Murine colonization model by Campylobacter coli DSPV458.

AIMS: To generate a murine experimental model of colonization by Campylobacter coli DSPV458. METHODS AND RESULTS: Twelve adult Balb/cCmedc female mice were housed in a treated group (T-G) and a control group (C-G) for 4 weeks. Both experimental groups received antibiotics for 5 days during the first week. The T-G was administered with 6.68log10 CFU of C. coli DSPV458 by oesophageal gavage. Necropsies were performed weekly to evaluate translocation and intestinal colonization in the spleen and liver and in the ileum and cecum respectively. Samples were cultured to quantify intestinal microbiota members. Faeces were cultured weekly for a C. coli DSPV458 count. Campylobacter coli DSPV458 was isolated from all the inoculated mice. The recovered level of C. coli DSPV458 was, on average, 6.9 log10 CFUg-1 , 8.0 log10 CFUg-1 and 1.6 log10 CFUg-1 in faeces, cecum and ileum respectively. Colonization by C. coli DSPV458 does not alter the normal clinical and physiological status. CONCLUSIONS: Campylobacter coli DSPV458 does not have an invasive capacity, and the model is suitable for evaluating strategies to reduce intestinal loads. SIGNIFICANCE AND IMPACT OF STUDY: Farm animals have an important impact on thermotolerant Campylobacter transmission to humans. Extremely few colonization models by C. coli have been reported to date. In food-producing animals, infection is mild or absent and thermotolerant Campylobacter colonize the intestines of animals. Colonization models are specific models that do not cause infection as they do not generally result in diarrhoea or other signs of disease. Therefore, this model will allow to evaluate the evolution of colonization by thermotolerant Campylobacter and the alternative tools development to antibiotics that limit their colonization in food-producing animals.

Multi-mycotoxin occurrence in feed, metabolism and carry-over to animal-derived food products: A review.

The world requests for raw materials used in animal feed has been steadily rising in the last years driven by higher demands for livestock production. Mycotoxins are frequent toxic metabolites present in these raw materials. The exposure of farm animals to mycotoxins could result in undesirable residues in animal-derived food products. Thus, the potential ingestion of edible animal products (milk, meat and fish) contaminated with mycotoxins constitutes a public health concern, since they enter the food chain and may cause adverse effects upon human health. The present review summarizes the state-of-the-art on the occurrence of mycotoxins in feed, their metabolism and carry-over into animal source foodstuffs, focusing particularly on the last decade. Maximum levels (MLs) for various mycotoxins have been established for a number of raw feed materials and animal food products. Such values are sometimes exceeded, however. Aflatoxins (AFs), fumonisins (FBs), ochratoxin A (OTA), trichothecenes (TCs) and zearalenone (ZEN) are the most prevalent mycotoxins in animal feed, with aflatoxin M1 (AFM1) predominating in milk and dairy products, and OTA in meat by-products. The co-occurrence of mycotoxins in feed raw materials tends to be the rule rather than the exception, and the carry-over of mycotoxins from feed to animal source foods is more than proven.

Protective effect of Lactiplantibacillus plantarum LP5 in a murine model of colonisation by Campylobacter coli DSPV458.

