Whole wheat foods are significant source of compounds exhibiting health-promoting properties. One of the most abundant class of phytochemicals in the wheat grain is represented by phenolic acids that are typically localized in the bran and germ portions. The objective of this study was to estimate the extent of variation for phenolic acids in durum wheat (T. turgidum L. ssp. durum). In addition, this study aimed at evaluating the anti-inflammatory activity of phenolic acids contained in whole-meal flour extracts. Phenolic acids were recovered from the whole meal flours of 65 durum cultivars and subsequently identified and quantified by HPLC-DAD analysis. Then, the anti-inflammatory activity of phenolic acids extracts was evaluated on LPS-stimulated HT-29 human colon cells by measuring the levels of interleukin 8 (IL-8) and transforming growth factor ?1 (TGF- ?1). A large variation for the content of phenolic acids was observed among genotypes and, on average, it accounted for 830 ?g/g dry weight. Whole meal flour extract significantly inhibited the secretion of the pro-inflammatory IL-8 mediator at 66 µg/mL of phenolic acids. Conversely, the secretion of the anti-inflammatory mediator TGF-?1 was not modified by addition of phenolic acids to HT-29 cells. Results showed that durum cultivars have different contents of phenolic acids, suggesting that a number of elite varieties could be used for breeding purposes. Moreover, results provide further insight into the health-related benefits of durum wheat foods as depending on the anti-inflammatory activity of phenolic acids.

Artichoke (Cynara cardunculus (L.) subsp. scolymus Hayek) is an important component of theMediterranean diet, and a good source of health-promoting compounds, including polyphenols.The main phenolic compounds of artichoke are 5-O-caffeoylquinic acid (chlorogenicacid), and 3,5-O-, 1,5-O-dicaffeoylquinic acids and its beneficial effects are linked totheir antioxidant activity that can be affected by their stability under gastro-intestinal conditions,during digestion. This study aimed to assess the antioxidant activity induced byartichoke head extract, and its main individual polyphenols, on a stimulated HT-29 cell line,using a DCFH-DA probe. In addition, influences of in vitro digestion on artichoke head andits individual polyphenols towards antioxidant activity,were tested. Artichoke extract showedhigher antioxidant activity than the individual polyphenols, even after in vitro gastrointestinaldigestion. In addition, in vitro digestion did not modify the antioxidant activity ofartichoke polyphenols, except for 1,5-O-dicaffeoylquinic acid,which proved to be the least active.

Flow cytometry is a useful tool that provides an accurate, objective and rapid evaluation of semen quality. The use ofthis technique could significantly improve the quality of buffalo semen samples used in artificial insemination. Thisstudy was carried out to evaluate, by flow cytometry, frozen-thawed buffalo spermatozoa quality parameters suchas sperm viability by SYBR-14 / propidium iodide staining; mitochondrial function by JC-1 potentiometric probe; spermchromatin stability (SCSA) by acridine orange; and acrosome reaction (AR) by FITC-PNA staining. Semen samples fromfive Italian Mediterranean buffalo bulls were used. Sperm viability was not different between bulls and ranged from33.4% to 43.6%. A consistent rate (55.1 ± 10.8%) of sperm cells showed high mitochondrial membrane potential (Dwhigh),with no significant differences between subjects. Sperm chromatin structure assay differed significantly between the fivebuffalo bulls; moreover, data showed high stability within each buffalo. DNA fragmentation indexes (DFI), such as %-DFI,X-DFI, SD-DFI, were 11.2 ± 8.6, 153.3 ± 24.6 and 81.6 ± 21.2, respectively. Regarding AR, the percentage ofacrosome-reacted live (ARL) and acrosome-reacted dead (ARD) spermatozoa was 0.3 ± 0.2 and 15.3 ± 5.5, respec-tively. This functional parameter differed significantly between buffalo bulls and showed high stability. Following to Ca2+ionophore A23187 for 3 h, AR significantly differed between subjects and was characterized by an increase in both ARL(10.8%) and ARD population (22.0%). This study indicates that flow cytometry could be a useful tool for a quickmultiparametric evaluation of sperm quality in buffalo. In particular, SCSA and AR resulted in sperm functionalparameters sensitive enough for the diagnosis of frozen-thawed semen fertilizing potential.

Ingestion of food is considered a major route of exposure to many contaminants including mycotoxins. The amount of mycotoxin resisting to the digestion process and potentially absorbable by the systemic circulation is only a smaller part of that ingested. In vitro digestion models turn useful for evaluating mycotoxins bioaccessibility during the intestinal transit and can be intended as a valuable tool for the assessment of mycotoxin bioavailability in food. In this paper we describe a study aimed at investigating toxicity of in vitro gastro-duodenal digests of mycotoxin contaminated bread collected along the digestion time-course. Toxicity tests were carried out on a sensitive RPMI lymphoid B cell line chosen as the most suitable lineage to assess toxicity retained by gastro-duodenal digests. In parallel, a chemical quantification of T-2 and HT-2 toxins contaminating the bread digests was accomplished during the gastric and duodenal transit. The digestive fluids undergoing chemical and toxicological analysis were collected at the beginning and end of gastric phase, and after completion of the duodenal phase. Results proved that a correlation between HT-2 content and toxicity did exist although a more persistent toxic activity was displayed in the later stage of the duodenal phase. This persistent toxicity might be explained by the co-occurrence of unknown HT-2-related conjugates or metabolites formed during digestion.

Methodologies, such as flow cytometry and computer assisted sperm analysis (CASA), provide objective, reproducible, rapid and multi-parametric evaluation of semen quality. In this study, semen samples collected from six stallions were analysed for viability (by propidium iodide), chromatin stability by sperm chromatin structure assay (SCSA) and mitochondrial membrane potential by JC-1 using flow cytometry. Total and progressive motility, average path velocity (VAP), curvilinear velocity (VCL) and straight-line velocity (VSL) were determined by CASA system. The cytofluorimetric analysis provided results with low intra-assay variability respect to motility analysis by CASA system. The data on viability and mitochondrial assessment were rather uniform between stallions. The SCSA was able to distinguish potential fertility levels between stallions. In fact statistical differences were found between stallions especially for %-DFI and SD-DFI parameter. The %-DFI parameter was negatively correlated with VCL parameter. The higher repeatability of %-DFI parameter respect to those of other SCSA parameters confirms the importance of this parameter notoriously related to fertility. In conclusion, the simultaneous assessment of different functional sperm parameters, by flow cytometry and CASA, may be allow to obtain detailed and repeatable evaluations of sperm quality in the stallion, usually not considered in breeding selection programs.

The increased consumption of whole wheat grains and whole wheat products has been associated with reduced risk of developing chronic diseases, such as cardiovascular disease, type 2 diabetes and colon cancer. These beneficial effects have been ascribed to the presence in whole wheat kernels of bioactive compounds which may vary for total content and composition among different wheat species and wheat varieties. In this work we present the profile of hydrophilic and lipophilic bioactive compounds of whole wheat semolina from five durum wheat elite cultivars. Whole semolina samples were analyzed to evaluate the total content and composition of phenolic acids (hydrophilic extract) and the total content and composition of carotenoids, tocopherols and tocotrienols (lipophilic extract). The total phenolic acid content was variable among the cultivars and ranged from 488 ?g/g to 1490 ?g/g whole flour. Among the detected compounds, ferulic acid was the most abundant, followed by sinapic acid and p-coumaric acid. Total carotenoid content varied from 2.64 µg/g whole flour and 4.75 µg/g whole flour and were mostly represented by lutein and zeaxanthin, while ?- e ?-carotene were present in trace amounts. Three different homologues of tocotrienols were detected (?, ? and ?), varying in a range between 18.3 and 28.6 µg/g whole flour, while tocopherols were detected in trace amounts. Duilio and Svevo cultivars, exhibited the highest content of hydrophilic and lipophilic bioactive compounds, respectively, and were selected to test the anti-inflammatory activity of extracts on human intestinal HT-29 cells. Preliminary experiments were carried out in order to assess the highest not cytotoxic concentration of lipophilic and hydrophilic extracts by using MTT test. Both extracts will be used to assess the anti-inflammatory activity on HT-29 cells stimulated by LPS mitogen, by quantification of IL-8. This research shows that whole wheat semolina flours of these five cultivars varied significantly in their contents of bioactive compounds and differences in their anti-inflammatory potential might suggest the possibility that durum wheat varieties could be selected based on potential health benefits.

