Marc Maresca

From the Gut to the Brain: Journey and Pathophysiological Effects of the Food-Associated Trichothecene Mycotoxin Deoxynivalenol

Mycotoxins are fungal secondary metabolites contaminating food and causing toxicity to animals and humans. Among the various mycotoxins found in crops used for food and feed production, the trichothecene toxin deoxynivalenol (DON or vomitoxin) is one of the most prevalent and hazardous. In addition to native toxins, food also contains a large amount of plant and fungal derivatives of DON, including acetyl-DON (3 and 15ADON), glucoside-DON (D3G), and potentially animal derivatives such as glucuronide metabolites (D3 and D15GA) present in animal tissues (e.g., blood, muscle and liver tissue). The present review summarizes previous and very recent experimental data collected in vivo and in vitro regarding the transport, detoxification/metabolism and physiological impact of DON and its derivatives on intestinal, immune, endocrine and neurologic functions during their journey from the gut to the brain.

Some food-associated mycotoxins as potential risk factors in humans predisposed to chronic intestinal inflammatory diseases.

Mycotoxins are fungal metabolites able to affect the functions of numerous tissues and organs in animals and humans, including intestinal and immune systems. However, the potential link between exposure to some mycotoxins and human chronic intestinal inflammatory diseases, such as celiac and Crohns diseases or ulcerative colitis, has not been investigated. Instead, several theories based on bacterial, immunological or neurological events have been elaborated to explain the etiology of these pathologies. Here we reviewed the literature on mycotoxin-induced intestinal dysfunctions and compared these perturbations to the impairments of intestinal functions typically observed in human chronic intestinal inflammatory diseases. Converging evidence based on various cellular and animal studies show that several mycotoxins induce intestinal alterations that are similar to those observed at the onset and during the progression of inflammatory bowel diseases. Although epidemiologic evidence is still required, existing data are sufficient to suspect a role of some food-associated mycotoxins in the induction and/or persistence of human chronic intestinal inflammatory diseases in genetically predisposed patients.

Both direct and indirect effects account for the pro-inflammatory activity of enteropathogenic mycotoxins on the human intestinal epithelium : Stimulation of interleukin-8 secretion, potentiation of interleukin-1β effect and increase in the transepithelial passage of commensal bacteria

Mycotoxins are fungal secondary metabolites responsible of food-mediated intoxication in animals and humans. Deoxynivalenol, ochratoxin A and patulin are the best known enteropathogenic mycotoxins able to alter intestinal functions resulting in malnutrition, diarrhea, vomiting and intestinal inflammation in vivo. Although their effects on intestinal barrier and transport activities have been extensively characterized, the mechanisms responsible for their pro-inflammatory effect are still poorly understood. Here we investigated if mycotoxin-induced intestinal inflammation results from a direct and/or indirect pro-inflammatory activity of these mycotoxins on human intestinal epithelial cells, using differentiated Caco-2 cells as model and interleukin 8 (IL-8) as an indicator of intestinal inflammation. Deoxynivalenol was the only mycotoxin able to directly increase IL-8 secretion (10- to 15-fold increase). We also investigated if these mycotoxins could indirectly stimulate IL-8 secretion through: (i) a modulation of the action of pro-inflammatory molecules such as the interleukin-1beta (IL-1beta), and/or (ii) an increase in the transepithelial passage of non-invasive commensal Escherichia coli. We found that deoxynivalenol, ochratoxin A and patulin all potentiated the effect of IL-1beta on IL-8 secretion (ranging from 35% to 138% increase) and increased the transepithelial passage of commensal bacteria (ranging from 12- to 1544-fold increase). In addition to potentially exacerbate established intestinal inflammation, these mycotoxins may thus participate in the induction of sepsis and intestinal inflammation in vivo. Taken together, our results suggest that the pro-inflammatory activity of enteropathogenic mycotoxins is mediated by both direct and indirect effects.