Thermotolerant Campylobacter species are the leading cause of foodborne bacterial diarrheal disease worldwide. Campylobacter coli, abundant in pigs and pork products, have been identified as a source of human infection. In this study, we propose the use of Lactiplantibacillus plantarum LP5 as a probiotic to reduce colonisation of this intestinal pathogen in a murine colonisation model of C. coli DSPV458. Six-week-old adult female Balb/cCmedc mice were housed in groups: Control, Campy and Pro-Campy. Control and Pro-Campy groups received antibiotics for 5 days and the Campy group for 12 days. Pro-Campy group was inoculated for 7 days with 8.78 log10 cfu total of L. plantarum LP5 suspended in De Man, Rogosa and Sharpe broth. All groups were inoculated with 6.72 log10 cfu of C. coli DSPV458 suspended in brain heart infusion broth. L. plantarum LP5 was recovered only in the Pro- Campy group. C. coli DSPV458 was recovered at higher levels in the Control and Campy groups. The differences with the Pro-Campy group were significant. As regards faeces, Control and Campy groups reached 7.41 and 7.84 log10 cfu/g, respectively, and the Pro-Campy group only 4.62 log10 cfu/g. In the caecum, Control and Campy groups reached 8.01 and 9.26 log10cfu/g, respectively, and the Pro-Campy group only 4.51 log10 cfu/g. In the ileum, Control and Campy groups reached 3.43 and 3.26 log10 cfu/g, respectively, and the Pro-Campy group did not show detectable levels. The reduction of C. coli DSPV458 in the Pro-Campy group compared to the Control group in faeces, caecum and ileum was 99.55, 99.98 and 100%, respectively. Animals were maintained under normal health conditions, and haematological parameters were within the standard values for Balb/cCmedc. The incorporation of a probiotic generated a protective effect in the mice colonisation model. The protective effect would also apply to intestinal colonisation by indigenous enterobacteria. Therefore, the strategy used in this study is of great importance to understand the protection mechanisms in a murine model, as well as its application in food-producing animals.

In vitro screening and in vivo colonization pilot model of Lactobacillus plantarum LP5 and Campylobacter coli DSPV 458 in mice.

The objective of this work was to determine the antibacterial effect of Lactobacillus plantarum strains of pork origin against Campylobacter coli strains, and to conduct experimental colonization pilot models in mice for both microorganisms. Inhibition assays allowed evaluation and selection of L. plantarum LP5 as the strain with the highest antagonistic activity against C. coli and with the best potential to be used in in vivo study. Adult 6-week-old female Balb/cCmedc mice were lodged in two groups. The treated group was administered with 9.4 log10CFU/2 times/wk of L. plantarum LP5. L. plantarum LP5 was recovered from the feces and cecum of the inoculated mice. However, when bacteria stopped being administered, probiotic counts decreased. Experimental colonization with C. coli was carried out in five groups of mice. All animals were treated with antibiotics in their drinking water to weaken the indigenous microbiota and to allow colonization of C. coli. Four groups were administered once with different C. coli strains (DSPV458: 8.49 log10CFU; DSPV567: 8.09 log10CFU; DSPV570: 8.46 log10CFU; DSPV541: 8.86 log10CFU, respectively). After 8 h, mice inoculated with different C. coli strains were colonized because the pathogen was detected in their feces. L. plantarum LP5 tolerated the gastrointestinal conditions of murine model without generating adverse effects on the animals. C. coli DSPV458 colonized the mice without causing infection by lodging in their digestive tract, thus generating a reproducible colonization model. Both models combined could be used as protection murine models against pathogens to test alternative control tools to antibiotics.

Bioinformatic analysis of post-transmission viral readaptation in Argentine patients with acute HIV-1 infection.

During the acute phase of HIV-1 infection, a strong readaptation occurs in the viral population. Our objective was to analyze the post-transmission mutations associated with escape to the cytotoxic immune response and its relationship with the progression of the infection. In this study, a total of 17 patients were enrolled during acute/early primary HIV infection and 8 subjects that were the HIV positive partner resulting in 8 transmission pairs. Genotyping of the genetic polymorphisms of HLA class I A and B was performed using PCR-SSOP. Viral RNA extraction was from plasma. 570 single Gag-gene amplifications were obtained by limiting-dilution RT-PCR. Epitope prediction was performed with NetMHC CBS prediction server for the 19 HLA-A and B alleles. Cytotoxic response prediction was performed by using the IEDB Analysis Resource. From our results, we deduce that the transmitted CTL / gag escape frequency in the founder virus was at least double compared to the post-transmission events. Additionally, by means of an algorithm that combines these frequencies, we observed that the founder viruses better adapted to the HLA A / B alleles of the recipient could contribute to a greater progression of the infection. Our results suggest that there is a large adaptation of HIV-1 to the HLA A / B alleles prevalent in our population. However, despite this adaptive advantage, the virus needs to make "readjustments" through new escape and compensatory mutations. Interestingly, according to our results, this readaptation could have a role in the progression of the infection.