Introduction. A healthy and balanced diet prevents the occurrence of many age-related diseases. On the basis of this claim, new types of food, defined novel foods, such as potato selenium, carrot, onion, tomato iodine, have been developed.Objectives. Considering the biological activities induced by silicon (antioxidant activity, increased mineralization of bone, development of connective tissue), the aim of this study was to obtain different silicon biofortificated vegetables (Tatsoi, Mizuna, Portulaca, Bietola and Cicoria) used as mix for the IV gamma product (ready to use), in order to have an useful food for post-menopausal women. In addition, the assessment of bioaccessibility of crops, by in vitro gastro-digestion process, and antioxidant activity on human intestinal cell line, were carried out.Methodology. For the biofortified plants production, a soilless system able to monitoring the effective absorption of the silicon was used. The plants were digested using the protocol described by Ferruzzi et al., (2001) in order assess the bioaccessibility. The antioxidant activity of digesta samples was evaluated on Caco-2 cell line using DCFH-DA probe.Results and conclusions. No modifications on the production and/or quality of the plant were found. A species-related accumulation of silicon was recorded with an mean value of 130 mg/kg fresh weight respect to average value blank of 22 mg/Kg. In addition, the silicon was bioaccessible in a range from 36 to 75% in relation to vegetable species studied. The biofortification significantly (p<0.001) improved the antioxidant activity induced by crops.

Olive oil production generates large amounts of recalcitrant compounds, the olive oil mill wastewater (OMWW), which represent one of the most contaminating effluents among those produced by the agrofood industries. Nowadays, this view has changed to one that recognizes the waste as a lowcost starting material rich in bioactive compounds, particularly biophenols, that can be extracted and applied as natural antioxidants for the food and pharmaceutical industries. The data reported in this paper indicate that the OMWW extracts, besides low molecular weight antioxidant phenolics such as tyrosol and hydroxytyrosol, also contain phenolics with a molecular weight in the range of 600-5000 Da, which exhibit efficient scavenging activities against hydroxyl and peroxyl radicals. This group of phenolics includes, besides verbascoside, isoverbascoside, and an oxidized form of verbascoside, a number of higher molecular weight phenolics arising from oxidative polymerization of hydroxytyrosol and caffeic acid. Overall, these higher molecular weight phenolics prove to be, in some in vitro tests, more efficient scavengers of hydrophilic hydroxyl radicals than hydroxytyrosol, which could be used for industrial applications as natural nontoxic antioxidants.

The raw materials used in the production of bakery products are the main source of sporeforming bacteria associated with the alteration of "bread rope" which occurs predominantly in humid hot weather and which results in significant economic losses in the bakery product sector (Valerio et al., 2012 , Int. J. Food Microbiol, 156, 278-285). The spores, surviving the cooking process, germinate and the vegetative cells start a degrading process of starch and bread proteins leading to the formation of slime. In the present study a screening test was carried out on 176 isolates from raw materials to evaluate which species could cause alteration in bread. Bacillus amyloliquefaciens, together with B. subtilis and B. pumilus, were the main species capable of causing the spoilage. Moreover, among the identified species, strains belonging to the B. cereus group account for 17%, of which 38% were found to be able to cause bread rope. The B. cereus group strains belong to the phylogenetic groups III and IV associated with high risk of intoxication, particularly those of group III, for which a high thermal resistance of the spores has been demonstrated during a test that simulates the thermal profile of the baking process (De Bellis et al., 2015, Int. J. Food Microbiol., 197, 30-39). To estimate the risk of bread spoilage during shelf-life, Symp Previus tool was used: cardinal parameters and growth / no growth boundaries of three strains were determined for three B. amyloliquefaciens strains isolated from bread with clear alteration symptoms, wheat and semolina. In addition, challenge tests were conducted by inoculating the spores of one strain in the mixing phase: various bread formulations were prepared to evaluate their effect on bacterial growth and the experimental results were compared with silica simulations. Finally, to estimate the probability of product contamination during shelf-life, two storage temperatures and a threshold of 5 log cfu / g were considered, a value which could cause alteration and / or represent a risk to consumer health. The characterization of the growth behavior of the three strains showed a difference in the contamination probability and subsequent alteration of the product, demonstrating the need to consider biological variability in predictive microbiological studies in order to obtain a realistic estimate of the risk of contamination. In conclusion, this study demonstrated the applicability of predictive microbiological instruments, known for assessing the risk of food contamination from pathogenic microorganisms, to the study of the behavior of alterative microorganisms to control the quality of food (Valerio et al. 2015 Food Microbiol., 45, 2-9).

In this study, the naturally debittered table olives cv Bella di Cerignola were studied in order to (i) characterizetheir phenolic composition; (ii) evaluate the polyphenols bioaccessibility; (iii) assess their absorption and transport, acrossCaco2/TC7. LC-MS/MS analysis has confirmed the presence of hydroxytyrosol acetate, caffeoyl-6?-secologanoside, andcomselogoside. In vitro bioaccessibility ranged from 7% of luteolin to 100% of tyrosol, highlighting the flavonoids sensitivity tothe digestive conditions. The Caco2/TC7 polyphenols accumulation was rapid (60 min) with an efficiency of 0.89%; the overallbioavailability was 1.86% (120 min), with hydroxytyrosol and tyrosol the highest bioavailables, followed by verbascoside andluteolin. In the cells and basolateral side, caffeic and coumaric acids metabolites, probably derived from esterase activities, weredetected. In conclusion, the naturally debittered table olives cv Bella di Cerignola can be considered as a source of bioaccessible,absorbable, and bioavailable polyphenols that, for their potential health promoting effect, permit inclusion of table olives as afunctional food suitable for a balanced diet.

Raw materials used in bread-making process may be a rich source of spore-forming bacteria whose presence after cooking may represent a spoilage concern for bakery industries and a risk to consumer health. The aim of this study was to investigate the toxigenic potential of 54 spore-forming bacterial strains isolated from bread ingredients and bread, mainly of the Bacillus genus, and their resistance to a thermal treatment reproducing the bread cooking process to ascertain if they could represent a health concern for consumers.The potential toxigenicity of the strains was evaluated by screening the cytotoxic activity on HT-29 cells using bacterial culture filtrates after growing bacterial cells in BHI and in the bread-based medium BEB. The results showed a high cytotoxic activity of B. cereus strains, although it was lower in BEB medium. PCR analyses detected the presence of genes involved in the production of NHE, HBL or CytK toxins in B. cereus strains, while none of the tested strains contained the gene for cereulide production. Production of NHE and HBL toxins was also confirmed by specific immunoassays only for B. cereus strains. Cytotoxic activity of 13 strains belonging to B. amyloliquefaciens (7 strains), Paenibacillus spp. (3) B. mojavensis (1), B. simplex (1) and B. pumilus (1) was also detected.Interestingly, B. cereus strains assigned to phylogenetic group IV exhibited a thermal resistance markedly lower than B. cereus group III; furthermore, B. amyloliquefaciens strains almost completely survived the heat treatment, but showed a low cytotoxic activity. It is also relevant that single strains belonging to B. mojavensis and B. simplex showed a cytotoxic activity higher after growth in BEB than in BHI and a spore resistance enough to survive the bread cooking process. In conclusion, our study indicates that spore-forming bacteria could represent a risk to consumer health related to strains able to produce toxic substances and to survive bread cooking conditions.

The aim of this study was to investigate the in vitro effects of the Fusarium fungus-derivedmycotoxin, zearalenone and its derivatives alpha-zearalenol and beta-zearalenol on motility parameters and theacrosome reaction of stallion sperm. Since the toxic effects of zearalenone and its derivatives are thought to resultfrom their structural similarity to 17beta-estradiol, 17beta-estradiol was used as a positive control for 'estrogen-like'effects.Methods: Stallion spermatozoa were exposed in vitro to zearalenone, alpha-zearalenol, beta-zearalenol or 17betaestradiolat concentrations ranging from 1 pM - 0.1 mM. After 2 hours exposure, motility parameters wereevaluated by computer-assisted analysis, and acrosome integrity was examined by flow cytometry after stainingwith fluoroscein-conjugated peanut agglutinin.Results: Mycotoxins affected sperm parameters only at the highest concentration tested (0.1 mM) after 2 hoursexposure. In this respect, all of the compounds reduced the average path velocity, but only alpha-zearalenolreduced percentages of motile and progressively motile sperm. Induction of motility patterns consistent withhyperactivation was stimulated according to the following rank of potency: alpha-zearalenol >17beta-estradiol >zearalenone = beta-zearalenol. The hyperactivity-associated changes observed included reductions in straight-linevelocity and linearity of movement, and an increase in the amplitude of lateral head displacement, while curvilinearvelocity was unchanged. In addition, whereas alpha- and beta- zearalenol increased the percentages of liveacrosome-reacted sperm, zearalenone and 17beta-estradiol had no apparent effect on acrosome status. In short,alpha-zearalenol inhibited normal sperm motility, but stimulated hyperactive motility in the remaining motile cellsand simultaneously induced the acrosome reaction. Beta-zearalenol induced the acrosome reaction withoutaltering motility. Conversely, zearalenone and 17beta-estradiol did not induce the acrosome reaction but inducedhyperactive motility albeit to a different extent.Conclusions: Apparently, the mycotoxin zearalenone has 17beta-estradiol-like estrogenic activity that enables it toinduce hyperactivated motility of equine sperm cells, whereas the zearalenol derivatives induce prematurecompletion of the acrosome reaction and thereby adversely affect stallion sperm physiology. The alpha form ofzearalenol still possessed the estrogenic ability to induce hyperactivated motility, whereas its beta stereo-isomerehad lost this property.

