Nolwenn Hymery

Natural Co-Occurrence of Mycotoxins in Foods and Feeds and Their in vitro Combined Toxicological Effects

Some foods and feeds are often contaminated by numerous mycotoxins, but most studies have focused on the occurrence and toxicology of a single mycotoxin. Regulations throughout the world do not consider the combined effects of mycotoxins. However, several surveys have reported the natural co-occurrence of mycotoxins from all over the world. Most of the published data has concerned the major mycotoxins aflatoxins (AFs), ochratoxin A (OTA), zearalenone (ZEA), fumonisins (FUM) and trichothecenes (TCTs), especially deoxynivalenol (DON). Concerning cereals and derived cereal product samples, among the 127 mycotoxin combinations described in the literature, AFs+FUM, DON+ZEA, AFs+OTA, and FUM+ZEA are the most observed. However, only a few studies specified the number of co-occurring mycotoxins with the percentage of the co-contaminated samples, as well as the main combinations found. Studies of mycotoxin combination toxicity showed antagonist, additive or synergic effects depending on the tested species, cell model or mixture, and were not necessarily time- or dose-dependent. This review summarizes the findings on mycotoxins and their co-occurrence in various foods and feeds from all over the world as well as in vitro experimental data on their combined toxicity.

Filamentous Fungi and Mycotoxins in Cheese: A Review

Important fungi growing on cheese include Penicillium, Aspergillus, Cladosporium, Geotrichum, Mucor, and Trichoderma. For some cheeses, such as Camembert, Roquefort, molds are intentionally added. However, some contaminating or technological fungal species have the potential to produce undesirable metabolites such as mycotoxins. The most hazardous mycotoxins found in cheese, ochratoxin A and aflatoxin M1, are produced by unwanted fungal species either via direct cheese contamination or indirect milk contamination (animal feed contamination), respectively. To date, no human food poisoning cases have been associated with contaminated cheese consumption. However, although some studies state that cheese is an unfavorable matrix for mycotoxin production; these metabolites are actually detected in cheeses at various concentrations. In this context, questions can be raised concerning mycotoxin production in cheese, the biotic and abiotic factors influencing their production, mycotoxin relative toxicity as well as the methods used for detection and quantification. This review emphasizes future challenges that need to be addressed by the scientific community, fungal culture manufacturers, and artisanal and industrial cheese producers.

Cytotoxicity and immunotoxicity of cyclopiazonic acid on human cells

In this study, in vitro cytotoxicity and immunotoxicity of the mycotoxin cyclopiazonic acid (CPA) was evaluated on human cells. To evaluate cytoxicity, several cellular targets were used (CD34+, monocytes, THP-1 and Caco-2). Monocytes were more sensitive to CPA than the THP-1 monocytic cell line after 48h of incubation in the tested conditions. Half maximal inhibitory concentration (IC50) were determined to be 8.5 × 10(-8) and 1.75 × 10(-7)M for monocytes and THP1, respectively, while IC50>1.25 × 10(-7)M was observed for Caco-2 and CD34+ cells. The CPA effect on macrophage differentiation was also examined at non-cytotoxic concentrations. The monocyte differentiation process was markedly disturbed in the presence of CPA. After 6 days of culture, CD71 expression was downregulated, while CD14 and CD11a expressions did not change. Moreover, activated macrophages showed a raised burst activity and TNF-α secretion. Overall, the results indicated that CPA exhibited toxicity on various human cellular models. Moreover, at non-cytotoxic concentrations, CPA disturbed human monocytes differentiation into macrophages. This work contributes to understanding the immunosuppressive properties of this food-related toxin.

Action mechanisms involved in the bioprotective effect of Lactobacillus harbinensis K.V9.3.1.Np against Yarrowia lipolytica in fermented milk