Yeast derivatives and wheat germ in the adult diet modulates fecundity in a tephritid pest.

Anastrepha fraterculus (Wiedemann), a pest of great economic importance in South America, needs urgently to be controlled by environmentally friendly methods such as the sterile insect technique for which mass rearing of insects is required. Because oogenesis takes place during the adult stage, mass-rearing facilities should provide the females a diet that maximizes egg production at the lowest cost. Accordingly, we investigated the effect of artificial protein sources in the adult diet (yeast derivatives of different cost but with similar amino acids profiles, and the addition of wheat germ) on fecundity. Additionally, we evaluated different ratios of yeast derivatives or wheat germ on ovary maturation, fecundity, and fertility as well as their association with the nutrient content of females. Females fed hydrolyzed yeast and yeast extract attained the highest fecundity level, and those fed brewer's yeast the lowest. Reducing the amount of hydrolyzed yeast, an expensive protein source, in the diet negatively affected fecundity and ovary maturation. Increasing the amount of brewer's yeast, a low-cost protein source, did not favor fecundity. The addition of wheat germ in the adult diet improved fecundity regardless of the yeast derivate considered. Percentage of egg hatch was not affected by the diet. Nutrient content of A. fraterculus females varied according to the adult diet provided and mating status. Our findings provide novel baseline information to understand the role of nutrition on reproductive performance of A. fraterculus females and are discussed in the context of resource allocation. They also provide valuable advances in the search for cost-effective adult diets at fruit fly mass rearing facilities.

Beauvericin and enniatin B effects on a human lymphoblastoid Jurkat T-cell model.

Several mycotoxins exert their effect on the immunological system; some are classified as immunotoxic. Jurkat T-cells were used to study toxic effects of beauvericin (BEA) and enniatin B (ENN B). Both are not legislated mycotoxins with increasing presence in feed and food. Concentrations studied were from 1 to 15 µM at 24, 48 and 72 h. Cell death by increasing the percentage of apoptotic/necrotic cells was: BEA > ENN B. IC50 values ranged from 3 to 7.5 µM for BEA. ENN B 15 µM decreased viability (21-29%). The percentage of apoptotic/necrotic cells was BEA > ENN B at 24 h but not at 48 h. Caspase-3&7 activation profile varied, although both mycotoxins increased this activation. No difference in ROS production for any mycotoxin was observed. Arrest in S phase for both mycotoxins was obtained. BEA increased the percentage of DNA in the tail (18% and 20%) with respect to the control, whereas not for ENN B. In summary, cytotoxicity of BEA involved mitochondrial alterations; while ENN B only at highest concentrations and time assayed. BEA had cell cycle disturbances and apoptotic and apoptotic/necrotic cells increased; for ENN B these were not evident. Different toxic responses in Jurkat T-cells may be involved in BEA and ENN B toxicity.

Predictors of Disordered Eating in Young Males.

Recent findings suggest that disordered eating (DE) symptomatology may be underestimated in the male population. The present study examined depressive symptomatology as a potential mediator of the relationships between body image dissatisfaction, strategies to change body weight and muscles, media pressure, and DE (emotional, restrained and emotional eating) in 260 male undergraduates who completed a self-reported questionnaire. Path analyses indicated that media influence and strategies to decrease body weight had direct positive effects on depressive symptomatology, which in turn predicted emotional eating. Media influence had a direct positive effect on emotional eating, whereas strategies to decrease body weight did not exhibit a direct effect on emotional eating. Therefore, the latter pathway was removed from the model. The link between media pressure, strategies to decrease body weight and emotional eating was partially mediated by depressive symptomatology. The present findings can inform the development and implementation of prevention and education programs for DE in schools and universities.

