Browsing by Author "Bettencourt, Ana"
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- Adverse Outcome Pathways Associated with the Ingestion of Titanium Dioxide Nanoparticles - A Systematic ReviewPublication . Rolo, Dora; Assunção, Ricardo; Ventura, Célia; Alvito, Paula; Gonçalves, Lídia; Martins, Carla; Bettencourt, Ana; Jordan, Peter; Vital, Nádia; Pereira, Joana; Pinto, Fátima; Matos, Paulo; Silva, Maria João; Louro, HenriquetaTitanium dioxide nanoparticles (TiO2-NPs) are widely used, and humans are exposed through food (E171), cosmetics (e.g., toothpaste), and pharmaceuticals. The oral and gastrointestinal (GIT) tract are the first contact sites, but it may be systemically distributed. However, a robust adverse outcome pathway (AOP) has not been developed upon GIT exposure to TiO2-NPs. The aim of this review was to provide an integrative analysis of the published data on cellular and molecular mechanisms triggered after the ingestion of TiO2-NPs, proposing plausible AOPs that may drive policy decisions. A systematic review according to Prisma Methodology was performed in three databases of peer-reviewed literature: Pubmed, Scopus, and Web of Science. A total of 787 records were identified, screened in title/abstract, being 185 used for data extraction. The main endpoints identified were oxidative stress, cytotoxicity/apoptosis/cell death, inflammation, cellular and systemic uptake, genotoxicity, and carcinogenicity. From the results, AOPs were proposed where colorectal cancer, liver injury, reproductive toxicity, cardiac and kidney damage, as well as hematological effects stand out as possible adverse outcomes. The recent transgenerational studies also point to concerns with regard to population effects. Overall, the findings further support a limitation of the use of TiO2-NPs in food, announced by the European Food Safety Authority (EFSA).
- 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.
- Analysis of the In Vitro Toxicity of Nanocelluloses in Human Lung Cells as Compared to Multi-Walled Carbon NanotubesPublication . Pinto, Fátima; Lourenço, Ana Filipa; Pedrosa, Jorge F.S.; Gonçalves, Lídia; Ventura, Célia; Vital, Nádia; Bettencourt, Ana; Fernandes, Susete N.; da Rosa, Rafaela R.; Godinho, Maria Helena; Louro, Henriqueta; Ferreira, Paulo J.T.; Silva, Maria JoãoCellulose micro/nanomaterials (CMNM), comprising cellulose microfibrils (CMF), nanofibrils (CNF), and nanocrystals (CNC), are being recognized as promising bio-nanomaterials due to their natural and renewable source, attractive properties, and potential for applications with industrial and economical value. Thus, it is crucial to investigate their potential toxicity before starting their production at a larger scale. The present study aimed at evaluating the cell internalization and in vitro cytotoxicity and genotoxicity of CMNM as compared to two multi-walled carbon nanotubes (MWCNT), NM-401 and NM-402, in A549 cells. The exposure to all studied NM, with the exception of CNC, resulted in evident cellular uptake, as analyzed by transmission electron microscopy. However, none of the CMNM induced cytotoxic effects, in contrast to the cytotoxicity observed for the MWCNT. Furthermore, no genotoxicity was observed for CNF, CNC, and NM-402 (cytokinesis-block micronucleus assay), while CMF and NM-401 were able to significantly raise micronucleus frequency. Only NM-402 was able to induce ROS formation, although it did not induce micronuclei. Thus, it is unlikely that the observed CMF and NM-401 genotoxicity is mediated by oxidative DNA damage. More studies targeting other genotoxicity endpoints and cellular and molecular events are underway to allow for a more comprehensive safety assessment of these nanocelluloses.
- Assessing the impact of TiO2 nanomaterials on intestinal cells: New evidence for epithelial translocation and potential pro-inflammatory effectsPublication . Rolo, Dora; Pereira, Joana F.S.; Gonçalves, Lídia; Bettencourt, Ana; Jordan, Peter; Silva, Maria João; Matos, Paulo; Louro, HenriquetaUnderstanding the potential impact of nanomaterials (NMs) on human health requires further investigation into the organ-specific nano-bio interplay at the cellular and molecular levels. We showed increased chromosomal damage in intestinal cells exposed to some of in vitro digested Titanium dioxide (TiO2) NMs. The present study aimed to explore possible mechanisms linked to the uptake, epithelial barrier integrity, cellular trafficking, as well as activation of pro-inflammatory pathways, after exposure to three TiO2-NMs (NM-102, NM-103, and NM-105). Using confocal microscopy, we show that all NMs, digested or not, were able to enter different types of intestinal cells. At the physiologically relevant concentration of 14 μg/mL, the digested TiO2-NMs did not compromise the transepithelial resistance, nor the levels of epithelial markers E-cadherin and Zonula occludens protein 1 (ZO-1), of polarized enterocyte monolayers. Nonetheless, all NMs were internalized by intestinal cells and, while NM-102 was retained in lysosomes, NM-103 and NM-105 were able to transverse the epithelial barrier through transcytosis. Moreover, 24 h exposure of 14 and 1.4 μg/mL digested NM-105, promoted interleukin IL-1β expression in activated M1 macrophages, indicating a potential pro-inflammatory action in the gut. Taken together, our findings shed light on the cell-specific nano-bio interplay of TiO2-NMs in the context of the intestinal tract and highlight transcytosis as a potential gateway for their systemic distribution. The potential proinflammatory action of digested NM-105 emphasizes the importance of pursuing research into the potential impact of NMs on human health and contribute to the weight of evidence to limit their use in food.