The use of lactic acid bacteria (LAB) as bioprotective cultures can be an alternative to chemical preservatives or antibiotic to prevent fungal spoilage in dairy products. Among antifungal LAB, Lactobacillus harbinensis K.V9.3.1Np showed a remarkable antifungal activity for the bioprotection of fermented milk without modifying their organoleptic properties (Delavenne et al., 2015). The aim of the present study was to elucidate the action mechanism of this bioprotective strain against the spoilage yeast Yarrowia lipolytica. To do so, yeast viability, membrane potential, intracellular pH (pHi) and reactive oxygen species (ROS) production were assessed using flow cytometry analyses after 3, 6 and 10days incubation in cell-free supernatants. The tested supernatants were obtained after milk fermentation with yogurt starter cultures either in co-culture with L. harbinensis K.V9.3.1Np (active supernatant) or not (control supernatant). Scanning-electron microscopy (SEM) was used to monitor yeast cell morphology and 9 known antifungal organic acids were quantified in both yogurt supernatants using high-performance liquid chromatograph (HPLC). Yeast growth occurred within 3days incubation in control supernatant, while it was prevented for up to 10days by the active supernatant. Interestingly, between 66 and 99% of yeast cells were under a viable but non-cultivable (VNC) state despite an absence of membrane integrity loss. While ROS production was not increased in active supernatant, cell physiological changes including membrane depolarization and pHi decrease were highlighted. Moreover, morphological changes including membrane collapsing and cell lysis were observed. These effects could be attributed to the synergistic action of organic acids. Indeed, among the 8 organic acids quantified in active supernatant, five of them (acetic, lactic, 2-pyrrolidone-5-carboxylic, hexanoic and 2-hydroxybenzoic acids) were at significantly higher concentrations in the active supernatant than in the control one. In conclusion, this study has provided new information on the physiological mechanisms induced by an antifungal LAB that could be used as part of the hurdle technology to prevent fungal spoilage in dairy products.

Individual and combined toxicological effects of deoxynivalenol and zearalenone on human hepatocytes in in vitro chronic exposure conditions

While numerous surveys highlighted the natural co-occurrence of mycotoxins in food, data about their toxicological combined effects is still limited. This is especially the case for chronic exposure conditions, although the latter are more representative of the mycotoxin risk associated with food consumption than acute exposure. In the present study, cell viability and gene expression levels of relevant hepatocyte-specific functions were evaluated for the HepaRG human liver cell line exposed to deoxynivalenol (DON) and/or zearalenone (ZEA) during 14, 28 and 42days at three subtoxic concentrations corresponding to i) the determined average exposure dose of French adult population, ii) the tolerable daily intake established by the Joint FAO/WHO Expert Committee and iii) the maximum level permitted by the European regulation in cereals intended for direct human consumption. For the latter, DON and DON+ZEA induced 90% cell mortality after 14days. In addition, depending on the considered toxin or mixture, doses and exposure periods, important variations of gene expression levels were observed. Despite the fact that in vitro conditions differ from the in vivo situation, the obtained results clearly highlighted that long-term toxicological effects of chronic exposure to mycotoxin combinations should be further investigated and, if necessary, taken into consideration at the regulatory level.

Influence of intraspecific variability and abiotic factors on mycotoxin production in Penicillium roqueforti

Penicillium roqueforti has the ability to produce secondary metabolites, including roquefortine C (ROQC) and mycophenolic acid (MPA). In a previous study, the presence of these mycotoxins, alone or in co-occurrence, has been reported in blue-veined cheese. A high variability of mycotoxin content has also been observed, although the majority of samples exhibited relatively low concentrations. The observed variability raises the question of the factors impacting ROQC and MPA production. In this context, the mycotoxigenic potential of 96 P. roqueforti strains (biotic factor) and the effect of some abiotic factors (pH, temperature, NaCl and O2 contents, and C/N ratio) on mycotoxin production were evaluated. A high intraspecific diversity, established via genotypic (RAPD) and phenotypic (FTIR) approaches, was observed. It was associated with mycotoxigenic potential variability and may thus explain part of the observed variability in mycotoxin content of blue-veined cheese. Moreover, a significant decrease of ROQC and MPA production was observed for conditions (temperature, C/N ratio, O2 and NaCl concentrations) encountered during cheese-making compared with optimal growth conditions. The results also highlighted that there was no significant effect of addition of ROQC amino-acid precursor on the production of both mycotoxins whereas a pH increase from 4.5 to 6.5 slightly reduced MPA but not ROQC production.