Reaction of zearalenone and α-zearalenol with allyl isothiocyanate, characterization of reaction products, their bioaccessibility and bioavailability in vitro.

This study investigates the reduction of zearalenone (ZEA) and α-zearalenol (α-ZOL) on a solution model using allyl isothiocyanate (AITC) and also determines the bioaccessibility and bioavailability of the reaction products isolated and identified by MS-LIT. Mycotoxin reductions were dose-dependent, and ZEA levels decreased more than α-ZOL, ranging from 0.2 to 96.9% and 0 to 89.5% respectively, with no difference (p⩽0.05) between pH 4 and 7. Overall, simulated gastric bioaccessibility was higher than duodenal bioaccessibility for both mycotoxins and mycotoxin-AITC conjugates, with duodenal fractions representing ⩾63.5% of the original concentration. Simulated bioavailability of reaction products (α-ZOL/ZEA-AITC) were lower than 42.13%, but significantly higher than the original mycotoxins. The cytotoxicity of α-ZOL and ZEA in Caco-2/TC7 cells was also evaluated, with toxic effects observed at higher levels than 75µM. Further studies should be performed to evaluate the toxicity and estrogenic effect of α-ZOL/ZEA-AITC.

A youth mentor-led nutritional intervention in urban recreation centers: a promising strategy for childhood obesity prevention in low-income neighborhoods.

B'More Healthy Community for Kids (BHCK) is an ongoing multi-level intervention to prevent childhood obesity in African-American low-income neighborhoods in Baltimore city, MD. Although previous nutrition interventions involving peer mentoring of youth have been successful, there is a lack of studies evaluating the influence of cross-age peers within interventions targeting youth. This article evaluates the implementation of the BHCK intervention in recreation centers, and describes lessons learned. Sixteen youth leaders delivered bi-weekly, interactive sessions to 10- to 14-y olds. Dose, fidelity and reach are assessed, as is qualitative information regarding what worked well during sessions. Dose is operationalized as the number of interactive sessions, and taste tests, giveaways and handouts per session; fidelity as the number of youth leaders participating in the entire intervention and per session and reach as the number of interactions with the target population. Based on a priori set values, number of interactive sessions was high, and number of taste tests, giveaways and handouts was moderate to high (dose). The number of participating youth leaders was also high (fidelity). Of the 14 planned sessions, the intervention was implemented with high/moderate reach. Data suggest that working with cross-age peers is a promising nutritional intervention for recreation centers.

Cytoprotective effect of resveratrol diastereomers in CHO-K1 cells exposed to beauvericin.

Beauvericin (BEA) causes cytotoxicity, lipid peroxidation and reactive oxygen species in CHO-K1 cells. Resveratrol (RSV) is a polyphenol with multiple biological properties, including antioxidant effects. RSV has two forms: trans and cis. The aims of this study were to determine the cytoprotective effect of trans-RSV and diastereomers mixtures (50:50 trans/cis-RSV and 70:30 trans/cis-RSV) incubated alone and in combination with BEA in ovarian (CHO-K1) cells. The results demonstrated that cell viability increases (from 9% to 77%) when they were exposed to low concentration of RSV. Moreover, when the cells were pre-treated with RSV and then exposed to BEA, a cytoprotective effect (from 25% to 76%) and a ROS production diminution (from 27% to 92%) were observed, with respect to cells exposed to BEA without previous RSV exposure. RSV pre-treatment decreased the MDA levels (from 15% to 37%) when it is compared with cells exposed only to BEA. Therefore, it can be concluded that RSV could reduce the toxicological risk produced by BEA when they are in combination.

Alternariol-induced cytotoxicity in Caco-2 cells. Protective effect of the phenolic fraction from virgin olive oil.