- Assessing the Safety of Polymeric Nanoparticulated Systems developed for Drug Delivery with Human OsteoblastsPublication . Dias, Kamila; Louro, Henriqueta; Gonçalves, Lídia; Bettencourt, Ana; Silva, Maria JoãoThe development of novel nanocarriers has been recognized as a promising approach to improve drug release profiles within the target sites, being the assessment of their biocompatibility and safety a critical point of the process. For this purpose, Poly(methyl methacrylate) (PMMA) and PMMA-Eudragit RL 100 (PMMA-Eud, 50:50) nanoparticles (NPs) were recently produced to target bone infections following prosthetic surgeries and a first toxicity screening on fibroblats was conducted. The objective of the present work was to further characterize the cellular interactions and the potential toxicity of those polymeric NPs, using human osteoblasts. PMMA and PMMA-Eud (50:50) NPs were produced by single emulsion evaporation methodology and their physicochemical properties (size distribution, surface charge, morphology and aggregation/agglomeration states) were assessed. Their safety was evaluated both in normal and differentiated MG63 cells through studies of cell uptake, cyto- and genotoxicity using several endpoints: cell viability, oxidative stress production, DNA and chromosome damage. The successful cellular uptake of PMMA and PMMA-Eud by osteoblast was confirmed. None of the NPs was cytotoxic or induced oxidative stress in differentiated cells, although a moderated toxicity was detected in undifferentiated cells. As to the genotoxic potential, both NPs induced primary DNA damage detected by the comet assay, especially in short-term exposure. Noteworthy, none of the NPs caused chromosome breakage/loss using the micronucleus assay that is recommended by OECD/ICH testing guidelines for pharmaceuticals. Our findings suggest that, despite PMMA and PMMA-Eud are promising nanocarriers for drug delivery, the primary genotoxicity observed in osteoblasts needs to be further investigated. In addition, the fact that NPs affect differently normal and differentiated osteoblasts suggests the need of using more sophisticated in vitro cellular systems to better mimic the complexity of the target tissues and thus minimize the utilization of in vivo models.
- Biocompatibility Assessment of Polymeric Nanoparticle Carriers for Drug Delivery in Human OsteoblastsPublication . Dias, Kamila; Louro, Henriqueta; Gonçalves, Lídia; Silva, Maria João; Bettencourt, AnaBone infections after prosthetic surgery represent a painful and costly process that should be prevented. The use of nanocarriers as efficient drug-delivery systems represents an innovative strategy to reduce systemic toxicity and avoid antimicrobial resistance. Still, great efforts are required to ensure the biocompatibility and safety of new nanocarriers before their medical application. In this work, the FDA-approved polymers poly(methyl methacrylate) (PMMA), and Eudragit RL 100 (Eud) were used to obtain two sets of nanoparticles, plain PMMA and PMMA-Eud (50:50). Their biocompatibility was assessed in a normal and differentiated human osteoblast cell line. The nanoparticles synthesis was conducted by a previously optimized methodology and their physicochemical properties, including size distribution, surface charge, morphology and aggregation/agglomeration states, were analysed during different preparation steps. Furthermore, the cell uptake and cytotoxicity as well as and their potential to produce reactive oxygen species (ROS) were also studied. Cell assays were performed following exposure of MG63 (ATCC®CRL-1427™) under normal and differentiation culture conditions, to a concentration range of both polymeric nanoparticles. The results of the uptake studies confirmed that both nanoparticles are internalized by osteoblasts. For both formulations, neither a significant decrease in cell viability nor oxidative stress induction was observed. The comparative analysis of data obtained in undifferentiated osteoblast and in those under differentiation conditions showed similar effects being the latter slightly less sensitive to the nanoparticles toxicity. Based on the reported data, PMMA and PMMA-Eud can be described as biocompatible nanomaterials and be further explored in a perspective of potential carriers for drug delivery, reducing adverse effects of conventional treatments. However, another key property i.e., genotoxicity, needs to be assessed before ensuring their safety for human health. This approach isn’t only relevant for understanding the nano-bio interface by disclosing how physicochemical features relate to toxicological events, but also to support the safe-by-design concept.