The first extracellular domain of the tumour stem cell marker CD133 contains an antigenic ganglioside-binding motif

Prominin 1/CD133 is a marker of transplantable cancer stem cells. We have generated anti-peptide antibodies against a N-terminal epitope of CD133 belonging to a ganglioside-binding domain. The labelling of colon cancer cells with these antibodies was inhibited by various gangliosides including GM1 and GD3, but not GT1b. CD133 immunolabelling progressively decreased to undetectable levels in post-confluent cultures, possibly through ganglioside-mediated epitope masking since the staining was partially recovered after chemical disruption of lipid rafts. We suggest that selected gangliosides could modulate the accessibility of CD133 and regulate cell-cell contacts involving CD133(+) stem cells at the earliest steps of tumour development.

Glycosphingolipid (GSL) microdomains as attachment platforms for host pathogens and their toxins on intestinal epithelial cells: activation of signal transduction pathways and perturbations of intestinal absorption and secretion.

Glycosphingolipid (GSL)-enriched microdomains are used as cellular binding sites for various pathogens including viruses and bacteria. These attachment platforms are specifically associated with transducer molecules, so that the binding of host pathogens (or their toxins) to the cell surface may result in the activation of signal transduction pathways. In the intestinal epithelium, such pathogen-induced dysregulations of signal transduction can elicit a severe impairment of enterocytic functions. In this study, we demonstrate that the interaction of a bacterial toxin (cholera toxin) and a viral envelope glycoprotein (HIV-1 gp120) with the apical plasma membrane of intestinal cells is mediated by GSL-enriched microdomains that are associated with G regulatory proteins. These microbial proteins induce a GSL-dependent increase of intestinal fluid secretion by two mechanisms: activation of chloride secretion and inhibition of Na+-dependent glucose absorption. Taken together, these data support the view that GSL-enriched microdomains in the apical plasma membrane of enterocytes are involved in the regulation of intestinal functions.

Deoxynivalenol inhibits the expression by goblet cells of intestinal mucins through a PKR and MAP kinase dependent repression of the resistin‐like molecule β

SCOPE The food-associated mycotoxin deoxynivalenol (DON) is known to affect intestinal functions. However, its effect on intestinal mucus is poorly characterized. METHODS AND RESULTS We analyzed the effects of DON on human goblet cells (HT29-16E cells) and porcine intestinal explants. Results showed that subtoxic doses of DON (as low as 1 μM) decreased mucin (MUC) production. qPCR analysis demonstrated that this inhibition was due to a specific decrease in the level of mRNA encoding for the intestinal membrane-associated (MUC1) and the secreted MUCs (MUC2, MUC3). Mechanistic studies demonstrated that DON effect relied on the activation of the protein kinase R and the mitogen-activated protein kinase p38 ultimately leading to the inhibition of the expression of resistin-like molecule beta, a known positive regulator of MUC expression. CONCLUSION Taken together, our results show that at low doses found in food and feed, DON is able to affect the expression and production of MUCs by human and animal goblet cells. Due to the important role of MUCs in the barrier function and in the interaction of commensal bacteria with the host, such effect could explain the observed modifications in the microbial diversity and the increased susceptibility to enteric infection following exposure to DON.

Interaction of curcumin with phosphocasein micelles processed or not by dynamic high-pressure

The binding of curcumin to native-like phosphocaseins (PC) dispersed in simulated milk ultrafiltrate at pH 6.6 was assessed by fluorescence spectrophotometry. Curcumin binds to native-like PC micelles with ∼1 binding site per casein molecule, and a binding constant of 0.6-5.6 × 10(4)M(-1). Dynamic high pressure (or ultra-high pressure homogenisation, UHPH) at 200 MPa did not affect the binding parameters of curcumin to processed PC. UHPH-processing of PC dispersions at 300 MPa was followed by a slight but significant (p=0.05) increase in the binding constant of curcumin to processed PC, which may result from the significant UHPH-induced dissociation of initial PC micelles into neo-micelles of smaller sizes, and from the corresponding 1.5-2-fold increase in micelle surface area. PC-curcumin complexes were resistant to pepsin but were degraded by pancreatin, providing the possibility of a spatiotemporally controlled release and protection of bound biomolecules. UHPH-processed PC did not induce TC7-cell damage or major inflammation as assessed by LDH release or IL-8 secretion, respectively, compared with native-like PC. PC micelles could provide a valuable submicron system to vectorise drugs and nutrients.