Effect of Penicillium roqueforti mycotoxins on Caco-2 cells: Acute and chronic exposure

Penicillium roqueforti is a common food and feed contaminant. However, it is also worldwide renowned for its use as a technological culture responsible for the typicity of blue-veined cheese. Members of the P. roqueforti species are also known to be able to produce secondary metabolites including mycophenolic acid (MPA) and roquefortine C (ROQ C) mycotoxins. In order to more closely simulate the reality of mycotoxin exposure through contaminated food consumption, this work investigated the toxicological effects of MPA and ROQ C not only in acute but also in chronic (i.e. 21-days continuous exposure) conditions on Caco-2 cells. Acute exposure to high MPA or ROQ C concentrations induced an increase of IL-8 secretion. Effects of 21-days continuous exposure on barrier integrity, based on concentrations found in blue-veined cheese and mean of blue cheese intake by French consumers, were monitored. Concerning exposure to ROQ C, no alteration of the intestinal barrier was observed. In contrast, the highest tested MPA concentration (780 μM) induced a decrease in the barrier function of Caco-2 cell monolayers, but no paracellular passage of bacteria was observed. This study highlighted that exposure to MPA and ROQ C average concentrations found in blue-veined cheese does not seem to induce significant toxicological effects in the tested conditions.

PR toxin toxicity on human cells: An in vitro study

T prevalence of invasive fungal infections (IFIs) has grown in last decades. With the increasing number of immunocompromised patients, Aspergillus fungi have become known as one of the major causes of human infection. The pathogenesis of invasive aspergillosis is intrinsically linked to the competency of the host immune system and its ability to recognize and destroy the fungus. Innate immunity plays an essential role in host defense against invasive aspergillosis. Developing new antifungal agents is restricted cause of eukaryotic nature of fungi. Therefore interest in application of natural immunostimulators to increase immune responses against pathogens has expanded. Honey is a natural substance produced by honey bees. Studies indicated that honey has different health-beneficial properties including gastro protective, hepatoprotective, reproductive, hypoglycemic, antioxidant, antihypertensive, antibacterial, anti-fungal, anti-inflammatory and wound healing effects. This work was designed to study the immunological effects of three Iranian honeys on some parts of innate immunity as well as survival rate in mice infected with Aspergillus fumigatus. For this purpose, mice were divided into 10 groups (honey alone, honey and infection, negative and positive controls) each containing 10 individuals. Mice were treated with honey (1.5 g/kg BW/orally) for 10 days. At day 6, Aspergillus fumigatus conidia (5×105/ml) were injected intravenously to the infection groups. The animals were sacrificed at day 11 and their peritoneal macrophages and spleen cells were cultured. Macrophage killing and nitric oxide production along with some proinflammatory cytokines (IL6, IL1β) were measured as well. TLR2 and TLR4 genes expression in macrophages was determined by semi-quantitative RT-PCR. Ten mice from infectious groups were kept for 30 days and survival rate was monitored. Based on the obtained results, all honeys could significantly increase killing of Aspergillus fumigatus conidia by macrophages and mean survival rate compared with control group (p<0.05). Honeys alone had immunomodulatory effect on proinflammatory cytokines, NO production and understudied genes. However by inducing infection, groups under honey treatment had higher cytokine and NO production and TLR2 gene expression compared to infection alone group (p<0.05). LPS stimuli had no significant effect on macrophages neither killing nor nitric oxide production (p>0.05). It is concluded that honey could boost the immune system and increase survival time in invasive aspergillosis; in order to illuminate the mechanism of action of honey on natural and adoptive immune responses during invasive aspergillosis, this study should be continued into the future.T use of embryonated egg as an alternative in the study of the pathogenesis of fungi is evolving, although murine models are “gold standard” however, these models are used to screen determinants of virulence among fungi species. This study was aimed at the study of the virulence potential of clinical (WM626) and environmental (EN028) isolate of Cryptococcus neoformans both belong to molecular types VNII in chick embryo infected via Chorio-allantioc membrane (CAM). At a concentration of 108 cfu per ml, the two strains had varying virulence potential on the embryo. Environmental strain EN028 achieved 100% mortality on day 5 of experiment whereas strain WM 626 did not cause 100% mortality. Histopathology of CAM from both strains revealed massive disruption of CAM cells in EN028 when compared to WM626. Also with decrease in the concentration of infectious does, infection without mortality was observed. Our study suggests that embryonated eggs is a useful alternative tool to pre-screen Cryptococcus neoformans strains to select strains for subsequent testing in murine models.