Alternariol Monomethyl-Ether Induces Toxicity via Cell Death and Oxidative Stress in Swine Intestinal Epithelial Cells.

Alternariol monomethyl-ether (AME), together with altenuene and alternariol, belongs to the Alternaria mycotoxins group, which can contaminate different substrates, including cereals. The aim of the present study was to obtain a deeper understanding concerning the effects of AME on pig intestinal health using epithelial intestinal cell lines as the data concerning the possible effects of Alternaria toxins on swine are scarce and insufficient for assessing the risk represented by Alternaria toxins for animal health. Our results have shown a dose-related effect on IPEC-1 cell viability, with an IC50 value of 10.5 µM. Exposure to the toxin induced an increase in total apoptotic cells, suggesting that AME induces programmed cell death through apoptosis based on caspase-3/7 activation in IPEC-1 cells. DNA and protein oxidative damage triggered by AME were associated with an alteration of the antioxidant response, as shown by a decrease in the enzymatic activity of catalase and superoxide dismutase. These effects on the oxidative response can be related to an inhibition of the Akt/Nrf2/HO-1 signaling pathway; however, further studies are needed in order to validate these in vitro data using in vivo trials in swine.

Yeast-Fermented Rapeseed Meal Extract Is Able to Reduce Inflammation and Oxidative Stress Caused by Escherichia coli Lipopolysaccharides and to Replace ZnO in Caco-2/HTX29 Co-Culture Cells.

(1) The present study tested in vitro the capacity of a fermented rapeseed meal extract to reduce medicinal ZnO, which will be banned at the EU level from 2023 onwards because of its potential to cause environmental pollution and the development of Zn resistance in gut bacteria. Rapeseed meal could be an important ZnO substitute as it has antioxidant/radical scavenging properties due to its content of bioactive compounds (e.g., polyphenols). (2) Protein array and flow cytometry were used to detect apoptosis, oxidative stress production, and inflammatory and signaling-related molecules in Caco-2 and goblet HT29-MTX co-culture cells challenged with Escherichia coli lipopolysaccharides and treated with ZnO and FRSM. (3) LPS induced cell death (21.1% vs. 12.7% in control, p < 0.005); apoptosis (16.6%); ROS production; and overexpression of biomarkers related to inflammation (63.15% cytokines and 66.67% chemokines), oxidative stress, and signaling proteins when compared to untreated cells. ZnO was effective in counteracting the effect of LPS, and 73.68% cytokines and 91.67% of chemokines were recovered. FRSM was better at restoring normal protein expression for 78.94% of cytokines, 91.67% of chemokines, and 61.11% of signaling molecules. FRSM was able to mitigate negative effects of LPS and might be an alternative to ZnO in pig diets.

Alterations in Essential Fatty Acids, Immunoglobulins (IgA, IgG, and IgM), and Enteric Methane Emission in Primiparous Sows Fed Hemp Seed Oil and Their Offspring Response.

This study shows the effects of dietary hemp seed oil on the milk composition, blood immunoglobulins (Ig), and enteric methane (E-CH4) production of primiparous sows, and their offspring's response at three time points. A bifactorial experiment was conducted for 21 days (d) on 18 primiparous sows (195 ± 3 days old). The sows were fed two diets: (i) a control diet (SO) based on soybean oil (1.6%), with an 18.82 n-6:n-3 polyunsaturated fatty acids (PUFA) ratio; (ii) an experimental diet (HO) based on hemp seed oil (1.6%), with a 9.14 n-6:n-3 PUFA ratio. The milk contained an elevated level of linoleic acids (LA), n-3 FA, and especially alpha-linolenic acids (ALA), while the n-6:n-3 ratio declined using hemp oil. The Ig concentration was higher in colostrum than in milk. In the first few hours, the IgG in the plasma of piglets was more than double that of maternal plasma IgG (+2.39 times). A period effect (p < 0.0001) for IgG concentration in the plasma of piglets was recorded (40% at 10 d, respectively 73% lower at 21 d than 12 h after parturition). However, the sow diet did not affect Ig (p > 0.05). The frequency of diarrhoea declined after about 7 d. The value of the rate of diarrhoea was 6.2% lower in the PHO group. We found a 4.5% decline in E-CH4 in the HO group. Applying multiple linear regression, feed intake, n-6:n-3 ratio, ALA, and lean meat were potential indicators in estimating E-CH4. In conclusion, sow dietary hemp seed oil increased lean meat %, milk EFA, and milk IgM. Significant changes in the other dependent variables of interest (body weight, plasma Igs in sows and offspring, E-CH4 production) were not recorded. There was reduced diarrhoea which shows that EFA could play a therapeutic role in the incidence of diarrhoea and in lowering of E-CH4 emission in sows and progeny. All dependent variables were significantly altered at different time points, except for fat concentration in milk and sow plasma IgG.