Squalamine: An Appropriate Strategy against the Emergence of Multidrug Resistant Gram-Negative Bacteria?

We reported that squalamine is a membrane-active molecule that targets the membrane integrity as demonstrated by the ATP release and dye entry. In this context, its activity may depend on the membrane lipid composition. This molecule shows a preserved activity against bacterial pathogens presenting a noticeable multi-resistance phenotype against antibiotics such as polymyxin B. In this context and because of its structure, action and its relative insensitivity to efflux resistance mechanisms, we have demonstrated that squalamine appears as an alternate way to combat MDR pathogens and by pass the gap regarding the failure of new active antibacterial molecules.

The ribotoxin deoxynivalenol affects the viability and functions of glial cells

Glial cells are responsible for maintaining brain homeostasis. Modification of the viability and functions of glial cells, including astrocytes and microglia, are associated with neuronal death and neurological diseases. Many toxins (heavy metals, pesticides, bacterial or viral toxins) are known to impact on brain cell viability and functions. Although recent publications suggest a potential link between environmental exposure of humans to mycotoxins and neurological diseases, data regarding the effects of fungal toxins on brain cells are scarce. In the present study, we looked at the impact of deoxynivalenol (DON), a fungal ribotoxin, on glial cells from animal and human origin. We found that DON decreased the viability of glial cells with a higher toxicity against microglial cells compared with astrocytes. In addition to cellular toxicity, DON affected key functions of glial cells. Thus, DON caused a biphasic effect on the neuroinflammatory response of microglia to lipopolysaccharide (LPS), while sublethal doses of DON increased the LPS‐induced secretion of TNF‐α and nitric oxide, toxic doses inhibited it. In addition to affecting microglial functions, sublethal doses of DON also suppressed the uptake of L‐glutamate by astrocytes. This inhibition was associated with a modification of the expression of the glutamate transporters at the plasma membrane. Our results suggest that environmental ribotoxins such as DON could, at low doses, cause modifications of brain homeostasis and possibly participate in the etiology of neurological diseases in which alterations of the glia are involved.

The Food-Associated Ribotoxin Deoxynivalenol Modulates Inducible NO Synthase in Human Intestinal Cell Model

The intestinal epithelium possesses active immune functions including the production of proinflammatory cytokines and antimicrobial molecules such as nitric oxide (NO). As observed with immune cells, the production of NO by the intestinal epithelium is mainly due to the expression of the inducible NO synthase (iNOS or NOS2). Epithelial immune functions could be affected by many factors including pathogenic microorganisms and food-associated toxins (bacterial and fungal). Among the various mycotoxins, deoxynivalenol (DON) is known to alter the systemic and intestinal immunity. However, little is known about the effect of DON on the production of NO by the intestinal epithelium. We studied the impact of DON on the intestinal expression of iNOS using the Caco-2 cell model. In line with its proinflammatory activity, we observed that DON dose-dependently up-regulates the expression of iNOS mRNA. Surprisingly, DON failed to increase the expression of iNOS protein. When testing the effects of DON on cytokine-mediated induction of iNOS, we found that very low concentrations of DON (ie, 1 µM) decrease the amount of iNOS protein but not of iNOS mRNA. We demonstrated that DONs effect on iNOS protein relies on its ability to activate signal pathways and to increase iNOS ubiquitinylation and degradation through the proteasome pathway. Taken together, our results demonstrate that although DON causes intestinal inflammation, it suppresses the ability of the gut epithelium to express iNOS and to produce NO, potentially explaining the increased susceptibility of animals to intestinal infection following exposure to low doses of DON.