antioxidant capacity as assessed by FRAP (R(2)=0.832) and flavonoids, neochlorogenic acid and vitamin C as assessed by ABTS (R(2)=0.831); while for plums these were selenium, total sugars, chlorogenic acid and vitamin C (R(2)=0.853), and selenium, chlorogenic acid and flavonoids for FRAP (R(2)=0.711). When considering Brassica and plum consumption in Luxembourg, it is estimated that both contribute to an antioxidant intake equivalent to 26 and 6 mg per day of ascorbic acid equivalents, respectively.

An improved 3D tetraculture system mimicking the cellular organisation at the alveolar barrier to study the potential toxic effects of particles on the lung.

BACKGROUND: Exposure to fine and ultra-fine ambient particles is still a problem of concern in many industrialised parts of the world and the intensified use of nanotechnology may further increase exposure to small particles. Complex in vitro coculture systems may be valuable tools to study particle-induced processes and to extrapolate effects of particles on the lung. A system consisting of four different human cell lines which mimics the cell response of the alveolar surface in vitro was developed to study native aerosol exposure (Vitrocell™ chamber). The system is composed of an alveolar type-II cell line (A549), differentiated macrophage-like cells (THP-1), mast cells (HMC-1) and endothelial cells (EA.hy 926), seeded in a 3D-orientation on a microporous membrane. RESULTS: The spatial distribution of the cells in the tetraculture was analysed by confocal laser scanning microscopy (CLSM), showing a confluent layer of endothelial and epithelial cells on both sides of the transwell. Macrophage-like cells and mast cells can be found on top of the epithelial cells. The cells formed colonies under submerged conditions, which disappeared at the ALI. To evaluate the response to oxidative stress, the dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used together with 2,2'-azobis-2-methyl-propanimidamide-dihydrochloride (AAPH) as inducer of oxidative stress. The tetraculture showed less induction of reactive oxygen species (ROS) production after being treated with a positive control compared to the monocultures of EA.hy 926, THP-1 and HMC-1. Submerged cultures showed elevated ROS and IL-8 levels compared to ALI cultures. The Vitrocell™ aerosol exposure system was not significantly influencing the viability. Using this system, cells were exposed to an aerosol of 50 nm SiO2-Rhodamine NPs in PBS. The distribution of the NPs in the tetraculture after exposure was evaluated by CLSM. Fluorescence from internalized particles was detected in CD11b-positive THP-1 cells only. CONCLUSION: The system can be used in conjunction with a native aerosol exposure system and may finally lead to a more realistic judgement regarding the hazard of new compounds and/or new nano-scaled materials in the future. The results for the ROS production and IL-8 secretion suggest that submerged exposure may lead to an overestimation of observed effects.

Polyphenol and glycoalkaloid contents in potato cultivars grown in Luxembourg.

The polyphenol (phenolic acids, flavanols and flavonols) and glycoalkaloid (α-chaconine and α-solanine) contents of potato tubers grown in Luxembourg were analyzed by UPLC-DAD and HPLC-MS/MS separately in peel (approx. 2mm), outer (approx. 1cm) and inner flesh. Polyphenol contents decreased from the peel via the outer to the inner flesh and differed among the cultivars. The cultivars Vitelotte and Luminella had the highest polyphenol contents (5202 and 572 µg/g dry weight (DW) in the outer flesh), whereas Charlotte and Bintje had the lowest contents (19.5 and 48.0 µg/g DW). Chlorogenic acid and its isomers (neo- and cryptochlorogenic acid) were the major polyphenols. Glycoalkaloid contents were highest in the peel and lowest in the inner flesh, values in the flesh were below guideline limits in all cultivars. In conclusion, potatoes contribute to the daily intake of polyphenols and their consumption, thereby, may have positive effects on health.

NanoSIMS50 - a powerful tool to elucidate cellular localization of halogenated organic compounds.