Dietary Grape Seed Meal Bioactive Compounds Alleviate Epithelial Dysfunctions and Attenuates Inflammation in Colon of DSS-Treated Piglets.

Inflammatory Bowel Diseases (IBD) are chronic inflammations associated with progressive degradation of intestinal epithelium and impairment of the local innate immune response. Restoring of epithelial integrity and of the mucosal barrier function, together with modulation of inflammatory and innate immune markers, represent targets for alternative strategies in IBD. The aim of our study was to evaluate the effects of a diet including 8% grape seed meal (GSM), rich in bioactive compounds (polyphenols, polyunsaturated fatty acids (PUFAs), fiber) on the markers of colonic epithelial integrity, mucosal barrier function, pro-inflammatory, and innate immunity in DSS-treated piglets used as animal models of intestinal inflammation. Our results have demonstrated the beneficial effects of bioactive compounds from dietary GSM, exerted at three complementary levels: (a) restoration of the epithelial integrity and mucosal barrier reinforcement by modulation of claudins, Occludin (OCCL) and Zonula-1 (ZO-1) tight junction genes and proteins, myosin IXB (MYO9B) and protein tyrosine phosphatase (PTPN) tight junction regulators and mucin-2 (MUC2) gene; (b) reduction of pro-inflammatory MMP-2 (matrix metalloproteinase-2) and MMP-9 (matrix metalloproteinase-9) genes and activities; and (c) suppression of the innate immune TLR-2 (Toll-like receptor-2) and TLR-4 (Toll-like receptor-4) genes and attenuation of the expression of MyD88 (Myeloid Differentiation Primary Response 88)/MD-2 (Myeloid differentiation factor-2) signaling molecules. These beneficial effects of GSM could further attenuate the transition of chronic colitis to carcinogenesis, by modulating the in-depth signaling mediators belonging to the Wnt pathway.

Zearalenone and the Immune Response.

Zearalenone (ZEA) is an estrogenic fusariotoxin, being classified as a phytoestrogen, or as a mycoestrogen. ZEA and its metabolites are able to bind to estrogen receptors, 17ß-estradiol specific receptors, leading to reproductive disorders which include low fertility, abnormal fetal development, reduced litter size and modification at the level of reproductive hormones especially in female pigs. ZEA has also significant effects on immune response with immunostimulatory or immunosuppressive results. This review presents the effects of ZEA and its derivatives on all levels of the immune response such as innate immunity with its principal component inflammatory response as well as the acquired immunity with two components, humoral and cellular immune response. The mechanisms involved by ZEA in triggering its effects are addressed. The review cited more than 150 publications and discuss the results obtained from in vitro and in vivo experiments exploring the immunotoxicity produced by ZEA on different type of immune cells (phagocytes related to innate immunity and lymphocytes related to acquired immunity) as well as on immune organs. The review indicates that despite the increasing number of studies analyzing the mechanisms used by ZEA to modulate the immune response the available data are unsubstantial and needs further works.

In Vitro Transcriptome Response to a Mixture of Lactobacilli Strains in Intestinal Porcine Epithelial Cell Line.

BACKGROUND: Food and feed supplements containing microorganisms with probiotic potential are of increasing interest due to their healthy promoting effect on human and animals. Their mechanism of action is still unknown. Using a microarray approach, the aim of this study was to investigate the differences in genome-wide gene expression induced by a mixture of three Lactobacillus strains (L. rhamnosus, L. plantarum, and L. paracasei) in intestinal porcine epithelial cells (IPEC-1) and to identify the genes and pathways involved in intestinal barrier functions. METHODS: Undifferentiated IPEC-1 cells seeded at a density of 2.0 × 105/mL in 24-wells culture plates were cultivated at 37 °C and 5% CO2 until they reached confluence (2⁻3 days). Confluent cells monolayer were then cultivated with 1 mL of fresh lactobacilli (LB) mixture suspension prepared for a concentration of approximately 3.3 × 107 CFU/mL for each strain (1 × 108 CFU/mL in total) for 3 h and analyzed by microarray using Gene Spring GX v.11.5. RESULTS: The functional analysis showed that 1811 of the genes modulated by LB treatment are involved in signaling (95% up-regulation, 121 genes with a fold change higher than 10). The most enhanced expression was registered for AXIN2 (axis inhibition protein 2-AXIN2) gene (13.93 Fc, p = 0.043), a negative regulator of ß-catenin with a key role in human cancer. LB affected the cellular proliferation by increasing 10 times (Fc) the NF1 gene encoding for the neurofibromin protein, a tumor suppressor that prevent cells from uncontrolled proliferation. The induction of genes like serpin peptidase inhibitor, clade A member 3 (SERPINA 3), interleukin-20 (IL-20), oncostatin M(OSM), granulocyte-macrophage colony-stimulating factor (GM-CSF), and the suppression of chemokine (C-X-C motif) ligand 2/macrophage inflammatory protein 2-alpha (CXCL-2/MIP-2), regulator of G-protein signaling 2 (RGS2), and of pro-inflammatory interleukin-18 (IL-18) genes highlights the protective role of lactobacilli in epithelial barrier function against inflammation and in the activation of immune response. CONCLUSION: Gene overexpression was the predominant effect produced by lactobacilli treatment in IPEC-1 cells, genes related to signaling pathways being the most affected. The protective role of lactobacilli in epithelial barrier function against inflammation and in the activation of immune response was also noticed.

