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- Evaluation of the cytotoxicity and genotoxicity of ingested titanium dioxide nanomaterials in intestinal cellsPublication . Gramacho, Ana Catarina; Martins, Carla; Assunção, Ricardo; Gonçalves, Lídia; Simão Bettencourt, Ana; Paula, Alvito; Silva, Maria João; Louro, HenriquetaMany products already available, namely in agriculture and food, contain nanomaterials (NMs) and thus human ingestion of these compounds is probable through consumer products or food chain. Although NMs have been extensively investigated in recent years, the studies have generated contradictory results, possibly due to differences in the physicochemical properties of the NMs studied and to other variables in the tested systems. Furthermore, the NMs properties have been recognized as being context-dependent, i.e. can be affected by the surrounding matrix. These secondary features may be potentially more relevant for determining the toxicological outcome. In particular, processes like digestion may modify the NMs characteristics leading to unexpected toxicity in intestine cells. This work aimed to investigate the nano-bio interactions of titanium dioxide NMs (from Joint Research Centre, Ispra) in the context of intestinal tract and digestion processes, to better understand key events that may be linked to an adverse outcome pathway (AOP). In vitro for digestion was simulated and the NMs secondary properties in the intestinal cell moiety were characterized after this process. The cytotoxic and genotoxic effects of digested NMs were determined after the in vitro exposure of human intestinal cells (Caco-2). In addition, the FPG-comet assay was used to analyze oxidative DNA lesions. The digestion products without the NM showed cytotoxic effects above the concentration of 10% in cell culture medium, leading to the need to reduce its concentration below this level. Therefore, initial dose-range studies set a working range of NM concentrations of 0.14 up to 14 µg/ml (0.5-4.5 % of digestion product), which relates also with the predicted levels of exposure of human intestinal cells in real life conditions. Under these conditions, the preliminary results suggest that the tested titanium dioxide NMs do not yield cytotoxic or genotoxic effects upon 24h of exposure of Caco-2 cells, directly after the digestion process. Future studies will investigate the subcellular localization of NMs, integrity of cell junctions, activation of stress signaling pathways and secretion of inflammatory cytokines, to allow an integrated approach to potential adverse effects of the NMs. By elucidating key events elicited by NMs, linking exposure to adverse outcomes, it is expected to contribute to the safety evaluation of NMs within an AOP landscape.
- Titanium dioxide nanomaterials - induced DNA damage in intestinal cells following simulated in vitro digestionPublication . Vieira, Adriana; Rolo, Dora; Vital, Nádia; Martins, Carla; Assunção, Ricardo; Alvito, Paula; Gonçalves, Lídia; Bettencourt, Ana; Silva, Maria João; Louro, HenriquetaIntroduction: The increased use of titanium dioxide nanomaterials (TiO2) in food products has raised oral exposure to those nanomaterials, with subsequent risks to human health, particularly genotoxicity and, ultimately, cancer development. In humans, the digestion process may modify the physicochemical properties of TiO2, thereby shaping the potential biological outcomes. Thus, such process should be considered when assessing their hazard upon oral exposure. This work aimed to investigate the genotoxic effects of three TiO2 (NM-102, NM-103 and NM-105, JRC repository) after the simulation of the human digestive process using the standardized INFOGEST in vitro digestion method. The secondary physicochemical properties and DNA damage levels, using the comet assay, were analysed in two intestinal cell lines exposed for 24h to digested or undigested TiO2. Results: An increase in the level of DNA strand breaks in two intestinal cell lines(Caco-2 and HT29-MTX-E12) was observed after exposure to digested NM-105, concomitantly with a decrease in its hydrodynamic size, comparatively to the undigested nanomaterial. Moreover, the digested NM-103 induced DNA damage in Caco-2 cells whereas the undigested nanomaterial did not. The FPG-modified comet assay also revealed an increase in oxidative DNA lesions upon treatment of Caco-2 with NM-103 and HT29-MTX-E12 with NM-102. Conclusions: One of the digested TiO2(NM-105) can be classified as potentially genotoxic in both cell lines, while the digested NM-103 induced an equivocal genotoxic response in Caco-2 cells. Therefore, the digestion simulation is of relevance to investigate the potential genotoxic effects of ingested nanomaterials.