The extra virgin olive oil (EVOO) has been associated to antioxidant effects. The mycotoxin alternariol (AOH) can contaminate olives. The aims of this work were to determine the cytotoxic effects and reactive oxygen species (ROS) produced by AOH, tyrosol and oleuropein (two polyphenols of olive oil) and a real EVOO extract in Caco-2 cells. The MTT assay and the ROS production by the H2-DCFDA probe were used. Results demonstrated that AOH reduces cellular proliferation depending on concentration, whereas tyrosol and oleuropein did not (12.5-100 µM). The combination of AOH + oleuropein (50 µM) increased cell proliferation (24%) whereas, AOH + tyrosol decreased (47%) it. Besides, AOH increased ROS generation depending on time and concentration. Oleuropein + AOH decreased ROS production. However, 25 µM of tyrosol increased 1.2-fold the ROS production. Respect to the EVOO extract, cytoprotective effect (151%) was evidenced, even with the combination EVOO extract + AOH (15%-55% respect to cells exposed to AOH alone). ROS generation was significantly reduced compared to ROS generation produced by 25 µM of AOH alone. The phenolic antioxidant of EVOO decreases cytotoxicity and ROS production in Caco-2 cells exposed to AOH. Thus, polyphenols of EVOO could contribute to diminish the toxicological risk that mycotoxins can produce to humans.

Cytotoxic and interactive effects of zearalenone, alpha-zearalenol and beta-zearalenol and formation of metabolites in HepG2 cells / Citotoxicidad, formación de metabolitos e interacción entre zearalenona, alpha-zearalenol y beta-zearalenol en células HepG2

Zearalenone (ZEA) is a secondary metabolite of Fusarium fungi. ZEA is a non-steroidal estrogenic mycotoxin which is rapidly absorbed and metabolized to α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) in the liver; therefore mixtures of these mycotoxins may be simultaneously present in biological systems and cause human health risk. The objectives of this study were: a) to compare the cytotoxicity of ZEA, α-ZOL and β-ZOL alone or in combination on human hepatoma (HepG2) cells using the MTT assay after 24, 48 and 72h of exposure, and b) to evaluate the interactions of these mycotoxins mixtures in HepG2 cell lines by the isobologram analysis. The IC50 values obtained for individual mycotoxins range from 70.0 to >100.0 µM, from 20.6 to 26.0 µM and from 38.4 to >100.0 µM in HepG2 cells for ZEA, α-ZOL and β-ZOL, respectively. Isobologram analysis provides a combination index (CI) value to determine the type of interaction that occurs. The interactions of ZEA and its metabolites showed slightly synergism (CI from 0.34±0.10 to 0.69±0.22) followed by additive effect (CI from 0.97±0.20 to 2.61±2.15) and turned into antagonism (CI from 1.29±0.18 to 7.77±2.27). The concentration of ZEA and its metabolites was determined with liquid chromatography coupled to the mass spectrometer detector-linear ion trap (LC-MS-LIT). No conversion of ZEA in α-ZOL and β-ZOL was detected. However other degradation products were detected (AU)