Intestinal Absorption and Antioxidant Activity of Grape Pomace Polyphenols.

The absorption and antioxidant activity of polyphenols from grape pomace (GP) are important aspects of its valorization as a feed additive in the diet of weaned piglets. This study aimed to evaluate the presence of polyphenols from GP both in vitro in IPEC cells and in vivo in the duodenum and colon of piglets fed with diets containing or not 5% GP and also to compare and correlate the aspects of their in vitro and in vivo absorption. Total polyphenolic content (TPC) and antioxidant status (TAS, CAT, SOD and GPx enzyme activity, and lipid peroxidation-TBARS level) were assessed in duodenum and colon of piglets fed or not a diet with 5% GP. The results of UV-Vis spectroscopy demonstrated that in cellular and extracellular medium the GP polyphenols were oxidized (between λmax = 276 nm and λmax = 627.0 nm) with the formation of o-quinones and dimers. LC-MS analysis indicated a procyanidin trimer possibly C2, and a procyanidin dimer as the major polyphenols identified in GP, 12.8% of the procyanidin trimer and 23% of the procyanidin dimer respectively being also found in the compound feed. Procyanidin trimer C2 is the compound accumulated in duodenum, 73% of it being found in the colon of control piglets, and 62.5% in the colon of GP piglets. Correlations exist between the in vitro and in vivo investigations regarding the qualitative evaluation of GP polyphenols in the cells (λmax at 287.1 nm) and in the gut (λmax at 287.5 nm), as oxidated metabolic products. Beside the presence of polyphenols metabolites this study shows also the presence of the unmetabolized procyanidin trimers in duodenum and colon tissue, an important point in evaluating the benefic actions of these molecules at intestinal level. Moreover the in vivo study shows that a 5% GP in piglet’s diet increased the total antioxidant status (TAS) and decreased lipid peroxidantion (TBARS) in both duodenum and colon, and increased SOD activity in duodenum and CAT and GPx activity in colon. These parameters are modulated by the different polyphenols absorbed, mainly by the procyanidin trimers and catechin on one side and the polyphenols metabolites on the other side.

Low level of ochratoxin A affects genome-wide expression in kidney of pig.

Ochratoxin A (OTA) is a mycotoxin produced by fungus belonging to Aspergillus and Penicillium genra. The aim of the present paper was to investigate if a low concentration OTA has toxic effect in pigs. Twelve piglets were fed with a control or an OTA (0.05 mg/kg feed) contaminated diet. After 30 days, animals were slaughtered and samples of blood and kidney were used for further analyses. The mycotoxin analyses showed a significant higher (6.25 times) concentration of OTA in the kidney of OTA intoxicated piglets than in control ones. While OTA has no effect on the urea and creatinine concentration, the microarray analysis of the effect of OTA on genome wide expression in the kidney of intoxicated piglets, revealed that approximately 105 different transcripts were significantly altered. As shown by the microarray results, 0.05 mg/kg of OTA can principally interfere with: i) canonical pathways (CD28 Signaling in T Helper Cells, Role of NFAT in Regulation of the Immune Response, Relaxin Signaling, IL-1 Signaling) ii) molecular and cellular function (cellular movement cellular function and maintenance, cellular growth and proliferation cellular assembly and organization, cell death and survival) etc. However, alteration of renal and urological system development and function and renal necrosis predicted through Ingenuity Pathway Analysis (IPA) were not supported by clinical pathological data. In conclusion, OTA toxicity was found even at low concentration of toxin, correlated with the activation of immune response pathways, oxidative stress response and early carcinogenic events. This effect need to be further investigated and analyzed in the context of human health.

Effects of a diet containing dried grape pomace on blood metabolites and milk composition of dairy cows.