Fumonisins (FBs), Fusarium mycotoxins common food contaminant, are a potent inducer of oxidative stress and lipid peroxidation in intestinal cells. In order to verify this toxic effect in intestine tract, the aim was to assess lipid peroxidation (as malondialdehyde MDA increased levels) on intestine rat samples exposed to chyme samples from in vitro digestion of FBs contaminated corn samples. Naturally (9.61 ± 3.2 ?g/gr), artificially (726 ± 94 ?g/gr) and spiked corn samples at EU permitted FBs levels were digested and added to luminal side of Ussing chamber for 120 min. Fumonisins-free corn sample was used as control. The MDA increase was observed just in 83% of intestine samples exposed at EU FBs levels and the digestion process seems to reduce this incidence (50% of samples). Malondialdehyde levels were FBs dose- and subject-related and ranged from 0.07 ± 0.01 to 3.59 ± 0.6 nmol/mg. Highest incidence and MDA % increment (I) were found when intestine tracts were exposed to chymes from artificially corn sample. The induction of lipid peroxidation induced by FBs could be due to interactions between FBs and intestinal membranes, with consequent modifications in membrane permeability and oxygen diffusion-concentration, as suggested by other authors.

This investigation shows the influence of the fermentation process started with the probiotic Lactobacillusparacasei LMG P-22043 (PF) on the fate of health promoting polyphenols in artichoke in comparison with notstarted fermented artichokes (NSF). The fermentation process did not influence significantly the polyphenolcompositions. However, low variations mainly related to the isomerization process of dicaffeoylquinic acids,probably due to the different pH values of the two samples, were observed. Further, the total polyphenolbioaccessibility of PF artichoke was lower (34%) than of NSF artichoke (64%) in small intestine. Moreover, bothpolyphenol isomerization and bioaccessibility influenced the antioxidant activity that was significantly lower forPF artichoke than for NSF ones. These effects were probably related to the bioprotective process exerted by theprobiotic strain on the food matrix, with the consequent limited release of polyphenols in the digestion liquids,making them available for further metabolism and absorption in the colon.

Table olives are a typical food of the Mediterranean diet and an important source of phenolic compoundswith high biological potential for human health. Their concentrations (?1-2% of FW) confer to table olivesantioxidant, anti-inflammatory, and antitumoral properties. The polyphenols content and composition in table olives can be affected by several factors, such as cultivars, climate, fruits ripeness, and, mainly, theprocessing methods. Among the de-bittering processes, the Greek method represents a spontaneous fermentation procedure that is driven by a mixed population of microorganisms, mainly consisting of yeastsand lactic acid bacteria (LAB). In this work, the effects of fermentation by autochthonous yeast and LAB starters on phenolics composition of Apulian table olives, Bella di Cerignola (BDC), Termite di Bitetto(TDB) and Cellina di Nardò (CEL) were studied in comparison with the commercial products. The samples were characterized by HPLC-DAD for their polyphenols composition; 18 compounds were identified andthe cultivar related effect was highlighted. The main identified phenolics were hydroxytyrosol, tyrosol, verbascoside and luteolin, followed by hydroxytyrosol-acetate detected in BDC and cyanidine-3-glucosideand quercetin in CEL. Further, the fermentation using selected LAB and yeasts influenced differently the composition and amount of polyphenols of the three cultivars, in particular the BDC amount was doubledcompared with the commercial sample. Instead, for TDB and CEL, the treatment did not influence the polyphenols composition. It is noteworthy that the use autochthonous microbial starter to drive table olivesfermentation process allows to maintain stable or increases polyphenols concentration in fermented table olives, significantly reducing the time necessary for de-bittering and improving organoleptic and sensorycharacteristics of the final product. Scavenger capacity in both DPPH and CAA assays, assessed the highest antioxidant effect for CEL with starters (21.7 mg Trolox eq/g FW; 8.5 ?mol hydroxytyrosol eq/100g FW).Moreover, the polyphenols were highly in vitro bioaccessible (>60%), although modifications in their profile, probably for combined effect of environment and microorganisms, were noted.Finally, fermented table olives are excellent source of health promoting compounds. Indeed, hydroxytyrosol and tyrosol are almost 8 times more than in olive oil for which a nutritional EU claim (No433/2012 of 23 May 2012) that attributes the protective effect from oxidative stress by polyphenols on blood lipids, has been established.

The purpose of this study was to assess the natural exposure of male horses (Equus caballus) to the mycotoxin zearalenone (ZEA) by using the ELISA test and to evaluate the effects of in vitro exposure of sperm cells to mycotoxin-containing urine extracts on sperm chromatin structure stability. Because of their occurrence in urine samples, ZEA and its derivatives were tested by sperm chromatin structure assay (SCSA) at natural levels detected by ELISA. Thirty-eight urine extracts of Italian (n = 11) and northeastern European (n = 27) horses were tested on frozen-thawed spermatozoa to evaluate the toxic effect of mycotoxin on their chromatin structure by flow cytometry. Different parameters of the DNA fragmentation index (DFI), such as the mean (X DFI), the percentage (%-DFI), and the standard deviation (SD-DFI), were analyzed. Urine samples showed a mean level of 32.3 ng/mL ZEA with significantly higher concentrations in northeastern European samples than in Italian samples, probably in relation to climatic and feeding differences. The toxic effects of ZEA-containing urine samples on SCSA parameters were found at low ZEA concentrations and were mainly observed in Italian samples. By using mycotoxin standards, ZEA, alpha-zearalenol, and beta-zearalenol proved to be more toxic compounds for sperm chromatin stability than other tested derivatives. A nongenomic mechanism of action can be hypothesized.

The purpose of this studywas to assess the natural exposure ofmale horses (Equus caballus) to the mycotoxin zearalenone (ZEA) byusing the ELISA test and to evaluate the effects of in vitro exposure of sperm cells to mycotoxin-containing urine extracts on spermchromatin structure stability. Because of their occurrence in urine samples, ZEA and its derivatives were tested by sperm chromatinstructure assay (SCSA) at natural levels detected by ELISA. Thirty-eight urine extracts of Italian (n = 11) and northeastern European (n= 27) horses were tested on frozen-thawed spermatozoa to evaluate the toxic effect of mycotoxin on their chromatin structure by flowcytometry. Different parameters of theDNA fragmentation index (DFI), such as the mean (¯X  DFI), the percentage (%-DFI), and thestandard deviation (SD-DFI), were analyzed. Urine samples showed a mean level of 32.3 ng/mL ZEA with significantly higherconcentrations in northeastern European samples than in Italian samples, probably in relation to climatic and feeding differences. Thetoxic effects of ZEA-containing urine samples on SCSA parameters were found at low ZEA concentrations and were mainly observedin Italian samples. By using mycotoxin standards, ZEA, a-zearalenol, and b-zearalenol proved to be more toxic compounds for spermchromatin stability than other tested derivatives. A nongenomic mechanism of action can be hypothesized.

Cadmium is a highly toxic heavy metal with negative effects on oocyte fertilization. The aim of this study was to analyse whether cadmium-induced impairment of fertilization is caused by mitochondria dysfunction and oxidative stress in the cumulus-oocyte complex (COC). Preliminarily, 19 trace element levels were measured in ovaries from juvenile and adult ewes and age-related cadmium ovarian bioaccumulation at nanomolar concentrations was found. COCs from juvenile and adult ewes, exposed during in vitro maturation to 1 nM or 100 nM CdCl2, and subjected to in vitro fertilization showed significantly lower fertilization rates in exposed COCs compared with controls. In vitro matured exposed and control COCs underwent confocal microscopy analysis of mitochondria activity and reactive oxygen species (ROS) levels and lipid peroxidation (LPO) assay at cumulus cell and oocyte level. In both age groups, cadmium at nanomolar concentrations induced cumulus-oocyte mitochondria over-activity and oxidative damage which were related to impaired oocyte fertilization. (C) 2017 Elsevier Inc. All rights reserved.