Persistent organic pollutants are widely distributed in the environment and lots of toxicological data are available. However, little is known on the intracellular fate of such compounds. Here a method applying secondary ion mass spectrometry is described that can be used to visualize cellular localization of halogenated compounds and to semi-quantitatively calculate concentrations of such compounds. Of the model compounds tested, TBBPA was homogenously distributed in the cell membrane of the H295R cells while PFOS accumulated in very distinct locations in the cell membrane. Relative intracellular concentrations of 4-OH-BDE69 and 4-OH-BDE121 in GH3.TRE were 61 % and 18 %, respectively, compared to the parent compounds. These differences may partly explain that observed effect concentrations for 4-OH-BDEs in in vitro experiments are usually lower than what would be expected based on receptor binding studies. NanoSIMS50 proved to be a powerful tool to describe the cellular distribution of halogenated compounds. The semi-quantitative data that can be obtained may help to further explain results from in vitro or in vivo experiments.

Atrazine and PCB 153 and their effects on the proteome of subcellular fractions of human MCF-7 cells.

Several man-made organic pollutants including polychlorinated biphenyls (PCBs) and several pesticides may exhibit endocrine disrupting (ED) properties. These ED molecules can be comparatively persistent in the environment, and have shown to perturb hormonal activity and several physiological functions. The objective of this investigation was to study the impact of PCB 153 and atrazine on human MCF-7 cells, and to search for marker proteins of their exposure. Cells were exposed to environmentally high but relevant concentrations of atrazine (200ppb), PCB 153 (500ppb), 17-ß estradiol (positive control, 10nM) and DMSO (0.1%, negative control) for t=36h (n=3 replicates/exposure group). Proteins from cell membrane and cytosol were isolated, and studied by 2D-DiGE. Differentially regulated proteins were trypsin-digested and identified by MALDI-ToF-ToF and NCBInr database. A total of 36 differentially regulated proteins (>|1.5| fold change, P<0.05) were identified in the membrane fraction and 22 in the cytosol, and were mainly involved in cell structure and in stress response, but also in xenobiotic metabolism. 67% (membrane) and 50% (cytosol) of differentially regulated proteins were more abundant following atrazine exposure whereas nearly 100% (membrane) and 45% (cytosol) were less abundant following PCB 153 exposure. Western blots of selected proteins (HSBP1, FKBP4, STMN1) confirmed 2D-DiGE results. This study emphasizes the numerous potential effects that ED compounds could have on exposed humans.

Carotenoid exposure of Caco-2 intestinal epithelial cells did not affect selected inflammatory markers but altered their proteomic response.

Carotenoid consumption has been linked to a number of beneficial health effects, including the reduction of chronic diseases such as cancer and cardiovascular complications. However, no data are available on their action on the intestinal epithelium, being exposed to the highest concentrations of carotenoids in the human body, and where they could act preventively on intestinal inflammatory diseases such as Crohn's disease and ulcerative colitis. The objective of the present study was to investigate whether lycopene and ß-carotene in micelles (M), at concentrations that could be reached via the diet (10-25 µg/ml) could aid in the reduction of TNF-α plus IL-1ß-induced inflammation of Caco-2 human epithelial cells. The impact on biomarkers of inflammation, including IL-8, NO and cyclo-oxygenase-2 (through PGE-2α), and the NF-κB and mitogen-activated protein kinase (MAPK) pathways of intracellular signalling cascades were evaluated compared with controls (empty M). Furthermore, proteomic analyses were conducted from total cellular protein extracts. The results revealed that isolated carotenoids had no statistical significant anti-inflammatory effect on the biomarkers observed, or on the regulation of NF-κB and MAPK. Nevertheless, analyses of the proteome suggested that fifteen proteins were significantly (P < 0·05, expression ratio >1·3) differentially regulated following ß-carotene exposure, participating mostly in metabolic activities including antioxidant mechanisms, such as glutathione S-transferase A1. Only one protein was differentially regulated by lycopene (profilin-1). To our knowledge, this is the first attempt to investigate pathways involved in the action of carotenoids on the intestinal epithelium.