BACKGROUND: The effect of a diet containing 15% grape pomace (GP) on the general health status and milk quality of dairy cows was assessed by plasma biochemistry and total polyphenol (TP) content, milk polyphenols, milk composition and milk protein fractions. RESULTS: Among the polyphenols measured by liquid chromatography-mass spectroscopy in GP, in feed containing GP (GP+) or not containing GP (GP-), gallic acid and epicatechin were present in the highest concentrations (67.58 and 19.23 µg mL-1 , respectively). Higher amounts of TP were also detected in the blood plasma of GP+ cows (114.06 and 83.93 mg GAE L-1 , respectively) but not in their milk (233.17 and 245.75 mg GAE L-1 , respectively). Also a significant increase was found for lactose and ß-lactoglobulin, although there was no effect on α-lactalbumin, albumin, secretory components and caseins. CONCLUSION: Inclusion of 15% GP in the diets of dairy cows is beneficial for overall normal blood constituent metabolism and helps to maintain cow health. The milk of cows fed with a GP diet preserves the normal levels of fat, protein and caseins, and has increased levels of components that make this milk a versatile ingredient material for the food industry (e.g. model whey powders, stability of lactose-rich powders). © 2016 Society of Chemical Industry.

Exposure to zearalenone mycotoxin alters in vitro porcine intestinal epithelial cells by differential gene expression.

The gut represents the main route of intoxication with mycotoxins. To evaluate the effect and the underlying molecular changes that occurred when the intestine is exposed to zearalenone, a Fusarium sp mycotoxin, porcine epithelial cells (IPEC-1) were treated with 10µM of ZEA for 24h and analysed by microarray using Gene Spring GX v.11.5. Our results showed that 10µM of ZEA did not affect cell viability, but can increase the expression of toll like receptors (TLR1-10) and of certain cytokines involved in inflammation (TNF-α, IL-1ß, IL-6, IL-8, MCP-1, IL-12p40, CCL20) or responsible for the recruitment of immune cells (IL-10, IL-18). Microarray results identified 190 genes significantly and differentially expressed, of which 70% were up-regulated. ZEA determined the over expression of ITGB5 gene, essential against the attachment and adhesion of ETEC to porcine jejunal cells and of TFF2 implicated in mucosal protection. An up-regulation of glutathione peroxidase enzymes (GPx6, GPx2, GPx1) was also observed. Upon ZEA challenge, genes like GTF3C4 responsible for the recruitment of polymerase III and initiation of tRNA transcription in eukaryotes and STAT5B were significantly higher induced. The up-regulation of CD97 gene and the down-regulation of tumour suppressor genes (DKK-1, PCDH11X and TC531386) demonstrates the carcinogenic potential of ZEA.

ω-3 PUFA rich camelina oil by-products improve the systemic metabolism and spleen cell functions in fattening pigs.

Camelina oil-cakes results after the extraction of oil from Camelina sativa plant. In this study, camelina oil-cakes were fed to fattening pigs for 33 days and its effect on performance, plasma biochemical analytes, pro-/anti-inflammatory mediators and antioxidant detoxifying defence in spleen was investigated in comparison with sunflower meal. 24 crossbred TOPIG pigs were randomly assigned to one of two experimental dietary treatments containing either 12% sunflower meal (treatment 1-T1), or 12.0% camelina oil-cakes, rich in polyunsaturated fatty acids ω-3 (ω-3 PUFA) (treatment 2-T2). The results showed no effect of T2 diet (camelina cakes) on feed intake, average weight gain or feed efficiency. Consumption of camelina diet resulted in a significant decrease in plasma glucose concentration (18.47%) with a trend towards also a decrease of plasma cholesterol. In spleen, T2 diet modulated cellular immune response by decreasing the protein and gene expression of pro-inflammatory markers, interleukin 1-beta (IL-1ß), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and interleukin (IL-8) and cyclooxigenase 2 (COX-2) in comparison with T1 diet. By contrast, T2 diet increased (P<0.05) in spleen the mRNA expression of antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase 1 (GPx1) by 3.43, 2.47 and 1.83 fold change respectively, inducible nitric oxide synthase (iNOS) (4.60 fold), endothelial nitric oxide synthase (eNOS) (3.23 fold) and the total antioxidant level (9.02%) in plasma. Camelina diet increased also peroxisome-proliferator activated receptor gamma (PPAR-γ) mRNA and decreased that of mitogen-activated protein kinase 14 (p38α MAPK) and nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB). At this level of inclusion (12%) camelina oil-cakes appears to be a potentially alternative feed source for pig which preserves a high content of ω-3 PUFA indicating antioxidant properties by the stimulation of detoxifying enzymes expression and the suppression of spleen pro-inflammatory markers.