The effects of fermentation by autochthonous microbial starters on phenolics composition of Apulian table olives, Bella di Cerignola (BDC), Termite di Bitetto (TDB) and Cellina di Nardò (CEL) were studied, highlighting also the cultivars influence. In BDC with starter, polyphenols amount doubled compared with commercial sample, while in TDB and CEL, phenolics remain almost unchanged. The main phenolics were hydroxytyrosol, tyrosol, verbascoside and luteolin, followed by hydroxytyrosol-acetate detected in BDC and cyanidine-3-glucoside and quercetin in CEL. Scavenger capacity in both DPPH and CAA assays, assessed the highest antioxidant effect for CEL with starters (21.7 mg Trolox eq/g FW; 8.5 ?mol hydroxytyrosol eq/100g FW). The polyphenols were highly in vitro bioaccessible (>60%), although modifications in their profile, probably for combined effect of environment and microorganisms, were noted. Finally, fermented table olives are excellent source of health promoting compounds, since hydroxytyrosol and tyrosol are almost 8 times more than in olive oil.

Ochratoxin A (OTA) is a renal mycotoxin and transplacental genotoxic carcinogen. The aim of this study was to evaluate the natural occurrence of OTA in equine blood samples and its placental transfer. For the assessment of OTA levels, serum samples were collected from 12 stallions, 7 cycling mares and 17 pregnant mares. OTA was found in 83% of serum samples (median value = 121.4 pg/mL). For the assessment of placental transfer, serum samples were collected from the 17 mares after delivery and from the umbilical cords of their foals, after foaling. Fourteen serum samples from pregnant mares contained OTA (median value = 106.5 pg/mL), but only 50% of their foals were exposed (median values = 96.6 pg/mL). HPLC analysis carried out on four serum samples (collected from two mares and their respective foals) supported the ELISA results on OTA placental transfer. This is the first report on the natural occurrence of OTA in horse serum samples and placental transfer in horses. © 2013 by the authors; licensee MDPI, Basel, Switzerland.

Le micotossine, frequenti contaminanti degli alimenti, rappresentano un serio problema per la salute umana e animale. Lo studio sulla tossicità indotta da naturali livelli di esposizione alle micotossine è necessario per una valutazione del rischio di tali sostanze. Recentemente è stato individuato per alcune micotossine, quali le fumonisine (FBs) e l'ocratossina A (OTA), l'induzione di stress ossidativo come principale e precoce meccanismo di tossicità in studi in vitro. Si è voluto verificare se questo meccanismo di tossicità fosse presente in altri organi target e fosse determinato da naturali livelli di esposizione. Nel caso delle FBs lo studio è stato condotto sull'intestino, principale target di esposizione. Si è utilizzato un modello che ha riprodotto fisiologicamente l'esposizione intestinale alle micotossine con gli alimenti; in particolare sono stati sottoposti a un processo di digestione di vitro (1) campioni di mais naturalmente o artificialmente contaminati da FBs e i campioni di chimo ottenuti sono stati utilizzati per una breve esposizione di porzioni di intestino (umano e di ratto) tramite la camera di Ussing. E' stata registrata una riduzione del parametro elettrico della corrente di cortocircuito in porzioni di intestino esposti a chimo senza FBs (conseguente all'osmolalità del chimo). Questa variazione del trasporto ionico non si è ritrovata in porzioni di intestino esposti a chimo contenente FBs (probabilmente per interferenza del trasporto ionico del Na+) con conseguente sbilancio idrico-salino. Sui campioni di intestino esposti ai diversi campioni di chimo, si è anche osservato un aumento dei livelli di malondialdeide (MDA), marker di tossicità dello stress ossidativo fortemente influenzato dall'effetto della digestione, dai livelli di FBs e dall'origine dei campioni intestinali (ratto e uomo). Questi risultati testimoniamo come lo stress ossidativo rappresenti anche in studi ex vivo su porzioni di intestino il principale meccanismo di tossicità (2, 3). Nel caso dell'OTA, l'induzione dello stress ossidativo, evidenziato in diversi modelli cellulari, è stato anche ritrovato in cellule mesenchimali staminali ottenute da cordone ombelicale prelevato da cagne, un utile modello in vitro per valutare la tossicità riproduttiva e dello sviluppo fetale. Dopo esposizione a livelli di OTA sovrapponibili a quelli serici riportati in bibliografia (da0.25 pM a 25 nM), si è osservato un effetto inibitorio dei parametri cinetici relativi alla proliferazione cellulare e al doubling time a tutte le concentrazioni di OTA testate. L'effetto tossico indotto dall'OTA sulla proliferazione era secondario all' induzione di un processo apoptotico, osservato tramite analisi confocale della condensazione e frammentazione della cromatina. Il danno cromatinico era conseguente a stress ossidativo, come evidenziato dall'aumento di fluorescenza generato dal danno ossidativo del DNA indotto dall'OTA e valutato con il kit 8-hydroxy-2'-deoxyguanosin

Whole wheat foods are significant source of compounds exhibiting health-promoting properties. One of the most abundant class of phytochemicals in the wheat grain is represented by phenolic acids that are typically localized in the bran and germ portions. The objective of this study was to estimate the extent of genetic variation for phenolic acids in durum wheat (T. turgidum L. ssp. durum). In addition, this study aimed at evaluating the anti-inflammatory activity of phenolic acids contained in whole-meal flour extracts. Phenolic acids were recovered from the whole meal flours of 65 durum cultivars and subsequently identified and quantified by HPLC-DAD analysis. Then, the anti-inflammatory activity of phenolic acids extracts was evaluated on LPS-stimulated HT-29 human colon cells by measuring the levels of interleukin 8 (IL-8) and transforming growth factor ?1 (TGF- ?1). A large variation for the content of phenolic acids was observed among genotypes and, on average, it accounted for 830 ?g/g dry weight. Whole meal flour extract significantly inhibited the secretion of the pro-inflammatory IL-8 mediator at 66 µg/mL of phenolic acids. Conversely, the secretion of the anti-inflammatory mediator TGF-?1 was not modified by addition of phenolic acids to HT-29 cells. Results showed that durum cultivars have different contents of phenolic acids, suggesting that a number of elite varieties could be used for breeding purposes. Moreover, results provide further insight into the health-related benefits of durum wheat foods as depending on the anti-inflammatory activity of phenolic acids.

Mycotoxins have become one of the most recognised feed chain contaminants, with hundreds of mycotoxins identified to date. Management of mycotoxins includes prevention, regulation, monitoring, decontamination, and animal treatments. Even with good management, unavoidably low levels of several mycotoxins can cause loss of feedstuffs, increased animal disease, reduced animal performance, and food residues. A promising approach to protect animals against the harmful effects of contaminated feed is based on the use of feed additives. These additives are defined as substances that, when included into contaminated feed, can adsorb or denature mycotoxins in the digestive tract of animals. Since 2009, they are officially allowed in the UE as technological feed additives. Mycotoxin adsorbents are the most studied additives and a variety of products are on the market claiming multi-toxin adsorption capacity. The efficacy of adsorbents in sequestering different mycotoxins has been poorly addressed. The aim of this study was the screening of commercial products for preparing a nutritional composition intended to reduce bioavailability of a large range of mycotoxins. 52 commercial products from 26 industrial partners, including minerals, yeast-based products and blend of components, were tested. Preliminary adsorption tests allowed the selection of 4 commercial products as effective in sequestering simultaneously aflatoxin B1, zearalenone, ochratoxin A and fumonisin B1. All products failed in adsorbing deoxynivalenol, but activated carbon. Adsorption experiments were performed with selected binders, at physiologically relevant pH values commonly found in the stomach and intestine, to determine adsorption parameters (capacity, affinity, chemisorption index). Mineralogical analysis (XRD) and ash content showed that 3 out of the 4 commercial products selected as best multi-toxin adsorbents (designated by the supplying companies as minerals) were organoclays. Organoclays are not suitable for feed ingredients due to toxicity of the interlayer quaternary alkylammonium ions. Two organoclays and one yeast cell wall product, out of 52 commercial products, were found toxic in 2 bioassays. In conclusion, multi-toxin adsorbents covering major mycotoxins are not commercially available. Most of them lack effectiveness towards trichotechenes. The identity/composition of commercial products could be counterfeit and misleading. Some commercial products can be even highly toxic in toxicity bioassays.

White table grape cv. Italia is a typical component of the Mediterranean diet and a source of phenolic compounds, particularly abundant in the skin portion. The aim of this study was to characterize the phenolic profile of the table grape skin and to assess its stability after the in vitro digestion process. The main phenolic compounds identified by the HPLC-DAD analysis were: procyanidin B1, caftaric acid, catechin, coutaric acid, quercetin 3-glucuronide and quercetin 3-glucoside. All compounds showed a good stability after in vitro digestion (from 43 to 80%). Moreover, the influence of grape skin polyphenols on the modulation of ROS and GSH levels was evaluated in basal and in stressed conditions on human intestinal cells (HT-29). In basal conditions, a higher polyphenol concentrations exerted pro-oxidant effect corresponding to high ROS level and low GSH content. This effect was probably due to the polyphenolic oxidation in cell culture condition with consequent production of hydrogen peroxide. Otherwise, in stressed conditions, grape skin polyphenols exerted antioxidant effects up to 1.3×10-6 ?g/g and restored the stress-related GSH reduction. The in vitro digestion process attenuated the biological effect of grape skin polyphenols on intestinal cell line (HT-29). In conclusion, grape skin polyphenols showed different behavior in relation to their concentrations and to the intracellular ROS levels.

