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Intestinal organoids, a reliable alternative model for studying the effects of foodborne nanoparticles in the gut: the titanium dioxide as proof of concept
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Edité par CCSD -
International audience. Daily oral exposure to inorganic nanoparticles (NP) from food additives used in ultra-processed food raises public health issues. Identifying hazards linked toNP ingestion requires long-term exposure in animal models, specific spaces and skills. Using this approach, oral exposure to the whitener and opacifyingagent titanium dioxide (TiO2) used in a wide range of products (foodstuffs, toothpaste, cosmetics, pharmaceutical tablets), led to gut barrier defectsincluding inflammation in rodents. Owing the in vivo constraints, organoids are proposed as an alternative to rodent studies but their use to assess theintestinal impacts of inorganic NPs remains to be evaluated. The aim of this study was to validate organoids as a reliable model for studying the effects offoodborne NPs in gut by comparing the impacts of food-grade TiO2 (fg-TiO2) on murine intestinal organoids with already reported in vivo data. Crypts fromsmall intestine of three wild-type C57bl/6 mice were purified, dissociated, and cells were cultured for organoid growth. Organoids were dissociated andseeded as a 2.5D culture, then exposed for 24h either to increasing doses of fg-TiO2 (0.1, 1, 10 or 100µg/mL) or to an IFN-γ/TNF-α cocktail (1 or 10 ng/mL)to check the organoids capacity to respond to a pro-inflammatory stimulus. Cytotoxicity was assessed by Lactate Dehydrogenase (LDH) releasequantification in supernatants. Gene expression of Toll Like Receptors (TLR), NFκB, cytokines and chemokines as well as markers expression of cellproliferation and differentiation, genotoxicity, antimicrobial peptides, permeability and oxidative stress were determined by qPCR. Cell apoptosis andgenotoxicity were also evaluated respectively by cleaved Caspase-3 and γH2AX quantification using immunofluorescence. Compared to control, nodifference in LDH secretion was observed following IFN-γ/TNF-α cocktail or fg-TiO2 exposure of intestinal organoids, concluding on the absence ofcytotoxicity for these treatments. A dose-dependent up-regulation of genes under control of pro-inflammatory mediators (Tlr4, Nfκb2 and Rela) wasobserved after exposure to the IFN-γ/TNF-α cocktail, showing the organoids as a functional and competent model to respond to inflammatory stresses.Organoids exposure to fg-TiO2 led to increased protein level of cleaved Caspase-3 together with up-regulation of Mki67 proliferation marker, suggestingepithelium renewal or restructuring. In addition, fg-TiO2-treated organoids showed a decrease in the expression of the stem cell marker Lgr5 and an increasein the expression of the mucin-producing gene Muc2 and the enterocyte and neuroendocrine differentiation markers Vill and Chga. Expression markers ofNFκB inflammatory response were decreased, while genes of TLR4 pathway and oxidative stress remained unchanged after fg-TiO2 treatment. In addition,fg-TiO2 increased expression of the DNA damage marker gene Gadd45A as well as γH2AX staining, while down-regulating genes encoding antimicrobialpeptides (Reg3g, S100a8) and tight junction proteins (Cldn1, Cldn7, Cldn15), suggesting genotoxic effects and possible impairments of epithelialpermeability and antimicrobial defenses. In accordance to reported in vivo data, the integrity of the gut barrier in terms of cell proliferation/differentiation,genotoxicity, innate defenses and epithelial permeability, was affected in murine gut organoids exposed for 24h to fg-TiO2. These results validated the use ofgut organoids as an alternative to in vivo experiments for screening the intestinal effects of inorganic NPs, which could be pertinent for read acrossassessment.
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