La zearalenona (ZEA) es un metabolito secundario producido por hongos del género Fusarium. ZEA es una micotoxina estrogénica no esteroidea que se metaboliza rápidamente en el hígado a α-zearalenol (α-ZOL) y β-zearalenol (β-ZOL); por lo tanto, mezclas de estas micotoxinas pueden estar presentes simultáneamente en un sistema biológico y causar un riesgo para la salud humana. Los objetivos de este estudio fueron: a) comparar la citotoxicidad de la ZEA, α-ZOL y β-ZOL de forma individual y en combinación en células hepáticas humanas (HepG2) usando el ensayo MTT tras una exposición de 24, 48 y 72h y b) evaluar la interacción de las mezclas de estas micotoxinas en células HepG2 mediante el análisis de las isobolas. Los valores de IC50 obtenidos en células HepG2 con las micotoxinas individuales van desde 70,0 a >100,0 µM, de 20,6 a 26,0 µM y de 38,4 a >100 µM para ZEA, α-ZOL y β-ZOL, respectivamente. El método de las isobolas proporciona el índice de combinación (IC) con el que se determina el tipo de interacción que se produce entre las micotoxinas. La interacción entre la ZEA y sus metabolitos mostró un ligero sinergismo (CI de 0,34±0,10 a 0,69±0,22), seguido de un efecto aditivo (CI de 0,97±0,20 a 2,61±2,15) que se acabó en antagonismo (CI de 1,29±0,18 a 7,77±2,27) dependiendo de la concentración y tiempo de exposición. La concentración de ZEA y sus metabolitos se determinó con cromatografía líquida acoplada a espectrometría de masas con trampa de iones lineal (LC-MS-LIT). No se detectó ninguna conversión de ZEA en α-ZOL y β-ZOL. Sin embargo se detectaron otros productos de degradación (AU)

Efectos tóxicos de alternariol por ensayos in vitro: revisión / Toxic effects of alternariol by in vitro assays: a review

Las micotoxinas son metabolitos secundarios producidos por hongos del genero Aspergillus, Penicillium, Fusarium y Alternaria. Las micotoxinas más abundantes son aflatoxinas (Aspergillus), ocratoxina A (Penicillium), fumonisinas, zearalenona, deoxinivalenol (Fusarium) y alternariol (Alternaria). De las especies de Alternaria, A. alternata es la especie productora más importante de micotoxinas. Todas están consideradas como contaminantes tóxicos que están presentes en alimentos de consumo diario. Esta revisión se centra en estudios in vitro relacionados con la respuesta y citotoxicidad a la micotoxina de Alternaria, alternariol (AOH). Para ello, se ha realizado una búsqueda bibliográfica de los artículos de AOH disponible en bases de datos como: Pubmed, Scopus, Science Direct y Current Contents en los últimos catorce años. Así pues, el objetivo de la revisión bibliográfica es evaluar los efectos de AOH investigados mediante ensayos in vitro (AU)

Mycotoxins are secondary metabolites produced by genera fungus of: Aspergillus, Penicillium, Fusarium and Alternaria. The most frequent mycotoxins are: aflatoxins (Aspergillus), ochratoxin A (Penicillium), fumonisins, zearalenone, deoxynivalenol (Fusarium) and alternariol (Alternaria). Among all Alternaria spp, A. Alternata is the most producer mycotoxin. All mycotoxins are considered toxic contaminants present in food of daily consumption. This review is based on in vitro studies where response and toxicity in cells of Alternaria mycotoxin, alternariol (AOH) have been carried out. In this sense a bibliographic search of AOH papers available in on-line data bases such as: Pubmed, Scopus, Science Direct and Current Contents in the last fourteen years, have been collected. The main objective of this bibliographic review is to evaluate the AOH effects detected in in vitro assay (AU)

Disturbance of antioxidant capacity produced by beauvericin in CHO-K1 cells.

Glutathione (GSH) levels, glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) as antioxidant defense system were evaluated in CHO-K1 cells after beauvericin (BEA) exposure. The effect of N-acetyl-cysteine (NAC) pre-treatment was assessed. GSH levels significantly decrease 18% and 29% after 5 µM of BEA in fresh medium and NAC pre-treatment, respectively compared to their controls. The GPx activity increased significantly from 35% to 66% in fresh medium and 20% in NAC pre-treatment. GR activity decreased after 5 µM of BEA up to 43% and 53% in fresh medium and NAC pre-treatment, respectively. The GST activity increased in fresh medium (from 61% to 89%) and decreased (from 22% to 35%) after NAC pre-treatment. Comparing BEA exposure in fresh medium and NAC pre-treatment, GSH levels, GPx activity and GST activity increased 716%, 458% and 206%, respectively respect to fresh medium; conversely no changes were observed in GR activity. In addition, NAC is an effective scavenger of BEA. GSH and related enzymes play an antioxidant role in the defense system of CHO-K1 cells exposed to BEA.