HeiferPlusTM is a new sperm sexing agent used to increase the percentage of heifer calves in dairy and beef cattle by enhancing the fertility and the motility of the X-chromosome bearing sperm. The aim of this study was to assess the influence of HeiferPlusTM treatment on sperm functional parameters of frozen-thawed bovine spermatozoa assessed by cytofluorimetric analysis such as sperm viability (by using propidium iodide probe), acrosome reaction (by using PNA-FITC probe), sperm chromatin stability (by using acridine orange probe) basal ROS determination (by using DCFH-DA probe) and mitochondrial activity (by using JC-1 probe). The HeiferPlusTM treatment induced a significant (p<0.05) reduction of: sperm viability, basal ROS level and mitochondrial function, while sperm chromatin stability, measured as %-DNA fragmentation index (DFI), mean DFI and SD-DFI was not affected by the treatment. Concerning acrosome reaction, it was already observed a significant increase (p<0.05) of reacted live spermatozoa after 1,5 h incubation in either the presence or absence of Ca2+ in the semen treated with HeiferPlusTM compared to 0 h, while in the untreated semen the increase of reacted live spermatozoa was observed only after 3h incubation in presence of Ca2+. The treatment seems to affect some functional parameter of bull spermatozoa and to anticipate acrosome reaction.

The gut is a possible target toward mycotoxin fumonisins (FBs) exposure. The study aims to investigatethe effects induced by FBs contaminated-corn chyme samples on functional parameters of human and ratintestine by using Ussing chamber. Fumonisins-contaminated corn and processed corn samples wereundergone to in vitro digestion process and then added to luminal side. A reduction (about 90%) of shortcircuit current (Isc lA/cm2) during exposure of human colon tissues to fumonisins-free corn chyme sampleswas observed, probably related to increased chyme osmolality. This hyperosmotic stress could drainwater towards the luminal compartment, modifying Na+ and Cl transports. The presence of FBs in cornchyme samples, independently to their concentration, did not affect significantly the Isc, probably relatedto their interference towards epithelial Na+ transport, as assessed by using a specific inhibitor (Amiloride).The rat colon tract represents a more accessible model to study FBs toxicity showing a similar functionalresponse to human. In the rat small intestine a significant reduction (about 15%) of Isc parameterduring exposure to uncontaminated or FBs contaminated corn chyme samples was observed; thereforesuch model was not suitable to assess the FBs toxicity, probably because the prevalent glucose and aminoacids electrogenic absorption overwhelmed the FBs influence on ionic transport.

Food industries are increasingly oriented towardnew foods to improve nutritional status and/or to combat nutritionaldeficiency diseases. In this context, siliconbiofortification could be an innovative tool for obtainingnew foods with possible positive effects on bone mineralization.In this paper, an alternative and quick in vitro approachwas applied in order to evaluate the potential healthpromotingeffects of five silicon-biofortified leafy vegetables(tatsoi, mizuna, purslane, Swiss chard and chicory) on bonemineralization compared with a commercial silicon supplement.The silicon bioaccessibility and bioavailability of thefive leafy vegetables (biofortified or not) and of the supplementwere assessed by applying a protocol consisting ofin vitro gastrointestinal digestion coupled with a Caco-2 cellmodel. Silicon bioaccessibility ranged from 0.89 to 8.18 mg/Land bioavailability ranged from 111 to 206 ?g/L of Si for bothvegetables and supplement. Furthermore, the bioavailablefractions were tested on a human osteoblast cell model following the expression of type 1 collagen and alkaline phosphatase.The results obtained highlighted that the bioavailablefraction of biofortified purslane and Swiss chard improved theexpression of both osteoblast markers compared with the supplementand other vegetables. These results underline the potentiallybeneficial effect of biofortified leafy vegetables andalso indicate the usefulness of in vitro approaches for selectingthe best vegetable with positive bone effects for further in vivoresearch.

Le Fumonisine (FBs) sono micotossine prodotte da funghi del genere Fusarium che contaminano mais e suoi derivati. La Fumonisina B1 (FB1) nell'uomo è correlata a cancro esofageo e difetti del tubo neurale e classificata da IARC come possibile cancerogeno (IARC 2003). La FB1 inibisce il metabolismo degli sfingolipidi competendo con le basi sfingoidi libere (sfinganina e sfingosina) e inducendo l'inibizione dell'enzima ceramide sintetasi.Scopo dello studio è stato valutare la stabilità delle FBs dopo un processo di digestione in vitro e l'influenza del chimo prodotto sulla funzionalità intestinale, utilizzando modelli ex vivo (colon umano e intestino di ratto) mediante la camera di Ussing. Campioni di mais a diverso livello di contaminazione di FBs, erano digeriti in vitro, mimando composizione fisiologica e pH dei fluidi intestinali nelle fasi della digestione orale, gastrica e intestinale, e l'esposizione naturale dell'intestino alle micotossine(Versantvoort et al. 2005).I livelli di FBs nel chimo ottenuto con digestione in vitro, determinati mediante LC-HRMS, hanno mostrato un'elevata stabilità delle FBs (> 60%). Inoltre, durante l'esposizione dei campioni di colon umano e di ratto al chimo di mais, è stata osservata una differente efficienza dei trasporti ionici in relazione alla presenza/assenza di FBs nel chimo. L'analisi dei parametri elettrici, dopo esposizione per 120 min. a campioni di mais non contaminato, mostrava la riduzione (fino al 90%) della corrente di cortocircuito (indice del trasporto ionico di membrana), variazione probabilmente legata all'elevata osmolalità del chimo e conseguente attivazione dei trasporti ionici. Al contrario, la presenza delle FBs, indipendentemente dalla concentrazione, non ha modificato in ambedue i modelli intestinali tale parametro. Le micotossine, presumibilmente hanno interferito con il trasporto del Na+, inibendo la risposta allo stress osmotico indotta dalla matrice (Minervini et al. 2014a). L'influenza delle FBs sul trasporto ionico intestinale potrebbe essere correlata ad alterazioni strutturali della membrana plasmatica. E' stato descritto che l'esposizione alla FB1 può indurre l'alterazione del bilancio dei lipidi di membrana e delle interazioni proteine-lipidi cruciali per la funzione delle proteine di membrana nel loro ruolo di trasporto o recettoriale (Devi Paila et al. 2010). Tale effetto delle FBs sulla membrana plasmatica è stato dimostrato da nostri studi in vitro sulla linea intestinale HT29. Infatti la FB1, inibendo il metabolismo sfingolipidico, ha indotto un aumento della microviscosità, e quindi della permeabilità della membrana, e un aumento della perossidazione lipidica (Minervini et al 2014b) secondaria all'interazione della FB1 con il doppio strato lipidico e alla formazione di radicali liberi (Yin et al. 1998). In conclusione l'esposizione a chimo di mais contaminato da FBs ha indotto modificazioni del trasporto ionico sia nel colon umano che di ratto. Questo è il primo st

Ochratoxin A (OTA) exposure during pregnancy in laboratory animals induces delayed/abnormal embryodevelopment. Foetal adnexa-derived mesenchymal stem cells (MSCs) could help evaluate the devel-opmental risk of exposure to chemicals in advanced gestational age. We tested the effects of OTA atconcentrations ranging from 2.5 × 10-4to 25 nM on growth parameters of canine umbilical cord matrix(UCM)-derived MSCs. The hypothesis that oxidative chromatin and DNA damage could underlie OTA-mediated cell toxicity was also investigated. After in vitro exposure, OTA significantly decreased celldensity and increased doubling time in a passage- and concentration-dependent manner and no exposedcells survived beyond passage 5. Significantly higher rates of cells showed condensed and fragmentedchromatin and oxidized DNA, as assessed by OxyDNA assay. These findings showed that in vitro exposureto OTA, at picomolar levels, perturbs UCM-MSC growth parameters through oxidative chromatin andDNA damage, suggesting possible consequences on canine foetal development.