- Cellular and molecular mechanisms of toxicity of ingested nanomaterialsPublication . Gramacho, Ana Catarina; Rolo, Dora; Martins, Carla; Assunção, Ricardo; Gonçalves, Lídia M.; Bettencourt, Ana; Alvito, Paula; Pereira, Joana; Jordan, Peter; Silva, Maria João; Louro, HenriquetaThe technology based on manufactured nanomaterials (NMs) has been pointed as key enabling technology, due to its potential to improve many products and processes, namely in agriculture, food and feed industry. Many of such products, already available, have NMs such as titanium dioxide nanomaterials (TiO2) and the oral exposure may occur either directly, through the consumption of products/pharmaceuticals containing NMs, or indirectly, through the ingestion of foods contaminated with NMs released from food-contact materials or even through concentration in the food chain due to environmental accumulation. Therefore, the gastrointestinal tract (GIT) appears to be a probable route of exposure to NMs and may lead to systemic exposure if the body barriers are surpassed. One major concern for public health is that NMs may produce adverse outcomes (AO) such as genotoxic effects that are associated with increased risk of cancer. 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 test systems. INSA has previously shown that NMs with the same chemistry, but differing in primary properties may yield different biological effects. Conversely, the NMs properties are context-dependent, i.e. can be affected by the surrounding matrix. These secondary features may be potentially more relevant for determining toxicological outcomes. In particular, processes like digestion may modify the NMs characteristics leading to unexpected toxicity in intestine cells. INGESTnano project aims to investigate the nano-bio interactions of NMs, at cellular and molecular level, in the context of intestinal tract and digestion processes, to better understand their potential negative impacts on human health with special reference to organ-specific cells. TiO2 has been selected as case-study to setup a workflow for addressing nanosafety concerns that may be in the future applied to other NMs to which GIT may be exposed. It is expected that this project will contribute to the safety evaluation of the TiO2 ingested, by elucidating key events (KE) elicited by these NMs and linking exposure to AO.
- Ingested nanomaterials: impact of digestion process in the physicochemical characteristics and biological consequences in intestinal cellsPublication . Vieira, Adriana; Vital, Nádia; Roque, Rossana; Gramacho, Ana Catarina; Rolo, Dora; Gonçalves, Lídia D.; Bettencourt, Ana; Martins, Carla; Assunção, Assunção; Alvito, Paula; Silva, Maria João; Louro, HenriquetaNanomaterials(NMs) provide a basis for key enabling technologies, in view of their potential to improve many products and processes, namely in food and feed industry. That is the case of titanium dioxide NMs(TiO2 NMs), presenting beneficial properties for a broad range of innovative applications such as food additives, toothpaste, pharmaceuticals, food products, etc., that may drive ingestion. The oral exposure can occur directly, by consumption of products/pharmaceuticals or foods containing NMs, or indirectly, through the ingestion of foods contaminated with NMs released from food-contact materials or environmental sources. As such, the gastrointestinal tract is the first site of contact of the ingested NMs, allowing a systemic exposure if the intestinal barriers is surpassed. This work aimed to investigate how the digestion process affects the physicochemical properties of three different TiO2 NMs(NM-102, NM-103 and NM-105) and their toxic effects on intestinal cells. After undergoing digestion through the standardized static INFOGEST 2.0 in vitro digestion method, the cytotoxicity of the TiO2 NMs was determined in Caco-2 and HT29-MTX-E12 intestinal cells, using the MTT assay. Furthermore, the cytokinesis-blocked micronucleus assay was used to investigate their genotoxicity in both cell lines in order to predict their carcinogenic potential. The results showed that, for one TiO2 NM(NM-105), the digestion caused changes in the hydrodynamic size of the NM and a more pronounced toxicity in HT29-MTX-E12 intestinal cells, as compared to the undigested one. The micronucleus assay suggests effects on the chromosomal integrity in the HT29-MTXE12 cells, for all the tested TiO2 NM especially after the in vitro digestion. Overall, we conclude that including the digestion prior to the in vitro bioassays for the safety evaluation of ingested NMs, allows integrating the physiological modifications that the NMs suffer in the organism, contributing to an improved hazard assessment of ingested NMs.
- Investigation of potential respiratory adverse effects of micro/nanofibrillated cellulose and cellulose nanocrystals using human lung cell lines.Publication . Pinto, Fátima; Ventura, Célia; Cadete, João; Lourenço, Ana Filipa; Pedrosa, Jorge F.S.; Vital, Nádia; Pereira, Joana F.S.; Matos, