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div>These observations can be compared with the results of a previous study in which we observed that intestinal morphological structures are also affected by CPF-exposure. We also found that CPF-exposure was associated with differences in protein expression in segments of the intestine. The immunohistochemical identification of claudin 4 seems to confirm Chiba et al.’s finding that claudin 4 is more strongly expressed in the small intestine than in the colon. Accordingly, one can hypothesize that CPF has a putative downregulating effect on claudin 4. Many studies have shown that disruption of TJs leads to a rise in epithelial permeability. For example, methotrexate induces ZO-1 dephosphorylation, which in turn is associated with a change in the protein’s localization in epithelial cells in the small intestine; this change may contribute to leakage of the intestinal barrier. Here, we used immunofluorescent staining to study protein localizations in epithelium because changes in permeability may modify protein addressing or induce protein internalization. Both ZO-1 and claudin 4 are reportedly involved in intestinal barrier dysfunction. We detected ZO-1 and claudin 4 in the rat ileum and colon at both D21 and D60. Exposure to CPF was associated with changes in the expression and/or localization of ZO-1 both in the ileum and colon. Surprisingly, CPF did not seem to impact on the intestinal distribution of claudin 4 - at least in immature animals. In D60 animals, ZO-1 and claudin 4 staining was much weaker in CPF1 rats than in CPF0 rats; this might be due to downregulated expression and/or altered localization. These observations could be related to Gearhart’s study that showed that chlorpyrifos inhibit kinesin-dependent microtubule motility and Grigoryan’s study that showed a tubulin polymerization disruption by chlorpyrifos oxon. As a consequence, proteins synthetized and involved in tight KU-0059436 junction formation might be restrained to the cytoplasmic space rather than moving along microtubules to tight junction space. The cholinergic signaling is also involved in the regulation of the barrier function. As described by Cameron and Perdue, HRP uptake as a marker of transepithelial transport is stimulated by the M3 muscarinic agonist bethanechol. In a similar way, the paracellular transport is also regulated by a cholinergic signaling. For example, carbachol, a cholinergic agonist, ameliorates LPSinduced intestinal epithelial tight junction damage by downregulating NF-kB and myosin light-chain kinase pathways. Chlorpyrifos oxon is known to inhibit acetylcholinesterase activity which increases the amount of acetylcholine in the synaptic space. Consequently, we could suspect an overstimulation of acetylcholine receptors leading to a rise in nerve transmission. But CPF oxon has been described to block muscarinic receptors, reducing the potentially protective effect of acetylcholine on gut barrier dysfunction. The results of our previous study of rat pups revealed that chronic CPF-exposure was also associated with a microbial dysbiosis at weaning. The same phenomenon was observed for mature animals at 60 days of age, and was associated with greater bacterial translocation to the liver. Bacterial toxins may be involved in the disruption of the cytoskeleton and thus leakage of macromolecules through the paracellular route. Moreover, CPF-induced dysbiosis was characterized by low counts of lactobacilli, which are involved in the relocation of occludin and ZO-1 to TJ complexes. In view of the fact that many foodstuffs are contaminated with pesticide residues, consumption of probiotics may help prevent dysbiosis and protect TJs. Dysbiosis and bacterial translocation may also stimulate the immune system and trigger the release of cytokines; this may also be a strong signal for the disruption of cell-cell contacts. These cytokines can directly affect cell-cell contacts and paracellular permeability in intestinal epithelial cells by modulating claudin expression and distribution. Bacterial translocation is known to contribute to the development of inflammation and various functional gastrointestinal disorders and chronic inflammatory diseases. It is also recognized that microbial dysbiosis exerts a strong influence on the immune system and intestinal permeability. On the basis of our present results, it is impossible to say whether CPF’s effect is direct or indirect.

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