L'ocratossina A (OTA) è una nefrotossina prodotta da funghi del genere Aspergillus e Penicillium con nota attività cancerogena, genotossica e teratogena. Riduzione della funzionalità riproduttiva è stata riportata nel suino (Birò et al 2003), ma non ci sono informazioni sui livelli di esposizione e sulla tossicità riproduttiva nell'equino. Scopo del lavoro è stato di valutare l'esposizione naturale degli equini all'OTA e il passaggio placentale, quantificando OTA nel siero raccolto da stalloni, cavalle cicliche e gravide e dal cordone ombelicale di puledri alla nascita. Inoltre, dopo esposizione in vitro di spermatozoi equini a livelli di OTA compresi tra 750 nM a 24 µM, si è valutata la vitalità (con il SYBR14/ioduro di propidio), la stabilità cromatinica utilizzando il test SCSA (con l'arancio di acridina), la produzione di ROS (utilizzando il 2'7'diclorofluorescin-diacetato) tramite analisi citofluorimetriche e la motilità spermatica tramite il sistema CASA (Giannoccaro et al 2010; Minervini et al 2010). Sono stati esaminati campioni di seme prelevati nella stagione riproduttiva (Aprile-Luglio) e non riproduttiva (Novembre-Gennaio). Le analisi citofluorimetriche sono state condotte dopo 2 ore (vitalità e SCSA) e dopo 30 min (ROS) di esposizione all'OTA. La motilità è stata analizzata dopo 30 min. L'OTA è stata ritrovata nell'83% dei campioni di siero di stalloni (valore mediano=121.4 pg/ml). In 14 campioni di siero su 17 raccolti da cavalle gravide, l'OTA è stata riscontrata con un livello simile (livello mediano=106.6 pg/ml), mentre è stata riscontrata solo nel 50% dei campioni serici prelevati dal cordone ombelicale, con un livello mediano pari a 96.6 pg/ml. La tossicità dell'OTA, dopo esposizione in vitro, è stata ritrovata solo in spermatozoi raccolti durante la stagione non riproduttiva. In entrambe le stagioni esaminate, la vitalità spermatica e la stabilità cromatinica non sono state influenzate da tutte le concentrazioni testate. Al contrario l'OTA ha indotto un significativo effetto pro-ossidante (evidenziato come aumento dei livelli di ROS) dopo esposizione in vitro a concentrazioni da 12 a 24 µM. Inoltre, un significativo aumento della motilità totale è stato registrato dopo esposizione per 30 minuti alle stesse concentrazioni. Gli equini sono risultati esposti a bassi livelli di OTA la cui presenza potrebbe rappresentare un rischio per l'efficienza riproduttiva di tale specie, dovuto ad un possibile accumulo nel plasma seminale e tossicità sui parametri spermatici, come riscontrato anche nel suino (Birò et al 2003). L'esposizione in vitro per brevi tempi all'OTA ha determinato un aumento dei livelli di ROS e di motilità solo negli spermatozoi equini raccolti nella stagione non riproduttiva. Questa maggiore sensibilità degli spermatozoi prelevati nella stagione non riproduttiva potrebbe essere dovuta a modificazioni morfologiche e funzionali presenti nelle due stagioni del cavallo conseguenti a fluttuazioni ormonali stagional

In this study, the quali-quantitative composition of hydrophilic (phenolic acids)and lipophilic (isoprenoids) extracts from whole-meal flour of five elite Italian durum wheatcultivars was determined. Significant differences in the content of bioactive compoundswere observed among the wheat extracts, in particular concerning the content of boundphenolic acids, lutein and ?-tocotrienols. The cultivars Duilio and Svevo showed the highestamount of phenolic acids and isoprenoids, respectively. Extracts were evaluated for theiranti-inflammatory activity on HT-29 human colon cells by measuring the levels ofinterleukin 8 (IL-8) and transforming growth factor ?1 (TGF-?1). Durum wheat extractssignificantly inhibited the secretion of the pro-inflammatory IL-8 mediator at 66 ?g/mLof phenolic acids and at 0.2 ?g/mL of isoprenoids. Conversely, the secretion of theanti-inflammatory mediator TGF-?1 was not modified by neither hydrophilic nor lipophilicextracts. These results provide further insight into the potential of durum wheat on humanhealth suggesting the significance of varieties with elevated contents of bioactive components.

Artichoke is a rich source of health promoting compounds such as polyphenols, important for their pharmaceutical and nutritional properties. In this study, the potential for bioavailability of the artichoke polyphenols was estimated by using both in vitro digestion and Caco-2 human intestinal cell models. In vitro digestive recoveries (bio-accessibility) were found to be 55.8% for total artichoke phenolics and in particular, 70.0% for chlorogenic acid, 41.3% for 3,5-O-dicaffeoylquinic acid, and 50.3% for 1,5-O-dicaffeoylquinic acid, highlighting potential sensitivity of these compounds to gastric and small intestinal digestive conditions. Uptake of artichoke polyphenols was rapid with peak accumulation occurring after 30 min with an efficiency of 0.16%, according to the poor uptake of dietary polyphenols. Some compounds, such as coumaric acid, caffeic acid and caffeic acid derivatives, were also detected in the basolateral side assuming an extra and intracellular esterases activities on chlorogenic acid. Only apigenin-7-O-glucoside was absorbed and transported through the Caco-2 monolayer demonstrating its bioavailability in the extent of 1.15% at 60 min. In addition, permeability coefficient (Papp=2.29 x 10-5 cm/sec), involving apical to basolateral transport of apigenin 7-O-glucoside, was calculated to facilitate estimation of absorption and transport through Caco-2 monolayer. Finally, the mono and dicaffeoylquinic acids present in artichoke heads, exert an antioxidant activity on human low density lipoprotein system correlated to their chemical structure. In conclusion, the utilized in vitro models, although not fully responding to the morphological and physiological features of human in vivo conditions, could be a useful tool for investigating mechanistic effects of polyphenols released from food matrix.

Artichoke (Cynara cardunculus L. subsp. scolymus Hayek) is an important component of the Mediterranean diet and a good source of health-promoting compounds well-known antioxidant and anti-inflammatory molecules, such as polyphenols(1). Recent evidences indicated that probiotic strains play an important role in modulating activities in the gut-associated lymphoid tissue. In this study the beneficial effects of artichoke was modulated by adding probiotic cells during in vitro digestion process. The artichoke intestinal digesta (containing a range from 0.033 to 67.13 ppm of total polyphenols), in presence or absence of the probiotic Lactobacillus paracasei strain LMG P-22043, was evaluated for antioxidant activity on human intestinal cell line (HT-29) using DCF-DA probe. Although the recovered bioaccessibility of total polyphenols did not change in the presence of L. paracasei (71± 17.6 % with respect to 66 ± 14.4%), the antioxidant effect was significantly (p<0.05) improved by the presence of probiotic cells (IC50 value= 1.0 ± 0.7 ppm of total polyphenols) respect to the digested artichoke (IC50= 2.7 ± 0.7ppm). These preliminary results indicate a possible interactive action between the strain and the active components of artichoke toward antioxidant activity in intestinal cells and suggest to further investigate similar interaction toward inflammatory mediators, considering the anti-inflammatory properties evidenced for other strains belonging to L. pacarasei (2).

Verbascoside (VB) is a bioactive polyphenol from olive oil mill wastewater with known antioxidant activity. Oxidative stress is an emerging problem in assisted reproductive technology (ART). Juvenile ART is a promising topic because, in farm animals, it reduces the generation gap and, in human reproductive medicine, it helps to overcome premature ovarian failure. The aim of this study was to test the effects of VB on the developmental competence of ovine prepubertal oocytes and the bioenergetic/oxidative stress status of fresh and vitrified oocytes. In fresh oocytes, VB exerted prooxidant short-term effects, that is, catalase activity increase and uncoupled increases of mitochondria and reactive oxygen species (ROS) fluorescence signals, and long-term effects, that is, reduced blastocyst formation rate. In vitrified oocytes, VB increased ROS levels. Prooxidant VB effects in ovine prepubertal oocytes could be related to higher VB accumulation, which was found as almost one thousand times higher than that reported in other cell systems in previous studies. Also, long exposure times of oocytes to VB, throughout the duration of in vitro maturation culture, may have contributed to significant increase of oocyte oxidation. Further studies are needed to identify lower concentrations and/or shorter exposure times to figure out VB antioxidant effects in juvenile ARTs.