Antioxidative mechanisms of whole-apple antioxidants employing different varieties from Luxembourg.

Many health beneficial functions of dietary ingredients, including antimutagenity and anticarcinogenity, have been discussed in relation to their antioxidant properties. In this study, antioxidative mechanisms of whole-apple antioxidants (from seven varieties) were investigated using the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging capacity assay, the ferric-reducing antioxidant power (FRAP) assay, and the ferrous iron(II) chelating activity assay. Results indicated the ability of primary antioxidants to act as hydrogen or electron donors, with considerable differences depending on variety, with ABTS and FRAP values ranging from 270 to 1,142 mg of vitamin C equivalents/100 g and from 695 to 3,143 µmol of Fe/100 g, respectively. However, varieties did not display measurable chelating activity except for Florina and Graham, exhibiting a weak activity (0.1-0.2 µg of EDTA equivalents/100 g). Correlation analyses showed that polyphenols were major primary antioxidants contributing to antioxidative mechanisms (r>0.99, P<.001), whereas their involvement as secondary antioxidants (i.e., as chelating compounds) was negligible. Our findings further showed that the intake of 100 g of apple fruits can provide antioxidants equivalent to approximately 270-1,140 mg of vitamin C, with highest antioxidant concentrations for the older varieties Grauapfel and Goldparmäne.

A difference gel electrophoresis study on thylakoids isolated from poplar leaves reveals a negative impact of ozone exposure on membrane proteins.

Populus tremula L. x P. alba L. (Populus x canescens (Aiton) Smith), clone INRA 717-1-B4, saplings were subjected to 120 ppb ozone exposure for 28 days. Chloroplasts were isolated, and the membrane proteins, solubilized using the detergent 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), were analyzed in a difference gel electrophoresis (DiGE) experiment comparing control versus ozone-exposed plants. Extrinsic photosystem (PS) proteins and adenosine triphosphatase (ATPase) subunits were detected to vary in abundance. The general trend was a decrease in abundance, except for ferredoxin-NADP(+) oxidoreductase (FNR), which increased after the first 7 days of exposure. The up-regulation of FNR would increase NAPDH production for reducing power and detoxification inside and outside of the chloroplast. Later on, FNR and a number of PS and ATPase subunits decrease in abundance. This could be the result of oxidative processes on chloroplast proteins but could also be a way to down-regulate photochemical reactions in response to an inhibition in Calvin cycle activity.

Two-year monitoring of Cryptosporidium parvum and Giardia lamblia occurrence in a recreational and drinking water reservoir using standard microscopic and molecular biology techniques.

Starting in 2006, a monitoring of Giardia lamblia and Cryptosporidium parvum occurrence was conducted for 2 years in the largest drinking water reservoir of Luxembourg (Esch-sur-Sûre reservoir) using microscopy and qPCR techniques. Parasite analyses were performed on water samples collected from three sites: site A located at the inlet of the reservoir, site B located 18 km downstream site A, at the inlet of the drinking water treatment plant near the dam of the reservoir and site C where the finished drinking water is injected in the distribution network. Results show that both parasites are present in the reservoir throughout the year with a higher occurrence of G. lamblia cysts compared to C. parvum oocysts. According to our results, only 25% of the samples positive by microscopy were confirmed by qPCR. (Oo)cyst concentrations were 10 to 100 times higher at site A compared to site B and they were positively correlated to the water turbidity and negatively correlated to the temperature. Highest (oo)cyst concentrations were observed in winter. In contrast, no relationship between the concentrations of (oo)cysts in the reservoir and rain events could be established. Though a correlation has been observed between both parasites and faecal indicators in the reservoir, some discrepancies highlight that the latter do not represent a reliable tool to predict the presence/absence of these pathogenic protozoa. In summer 2007, the maximal risk of parasite infection per exposure event for swimmers in the reservoir was estimated to be 0.0015% for C. parvum and 0.56% for G. lamblia. Finally, no (oo)cysts could be detected in large volumes of finished drinking water.