Effect of ciprofloxacin antibiotic on the partial-nitritation process and bacterial community structure of a submerged biofilter.

A partial-nitritation bench-scale submerged biofilter was used for the treatment of synthetic wastewater containing a high concentration of ammonium in order to study the influence of the antibiotic ciprofloxacin on the partial-nitritation process and biodiversity of the bacterial community structure. The influence of ciprofloxacin was evaluated in four partial-nitritation bioreactors working in parallel, which received sterile synthetic wastewater amended with 350 ng/L of ciprofloxacin (Experiment 1), synthetic wastewater without ciprofloxacin (Experiment 2), synthetic wastewater amended with 100 ng/L of ciprofloxacin (Experiment 3) and synthetic wastewater amended with 350 ng/L of ciprofloxacin (Experiment 4). The concentration of 100 ng/L of antibiotics demonstrated that the partial-nitritation process, microbial biomass and bacterial structure generated by tag-pyrosequencing adapted progressively to the conditions in the bioreactor. However, high concentrations of ciprofloxacin (350 ng/L) induced a decay of the partial-nitritation process, while the total microbial biomass was increased. Within the same experiment, the bacterial community experienced sequential shifts with a clear reduction of the ammonium oxidation bacteria (AOB) and an evident increase of Commamonas sp., which have been previously reported to be ciprofloxacin-resistant. Our study suggests the need for careful monitoring of the concentration of antibiotics such as ciprofloxacin in partial-nitritation bioreactors, in order to choose and maintain the most appropriate conditions for the proper operation of the system.

Interaction effects of Fusarium enniatins (A, A1, B and B1) combinations on in vitro cytotoxicity of Caco-2 cells.

Foodstuff is usually contaminated by more than one mycotoxin, however toxicological data are lacking as regards the effects in combinations compared to their individual effect. This study investigated the in vitro effects of enniatins (ENs) A, A1, B and B1, alone and in combinations, on Caco-2 cells viability by MTT assay after 24h of exposure. Cells were treated with concentrations ranging from 0.9 to 15.0µM, individually and in combination of two, three and four mycotoxins. Dose-response curves were generated for each mycotoxin and the isobologram method was used to determine the interactive effects of tested mixtures. Tested ENs produced significant cytotoxic effects both individually and in combination in a dose-dependent manner. IC50 values obtained for all individually tested mycotoxins ranged from 1.3 to >15µM. In ENs combination tests, synergistic effect in Caco-2 viability are observed for EN B+EN A1, EN B1+EN A1 and EN A+EN A1+EN B (CI=0.33-0.52). All other combinations showed additive effect at medium and high affected fraction with exception of lower fraction affected and the EN B+EN B1 mixture that produced antagonistic effect (CI=1.76-10.36). The use of combination index-isobole method could help to better understand the potential interaction between co-occurring mycotoxins and may contribute to their risk assessment.

Reactive oxygen species involvement in apoptosis and mitochondrial damage in Caco-2 cells induced by enniatins A, A1, B and B1.