An integrated device for real-time monitoring of glucose and phenols absorption, that consists of a sensors/biosensors system (SB) and a Caco-2TC7 human intestinal cell culture, is described in this study. The SB is composed of a glucose oxidase-based biosensor, a sentinel platinum sensor, a laccase/tyrosinase-based biosensor and a sentinel carbon sensor, all located in the basolateral compartment (BC) of a cell culture plate. Caco-2TC7 cells, differentiated on culture inserts, separated the apical compartment that simulates the intestinal lumen, from the BC which represented the bloodstream. The system recorded currents relative to glucose (1mM) absorption, obtaining bioavailability values (5.1%) comparable to HPLC analysis (4.8%). Phloridzin and phloretin, specific phenolic inhibitors of SGLT1 and GLUT2 glucose transporters, reduced the glucose transport of almost 10 times. They were minimally absorbed in the BC with a bioavailability of 0.13% and 0.49% respectively. The hypoglycemic potential of blueberry and pomegranate juices was also studied. In particular, the amount of glucose absorbed through the Caco-2TC7 monolayer was 8? for pomegranate and 1.7? for blueberry, demonstrating the potential hypoglycemic effect of the juices. Polyphenols absorption was also monitored by the SB and an increase was recorded during the first 50min in presence of both blueberry and pomegranate juices, then a constant decrease occurred. The proposed device has been developed as innovative tool for the dynamic monitoring of natural compounds effects on glucose absorption, in order to manage postprandial hyperglycemia.

Olive oil processing industries generate substantial quantities of phenolic-rich by-products, which could be valuable natural sources of antioxidants. This work is focused on the recovery and structural characterisation of antioxidant compounds from olive mill wastewater (OMWW), a polluting by-product of the olive oil production process. Phenolics were extracted from the waste material using a membrane technology coupled to a low-pressure gel filtration chromatography on a Sephadex LH-20. The LH-20 fraction was, in turn, characterized for its phenolic composition by HPLC-DAD-MS/MS analyses. Verbascoside, isoverbascoside, ?-hydroxyverbascoside, ?-hydroxyisoverbascoside, and various oxidized phenolics were identified. Uptake of verbascoside, purified from the LH-20 fraction, by HT-29 human colon carcinoma cell lines, an established model system for studying drug transport properties, was also assayed.

Fruits and vegetables contain a number of components (polyphenols, carotenoids, vitamins, folate, minerals such as magnesium, calcium, silicon and other compounds) that could affect the bone health. Considering the biological activities induced by silicon (antioxidant activity, increased mineralization of bone, development of connective tissue), this study aims to compare the biological activity induced by the silicon biofortificated and unbiofortificated vegetables (tatsoi, mizuna, purslane, Swiss chard and chicory) respect to the commercial silicon supplement. In addition, the assessment of silicon bioaccessibility, bioavailability and biological activity in human intestinal cell line (Caco-2) and human osteoblasts (hOBs) were performed. After in vitro gastrointestinal digestion of plant edible leaves and the silicon supplementation, the silicon bioaccessibility ranged from 7.01 to 13.22 mg/L. On Caco-2 cell line, the antioxidant activity of the bioaccessible fraction was assessed as reduction of induced ROS levels and ranged from 25 to 44% respect to stimulated control. The silicon biofortification significantly (p<0.001) improved the antioxidant activity induced by the vegetables. The bioavailability of digested samples, evaluated on Caco-2 differentiated cell line, ranged from 237 to 440 µg/L with percentages ranged from 27% to 61% in relation to the considered vegetable species. The expression of collagen type 1 and alkaline phosphatase induced by the bioavailable fraction after 12 and 36 h of treatment was evaluated in hOBs by real-time PCR. The bioavailable fraction of tatsoi, purslane and Swiss chard (biofortificated leafy vegetables), improved (after 36 hours of incubation) the expression of collagen type 1 and alkaline phosphatase with respect to the silicon supplement and unbiofortifated vegetables in hOBs.

Verbascoside (VB) is a phenolic antioxidant present in different plants, but, as reported for other polyphenols, could be unstable at different pH conditions. The aim of this work was to assess how chemical modification of VB, occurring during incubation at pH values similar to gastric (pH 3) and intestinal (pH 7) conditions, may impact antioxidant activity in two human intestinal cells lines. Quali-quantitative approaches were performed in order to evaluate the specific interactions between VB and its derivatives. HPLC analysis was performed to assess possible VB transformation into derivative products. The antioxidant activity of a mixture of VB, Isoverbascoside (IsoVB) and oxidized products (obtained after incubation at pH 7), or purified VB and IsoVB, was assessed in two human intestinal cell lines (HT-29 and Caco-2) using a DCFH-DA probe. VB was stable at pH 3 with a recovery of ~100% after 24 hrs. In contrast, VB was unstable at pH 7, with a loss of 62.4%, transforming into IsoVB and other oxidative products. Cellular antioxidant assays found that the mixture of oxidized VB products resulted less active (EC50 ranging from 2.7 to 3.4 ?M) than VB (EC50 ranging from 0.24 to 0.29 ?M), IsoVB (EC50 ranging from 0.85 to 1.4 ?M), and VB+IsoVB (EC50 ranging from 0.12 to 0.21 ?M). Cellular uptake of IsoVB was found to differ between cell lines, with higher uptake by Caco-2 compared to HT-29. Both graphical and mathematical methods identified different interactions between VB and IsoVB on the two cell lines. In conclusion, theantioxidant activity of VB can be modified by the conditions present in intestinal environment.

The effects of verbascoside (VB), added at nanomolar concentrations during in vitro maturation (IVM) of juvenile sheep oocytes, on in vitro embryo development and its mechanisms of action at the oocyte level were analyzed. Developmental rates, after IVM in the presence/absence of VB (1nM for 24h; 1nM for 2h; 10nM for 2h), were evaluated. The bioenergetic/oxidative status of oocytes matured after IVM in the presence/absence of 1nM VB for 24h was assessed by confocal analysis of mitochondria and reactive oxygen species (ROS), lipid peroxidation (LPO) assay, and quantitative PCR of bioenergy/redox-related genes. The addition of 1nM VB during 24h IVM significantly increased blastocyst formation and quality. Verbascoside reduced oocyte ROS and LPO and increased mitochondria/ROS colocalization while keeping mitochondria activity and gene expression unchanged. In conclusion, supplementation with nanomolar concentrations of VB during IVM, in the juvenile sheep model, promotes embryo development by protecting the oocyte against oxidative stress.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interferencewith the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicitywas reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitiveanimal species, has been reported. The aim of the present study was to assess the in vitro toxicity ofFB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability(SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm motility by CASA system.Fumonisin B1 did not affect viability of fresh spermatozoa after 2 h exposure up to 25 lM. Damageon sperm chromatin structure was observed only in one frozen sample after exposure up to2.5  105 lM FB1 without associated increase of ROS. Increase of ROS, at FB1 levels up to2.5  104 lM, was found on another frozen-thawed sperm sample, may be as a consequence of seminalplasma removal. At 7.5 and 15 lM, FB1 induced reduction of total and progressive motility.

Ochratoxin A (OTA) is one of the most frequently occurring mycotoxins in several food products and, under experimental conditions, it causes a wide spectrum of toxic effects, including teratogenicity, carcinogenicity, nephrotoxicity, and immunotoxicity. In laboratory animals, the exposure to OTA during pregnancy may interfere with embryonic development and may induce embryo lethality, growth delay and teratogenic effects. An alternative approach to in vitro predict the developmental risk of chemicals has been offered by mesenchymal stem cells (MSCs) isolated from fetal adnexa. The aim of this work is to study OTA effects at nanomolar concentrations, corresponding to levels detected in human placenta, on proliferation of canine umbilical cord matrix MSCs (UCM-MSCs). Cells were plated in 12-well dishes to perform doubling time (DT) analysis, cellular density and viability from passage (P) 1 to P7, by using Trypan blue dye exclusion test. OTA was added at concentrations ranging from 0.01ng/ml to 10ng/ml. Two experimental protocols of OTA treatment were performed: 1) continue exposure model; 2) alternate exposure model, in which cultures were performed for 3 days in standard medium followed by 4 days in presence of OTA. Control cultures received the same amount of OTA solvent (DMSO). After OTA exposure, cell proliferation was observed until P3, in contrast to control samples in which cells proliferated until P7. In both exposure models, DT values were higher than those found in control samples. In particular, lowest OTA concentrations (0.01-0.1ng/ml) significantly increased (P<0.0001) DT values compared with controls in both exposure models, with consequent inhibition of cell proliferation. No differences were observed between continue and alternate exposure models, leading to suppose that the cell damage early occurs and cannot be reversed independently of exposure model. At all passages, OTA treatment did not influence cell viability. In addition, changes in osteo- and neuron-like morphology were observed in treated cells at P3, in contrast with controls that maintained fibroblast-like morphology. The action mechanism of OTA on cell proliferation, at nanomolar concentrations, is not clear. It has been reported in renal epithelial cells that OTA may interact with cellular key targets, such as mitochondria, and interfere with Ca2+, pH and energy homeostasis, leading to the disturbance of cell signaling/proliferation and apoptosis. Moreover, OTA, in renal epithelial cells, may activate MAP-kinase, which cause cell dedifferentiation and transformation: this evidence could explain the morphological changes observed also in UCM-MSCs. In conclusion, OTA, at nanomolar concentrations, modulates physiological signals influencing cell growth, self-renewal and differentiation in UCM-MSCs as it may occur during embryo development.