Total phenolics, flavonoids, anthocyanins and antioxidant activity following simulated gastro-intestinal digestion and dialysis of apple varieties: Bioaccessibility and potential uptake.

In the present study, an in vitro model simulating gastrointestinal (GI) digestion, including dialysability, was adapted to assess free soluble polyphenols from apples (four varieties). Results indicated that polyphenol release was mainly achieved during the gastric phase (ca. 65% of phenolics and flavonoids), with a slight further release (<10%) during intestinal digestion. Anthocyanins present after the gastric phase (1.04-1.14mg/100g) were not detectable following intestinal digestion. Dialysis experiments employing a semipermeable cellulose membrane, presenting a simplified model of the epithelial barrier, showed that free soluble dialysable polyphenols and flavonoids were 55% and 44% of native concentrations, respectively, being approximately 20% and 30% lower than that of the GI digesta. Similar results were found for the antioxidant capacity of dialysable antioxidants, being 57% and 46% lower compared to total antioxidants in fresh apples (FRAP and ABTS test, respectively). It is suggested that some polyphenols are bound to macromolecular compounds that are non-dialysable, that the presented method allowed the study of free soluble polyphenols available for further uptake, and that both chemical extraction and concentrations in final digesta would overestimate polyphenol availability.

Development of a multi-class method for the quantification of veterinary drug residues in feedingstuffs by liquid chromatography-tandem mass spectrometry.

A simple multi-residue method was developed for detecting and quantifying 33 analytes from 13 classes of antibiotics (tetracyclines (3), quinolones (7), penicillins (3), ionophore coccidiostats (7), macrolides (3), sulfonamides (1), quinoxalines (2), phenicols (2), lincosamides (1), diaminopyrimidines (1), polypeptides (1), streptogramins (1) and pleuromutilins (1)) in animal feeds. Extraction and clean-up procedures were optimized with spiked piglet feed. Samples were extracted by ultrasonic-assisted extraction with a mixture of methanol/acetonitrile/McIlvaine buffer at pH 4.6 (37.5/37.5/25, v/v/v) containing 0.3% of EDTA-Na(2), followed by a clean up using a dispersive solid-phase extraction (d-SPE) with PSA (primary secondary amine). Detection of antibiotics was achieved by liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) within 28 min using both positive and negative ESI mode. Average recoveries ranged from 51% (oxytetracycline) to 116% (tilmicosin) with associated relative standard deviations of 7.3% and 9.0% and an overall mean of 87%. Limits of quantification ranged from 3.8 ngg(-1) (lincomycin) to 65.0 ngg(-1) (bacitracin). Following optimization, the method was further verified for bovine and lamb feeding stuffs; negative matrix effects were evaluated and overcome by a standard addition method.

Effects of the endocrine disruptors atrazine and PCB 153 on the protein expression of MCF-7 human cells.

Polychlorinated biphenyls (PCBs) and a number of pesticides can act as endocrine disrupting compounds (EDCs). These molecules exhibit hormonal activity in vivo, and can therefore interact and perturb normal physiological functions. Many of these compounds are persistent in the environment, and their bioaccumulation may constitute a significant threat for human health. Physiological abnormalities following exposure to these xenobiotic compounds go along with alterations at the protein level of individual cells. In this study, MCF-7 cells were exposed to environmentally relevant concentrations of atrazine, PCB153 (100 ppb, respectively), 17-beta estradiol (positive control, 10 nM) and a negative control (solvent) for t = 24 h (n = 3 replicates/exposure group). After trizol extraction and protein solubilization, protein expression levels were studied by 2D-DIGE. Proteins differentially expressed were excised, trypsin-digested, and identified by MALDI-ToF-ToF, followed by NCBInr database search. 2D-DIGE experiments demonstrated that 49 spots corresponding to 29 proteins were significantly differentially expressed in MCF-7 cells (>1.5-fold, P < 0.05, Student's paired t test). These proteins belonged to various cellular compartments (nucleus, cytosol, membrane), and varied in function; 88% of proteins were down-regulated during atrazine exposure, whereas 75% of proteins were up-regulated by PCB153. Affected proteins included those regulating oxidative stress such as superoxide dismutase and structural proteins such as actin or tropomyosin, which may explain morphological changes of cells already observed under the microscope. This study highlights the susceptibility of human cells to compounds with endocrine disrupting properties.

