The applicability of in vitro models of the intestinal barrier for the risk assessment of engineered nanomaterials used as food additives
- Autores: Alba García Rodríguez
- Directores de la Tesis: Ricardo Marcos Dauder (dir. tes.), Constanza Cortés Crignola (codir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2018
- Idioma: español
- Materias:
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- Resumen
Nano-technological approaches are allowing the development of deliberately engineered nanomaterials (ENMs), presenting promising new applications for many industrial fields. Especially, ENMs possess unique properties and novel uses in food or food packaging materials such as the enhancement of texture, colour, flavour, nutrient stability and food packaging safety.
Despite their innovative properties, there is an increasing concern about the possibility that human exposure to TiO2NPs may lead to significant adverse health effects. The International Agency for Research on Cancer (IARC) classified TiO2 as a human carcinogen group 2B because there was enough evidence that nano-TiO2 may cause lung cancer by inhalation. Although oral exposure was also debated by IARC, the final report was inconclusive due to non-existing standardized procedures for nano- TiO2 risk assessment. Due to the potential adverse effects of this ENMs and the lack of information regarding toxicological aspects over the oral exposure, in this Thesis we have carried out in vitro studies on the biological effects of TiO2NPs.
For the aforementioned purpose, we set up and characterized, for the first time in our laboratory, an epithelial in vitro model that closely mimics the human small intestine. Thus, in our first study, we defined the best culture conditions for the alreadydescribed model, Caco-2/HT29/Raji-B. From our integrity and permeability findings, we confirmed that the best Caco-2/HT29 cell ratio is 90:10, respectively, as TEER values, paracellular LY permeability and the mucus shed formed correlated well with other studies. We also were able to detect the induction of M-like cells by TEM. Moreover, in order to monitor the proper barrier formation, we proposed a set of genes related to the cell junctional complexes, brush border enzymes, mucus shed components and M-cell markers. Finally, we tested the goodness of our epithelial in vitro model by exposing it to both TiO2NPs and SiO2NPs for 24 h. Our confocal results evidenced the potential adverse effects of TiO2NPs and SiO2NPs on the intestinal epithelium, as NPs internalization and NPs-cell nucleus interaction were observed.
Because of the heightened interest in the identification, validation and standardization of the effects associated to exposures to new ENMs, our second study aimed to assess the effects of three different shapes of TiO2NPs (spheres, rods and wires) on the Caco-2/HT29 barrier. Our results demonstrated that the three types of TiO2NPs have the ability to impair the membrane’s integrity, translocate through the mucus shed and internalize in the cells, reaching the nucleus. Taking into account our confocal images results, we hypothesize that due to their shapes, nano-wires are more likely to cross paracellularly, while nano-spheres and nano-rods used intracellular passage to cross the intestinal epithelium. Despite previous evidence that relate the capability of TiO2NPs to produce ROS, we have not detected oxidatively DNA damage.
However, and in accordance with the confocal images showing a great amount of NPscell nucleus events, we detected a slight but significant general DNA damage in the barrier’s cells.
Finally, the third study was performed under the framework of an international mention carried out in the Biomedical Engineering Department at the Binghamton University (Binghamton, NY, USA). Nutrient absorption is one of the main and most important functions of the small intestine. Thus, to understand and evaluate whether ENPs can trigger physiological potential pathologies, the activity of the intestinal alkaline phosphatase (IAP), aminopeptidase-N (APN) and Na+/K+ ATPase enzymes were measured after exposing the Caco-2/HT29-MTX barrier to TiO2NPs and SiO2NPs for 4 h. Moreover, and in order to further mimic the physiological conditions of a real digestion, the Caco-2/HT29-MTX barrier was exposed to both NPs previously digested and co-cultured with both Escherichia coli and Lactobacillus rhamnosus, as examples of commensal microbiota.