The gastrointestinal tract is the main target ofexposure to mycotoxin fumonisin B1 (FB1), common naturalcontaminant in food. Previous studies reported thatproliferating cells are more sensitive than confluent cells tothe toxic effect of FB1. This study aims to investigate, bydose- and time-dependent experiments on human colonproliferating intestinal cell line (HT-29), the modificationsinduced by FB1 at concentrations ranging from 0.25 to69 lM. The choice of highest FB1 concentration consideredthe low toxicity previously reported on intestinal celllines, whereas the lowest one corresponded to the lowerFBs levels permitted by European Commission Regulation.Different functional parameters were tested such as cellproliferation, oxidative status, immunomodulatory effectand changes in membrane microviscosity. In addition FB1-FITC localization in this cell line was assessed by usingconfocal laser scanning microscopy. Lipid peroxidationinduction was the main and early (12 h) effect induced byFB1 at concentrations ranging from 0.5 to 69 lM, followedby inhibition of cell proliferation (up to 8.6 lM), theimmunomodulatory effect (up to 17.2 lM), by assessingIL-8 secretion, and increase in membrane microviscosity(up to 34.5 lM). The toxic effects observed in differentfunctional parameters were not dose-dependent and couldbe the consequence of the FB1 intracytoplasmatic localizationas confirmed by confocal microscopy results. Thedifferent timescales and concentrations active of differentfunctional parameters could suggest different cellular targetsof FB1.

Fusarium is a genus frequently contaminating different agricultural products and is also a renown producer of Trichothecenes1. This group of mycotoxins can be further subdivided into subgroups with T-2/HT-2 toxins and deoxynivalenol being the main representatives of the type A and B respectively. In general Trichothecenes are responsible for a wide variety of toxic effects in humans and animals. Within the type class A, in particular, T-2 and HT-2 toxins represent the most acutely toxic compounds2 and have been lately reviewed by EFSA3. In human health risk assessment, ingestion of contaminated food is considered the major route of human exposure to these compounds although only a smaller amount will be further available for absorption during its transit along the gut4. In vitro digestion models have been used to evaluate mycotoxins bioaccessibility during the intestinal transit. As a result, the bioaccessibility can be calculated representing a measure for the assessment of mycotoxin bioavailability in food. Some investigations have been already carried out in the past by our group in order to assess T-2 and HT-2 bioaccessibility in bread chosen as reference food model5 In this note we describe a study aimed to investigate toxicity of gastro-duodenal digests of mycotoxins contaminated bread samples collected along the digestion time-course, assayed on RPMI lymphoid B cell line, as a sensitive cellular model. In parallel, a chemical quantification of T-2 and HT-2 toxins contaminating the bread digests was accomplished along the gastric and duodenal phase. The digestive fluids undergoing chemical and toxicological analysis, were collected at the beginning and end of the gastric phase, and after completion of the duodenal phase Results proved that a correlation between HT-2 content and toxicity did exist in digestive fluids although a more persistent toxic activity was noticed in the later stage of the duodenal phase.

Fifty-four spore-forming bacterial strains isolated from bread ingredients and bread, mainly belonging to the genus Bacillus (including Bacillus cereus), together with 11 reference strains were investigated to evaluate their cytotoxic potential and heat survival in order to ascertain if they could represent a risk for consumer health. Therefore, we performed a screening test of cytotoxic activity on HT-29 cells using bacterial culture filtrates after growing bacterial cells in Brain Heart Infusion medium and in the bread-based medium Bread Extract Broth (BEB). Moreover, immunoassays and PCR analyses, specifically targeting already known toxins and related genes of B. cereus, as well as a heat spore inactivation assay were carried out. Despite of strain variability, the results clearly demonstrated a high cytotoxic activity of B. cereus strains, even if for most of them it was significantly lower in BEB medium. Cytotoxic activity was also detected in 30% of strains belonging to species different from B. cereus, although, with a few exceptions (e.g. Bacillus simplex N58.2), it was low or very low. PCR analyses detected the presence of genes involved in the production of NHE, HBL or CytK toxins in B. cereus strains, while genes responsible for cereulide production were not detected. Production of NHE and HBL toxins was also confirmed by specific immunoassays only for B. cereus strains even if PCR analyses revealed the presence of related toxin genes also in some strains of other species. Viable spore count was ascertained after a heat treatment simulating the bread cooking process. Results indicated that B. amyloliquefaciens strains almost completely survived the heat treatment showing less than 2 log-cycle reductions similarly to two strains of B. cereus group III and single strains belonging to Bacillus subtilis, Bacillus mojavensis and Paenibacillus spp. Importantly, spores from strains of the B. cereus group IV exhibited a thermal resistance markedly lower than B. cereus group III with high values of log-cycle reductions. In conclusion, our results indicate that spore-forming bacteria contaminating bread ingredients and bread could represent a source of concern for consumer health related to the presence of strains, such as strains of B. cereus group III and single strains of other species, showing the ability to produce toxic substances associated to a thermal resistance enough to survive the bread cooking conditions.

Olive oil processing industries generate substantial quantities of phenolic-rich byproducts, which could be valuable natural sources of antioxidants. This work is focused on the recovery and structural characterization of antioxidant compounds from olive mill wastewater (OMWW), a polluting byproduct of the olive oil production process. Phenolics were extracted from the waste material using a membrane technology coupled to low-pressure gel filtration chromatography on a Sephadex LH-20.The LH-20 fraction was, in turn, characterized for its phenolic composition by HPLC-DAD-MS/MS analyses. Verbascoside, isoverbascoside, ?-hydroxyverbascoside, ?-hydroxyisoverbascoside, and various oxidized phenolics were identified. Uptake of verbascoside, purified from the LH-20 fraction, by HT-29 cells, an established model system for studying drug transport properties, was also assayed. Finally, the antioxidant activities of the LH-20 fraction and verbascoside were characterized by two different techniques. Individual verbascoside was more active as a scavenger of reactive oxygen species and as a chemopreventive agent protecting low-density lipoproteins from oxidative damage than the LH-20 fraction.

Verbascoside (VB), a bioactive polyphenol from olive mill wastewater with known antioxidant activity, was shown to act as a pro-oxidant molecule, by impairing energy/redox status and embryo developmental competence of prepubertal ovine oocytes when added at micromolar concentrations in a continuative 24-h in vitro maturation (IVM) exposure protocol (1). The aim of the present study was to determine whether a lower (nanomolar) VB concentration and a shorter exposure time (2 v. 24h) during IVM may improve the maturation rates of prepubertal ovine oocytes and their subsequent embryonic development in vitro. Cumulus-oocyte complexes derived from the ovaries of slaughtered 1-mo-old prepubertal sheep oocytes underwent IVM in TCM 199 with 10% oestrus sheep serum, 0.1IUmL(-1) of FSH/LH, and 100M cysteamine, in 5% CO2 in air at 38.5°C for 24h. Based on our previous results (Dell'Aquila et al. 2014 Biomed. Res. Int. 2014, 878062), VB was added in the IVM medium at 1.03nM, and 2 incubation times (24 and 2h) were tested. In the 2-h exposure group, after 2h of exposure to VB, oocytes were washed and cultured up to 24h without VB. A group of oocytes were cultured in absence of VB, as controls. Matured oocytes were fertilized with frozen-thawed ram semen in SOF medium for 22h and zygotes were cultured in vitro for 8 days. Metaphase II (MII) cleavage and blastocyst rates were analysed by Chi-squared test. Embryo quality was evaluated by staining and total cell count of the blastocyst and analysis of variance (ANOVA) was applied. Differences were considered to be significant when P<0.05. Compared to controls, VB treatment at the concentration of 1.03nM and 24h of exposure had no effect on MII rates (196/268, 73% v. 226/323, 70% MII/cultured oocytes; P>0.05). However, this treatment allowed to obtain significantly higher rates of cleaved embryos/MII oocytes (156/196, 80% v. 165/226, 73%; respectively; P<0.05), blastocyst yield/cleaved embryos (59/156, 38% v. 45/165, 27%, respectively; P<0.05), and total blastocyst cell numbers (108.62±19.87 v. 89.61±26.32, respectively; P<0.05) compared to control oocytes. The VB treatment at the same concentration but for 2h induced only significantly higher cleavage rate (196/210, 93% v. 165/226, 73%; P<0.05). In conclusion, our results showed that VB treatment at 1.03nM during 24h of IVM exerted a positive effect on in vitro embryo development of prepubertal ovine oocytes by increasing the blastocyst yield and their quality. The hypothesis that VB at nanomolar concentrations may improve cumulus-oocyte energy/redox status is under investigation.