The cytotoxic effects, the generation of reactive oxygen species (ROS) and lipid peroxidation (LPO) as well as the cell cycle disruption, the induction of apoptosis and changes in mitochondrial membrane potential (ΔΨm) as a function of increasing time have been determined in human colorectal adenocarcinoma (Caco-2) cells after exposure to enniatins (ENs) A, A1, B and B1. IC50 values obtained by the MTT and Neutral Red assay, after 24, 48 and 72 h of exposure ranged from 0.5±0.1 to >15 µM. A significant increase (p≤0.05) in ROS generation and LPO production, as determined by the fluorescent probe H2-DCFDA and TBARS method respectively, was observed for all mycotoxins tested at 3.0 µM concentration. The highest increase in ROS generation (2.6 fold higher than control) and LPO production (111%, as compared to control) was observed with EN A. Cell cycle was significantly arrested at G2/M phase after 24 h of exposure to EN A, A1, B1, whereas after 72 h of exposure an arrest in S phase was observed almost for all mycotoxins tested. Moreover, after 24 and 48 h of exposure, ENs increased the early apoptotic cells, whereas after 72h of exposure necrosis was observed. In addition the loss of ΔΨm was produced on Caco-2 cells after ENs exposure. ENs A, A1, B and B1 cytotoxicity involved early ROS generation that induced LPO oxidative damage, apoptosis and necrosis via the mitochondrial pathway. ENs A, A1 and B1 induced DNA damage. However the same effects cannot be proposed for EN B. Further studies on the toxicological effects induced by ENs A, A1, B and B1 are needed.

Beauvericin-induced cytotoxicity via ROS production and mitochondrial damage in Caco-2 cells.

The cytotoxicity of beauvericin (BEA) on human colon adenocarcinoma (Caco-2) cells was studied as a function of time. Moreover, the oxidative damage and cell death endpoints were monitored after 24, 48 and 72 h. After BEA exposure, the IC50 values ranged from 1.9 ± 0.7 to 20.6 ± 6.9 µM. A decrease in reduced glutathione (GSH; 31%) levels, as well as an increase in oxidized glutathione (GSSG, 20%) was observed. In the presence of BEA, reactive oxygen species (ROS) level was highly increased at an early stage with the highest production of 2.0-fold higher than the control that was observed at 120 min. BEA induced cell death by mitochondria-dependent apoptotic process with loss of the mitochondrial membrane potential (ΔΨm; 9% compared to the control), increase in LPO level (from 120% to 207% compared to the control) and reduced G0/G1 phase, with an arrest in G2/M, in a dose and time-dependent manner. Cell proliferation, apoptosis and ΔΨm determined, were in a dose- time-dependent manner. Moreover, DNA damage was observed after 12.0 µM concentration. This study demonstrated that oxidative stress is one of the mechanism involved in BEA toxicity, moreover apoptosis induction and loss of ΔΨm contribute to its cytotoxicity in Caco-2 cells.

Cryopreservation of aoudad (Ammotragus lervia sahariensis) sperm obtained by transrectal ultrasound-guided massage of the accessory sex glands and electroejaculation.

This study examines (1) the effectiveness of transrectal, ultrasound-guided massage of the accessory sex glands (TUMASG) combined with electroejaculation for obtaining aoudad (Ammotragus lervia sahariensis) sperm samples for cryopreservation, and (2) the effectiveness of a Tris-citric acid-glucose-based medium (TCG; usually used for freezing ibex sperm) and a TES-Tris-glucose-based medium (TTG; typically used in the cryopreservation of mouflon sperm) as sperm extenders. After TUMASG, just one to three electrical pulses were required for ejaculation to occur in five of the six animals studied; one ejaculated after TUMASG alone. Transrectal, ultrasound-guided massage of the accessory sex glands would therefore appear to be useful in obtaining sperm samples from this species, requiring few subsequent electrical electroejaculation stimuli and sometimes none at all. After thawing, the membrane integrity (assessed by nigrosin-eosin staining) of sperm extended with TTG was greater than that of sperm extended with TCG (P < 0.05). The total percentage of sperm showing an intact acrosome, as assessed by fluorescein isothiocyanate-conjugated peanut (Arachis hypogea) agglutinin, was also higher in the TTG-extended sperm (P < 0.05), and the percentage of dead sperm with a damaged acrosome was lower (P < 0.05). No differences were seen between TCG and TTG in terms of apoptotic manifestations (DNA damage, caspase activity, mitochondrial membrane potential, and plasmalemma stability). Therefore, TTG appears to be a better extender than TCG for cryopreserving aoudad sperm.