Gene expression changes related to the production of phenolic compounds in potato tubers grown under drought stress.

Polyphenols represent a large family of plant secondary metabolites implicated in the prevention of various diseases such as cancers and cardiovascular diseases. The potato is a significant source of polyphenols in the human diet. In this study, we examined the expression of thirteen genes involved in the biosynthesis of polyphenols in potato tubers using real-time RT-PCR. A selection of five field grown native Andean cultivars, presenting contrasting polyphenol profiles, was used. Moreover, we investigated the expression of the genes after a drought exposure. We concluded that the diverse polyphenolic profiles are correlated to variations in gene expression profiles. The drought-induced variations of the gene expression was highly cultivar-specific. In the three anthocyanin-containing cultivars, gene expression was coordinated and reflected at the metabolite level supporting a hypothesis that regulation of gene expression plays an essential role in the potato polyphenol production. We proposed that the altered sucrose flux induced by the drought stress is partly responsible for the changes in gene expression. This study provides information on key polyphenol biosynthetic and regulatory genes, which could be useful in the development of potato varieties with enhanced health and nutritional benefits.

Proteomic and enzymatic response of poplar to cadmium stress.

This study highlights proteomic and enzymatic changes in roots and leaves of actively growing poplar plants upon a cadmium stress exposure. Proteomic changes in response to a short-term (14 days), as well as a longer term (56 days) treatment are observed between the different organs. In leaves, stress-related proteins, like heat shock proteins, proteinases and pathogenesis-related proteins increased in abundance. A response similar to a hypersensitive response upon plant-pathogen interaction seemed to be induced. Concerning roots it appeared that the metabolic impact of cadmium was more deleterious than in leaves. This is evidenced by the early increase in abundance of many typical stress-related proteins like heat shock proteins, or glutathione-S-transferases, while most proteins from the primary metabolism (glycolysis, tricarboxylic acid cycle, nitrogen metabolism, sulfur metabolism) were severely decreased in abundance. Additionally the impact of cadmium on the glutathione metabolism could be assessed by activity assays of several important enzymes. Cadmium treatment had an inhibitory effect on glutathione reductase and ascorbate peroxidase in leaves, but not in roots. Conversely, glutathione-S-transferase showed a higher activity (and abundance) in roots but not in leaves.

Quantification of Giardia transcripts during in vitro excystation: interest for the estimation of cyst viability.

The aim of the present study was to evaluate the potential of transcript quantification as an indicator of Giardia cyst viability. The variations of beta-giardin, EF1A and ADHE mRNAs were quantified during excystation by real-time RT-PCR assays and compared with the percentages of viability estimated using propidium iodide staining and in vitro excystation. The first experiments were performed with purified G. duodenalis assemblage B cysts. When 55% of excysting protozoa were observed, the increase of the selected transcripts ranged from 0.40+/-0.13 to 0.97+/-0.11 log10 after 1h of incubation in excystation medium. Purified cysts were also stored at 4 degrees C for up to 56 days and analysed at several sampling times. Significant correlations were observed between the variations of the selected mRNAs and the percentages of viability estimated with staining and excystation methods. Among the three transcripts, beta-giardin appeared to be the most appropriate to study the viability of Giardia cysts concentrated from wastewater samples.