Percorrer por autor "Gramacho, A."
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- Analysis of the Characteristics and Cytotoxicity of Titanium Dioxide Nanomaterials Following Simulated In Vitro DigestionPublication . Bettencourt, Ana; Gonçalves, L.; Gramacho, A.; Vieira, A; Rolo, D.; Martins, Carla; Assunção, Ricardo; Alvito, Paula; Silva, Maria João; Louro, HenriquetaSeveral metallic nanomaterials (NMs), such as titanium dioxide nanomaterials (TiO2), present beneficial properties with a broad range of innovative applications. The human population is exposed to TiO2, particularly by ingestion, due to its increasing use as a food additive and inclusion in dietary supplements and food packaging materials. Whether this oral exposure may lead to adverse local or systemic outcomes has been the subject of research, but studies have generated contradictory results, reflecting differences in the physicochemical properties of the TiO2 studied, effects of the surrounding matrix, and modifications during digestion. This work aimed to investigate the toxic effects of three different TiO2 NMs (NM-103, NM-103 and NM-105) on the gastrointestinal tract cells, Caco-2 and HT29-MTX-E12, after the use of the standardized static INFOGEST 2.0 in vitro digestion method to mimic human digestion of TiO2, contributing to hazard assessment. The results show that, for one of the digested TiO2 NMs studied (NM-105), a more pronounced toxicity occurs after exposure of HT29-MTX-E12 intestinal cells, as compared to undigested NM, concomitantly with subtle changes in characteristics of the NM. Thus, the inclusion of the digestion simulation in the safety evaluation of ingested NMs through in vitro bioassays can better integrate the modifications that NMs suffer in the organism. It is expected that such an approach will reduce uncertainties in the hazard assessment of ingested NMs for human health.
- Applicability of the harmonized in vitro digestion method to titanium dioxide nanomaterialsPublication . Alvito, Paula; Assunção, Ricardo; Gramacho, A.; Silva, M.J.; Louro, H.; Martins, C.Titanium dioxide nanomaterials (TiO2 NMs) have a high potential for ingestion by human populations, due to their increasing use as food additives, inclusion in dietary supplements and food packaging materials. Whether this oral exposure may lead to adverse local or systemic outcomes, has been the subject of research. In vitro studies have generated contradictory results, possibly due to differences in the physicochemical properties of the TiO2 NMs studied, which can be additionally affected by the surrounding matrix and interactions during digestion. INGESTnano is a national project aimed at investigating the nano-bio interactions of nanomaterials on intestinal cells, at the cellular and molecular levels, after the digestion process, to better understand their potential impacts on human health. Three TiO2 NMs were selected as case-studies to setup a workflow for addressing nanosafety concerns of ingested NMs, while considering the nano-bio interactions under physiological conditions. As an alternative to in vivo testing, this project is focused on the use of the harmonized in vitro digestion method for simulating the human digestion of NMs. This digestion comprises three compartments: a) mouth (pH 7.0, alpha-amylase, salts); b) stomach (pH 3, HCl, pepsin, salts) and c) small intestine (pH 7.5, bile salts, pancreatin, salts). The final product of digestion is tested in bioassays using intestinal cells, to ascertain its toxicity. A high level of toxicity of the final digestion product challenged the applicability of the digestion product in the biological assays. The results revealed that digestion products without the NM showed cytotoxic effects above the concentration of 10% in cell culture medium. Several modifications to the initial protocol were thus performed to overcome this issue. The results suggested that the addition of bile salts accounted for most of the toxicity observed. The applicability of the harmonized in vitro digestion method is discussed in view of its potential use as a tool for addressing the toxicity of ingested NMs or other food contaminants, mimicking the physiological processes, in alternative to animal models.
- Nanomaterials in foods and the standardized static in vitro digestion method: contributing to the study of the potential toxic effectsPublication . Martins, Carla; Assunção, Ricardo; Gramacho, A.; Alvito, Paula; Silva, Maria João; Louro, H.The use of nanoparticles (e.g. titanium dioxide) in commercial food products to modify some properties, such as brightness and whiteness, increased in the last years and is nowadays widespread. Despite the inhalation of nanoparticles is already a topic of concern, the potential adverse health effects due to ingestion still presents gaps of knowledge. In fact, gastrointestinal tract is the first interface between the body and the external environment and consequently could represent a target organ for compounds present in food that could exert toxic effects. The in vitro digestion models used to simulate the human digestion may contribute to fill these gaps. A standardized in vitro digestion model (IVD) was developed within the INFOGEST COST Action. This method considers a three-compartment model, simulating the digestion in the mouth, stomach and small intestine, applying standardized parameters such as pH, enzymatic activity and incubation periods. Within the scope of INGESTnano project (PTDC/SAU-PUB/29841/2017), and under physiological conditions, three TiO2 nanomaterials (NMs) were selected to setup a workflow to address the nanosafety regarding ingested NMs. The IVD was used to simulate the human gastrointestinal digestion of the NMs and the final product was used to test and ascertain NMs toxicity, using cell lines as intestinal models. The use of this standardized IVD model presented some challenges such as the high level of toxicity of the final digestion product for the bioassays. Several modifications to the initial protocol were investigated to overcome this issue. The results suggested that the addition of bile salts accounted for the majority of the observed toxicity. The applicability of the harmonized in vitro digestion method will be discussed in view of its potential use as a tool for addressing the toxicity of ingested NMs or other food contaminants, mimicking the physiological processes.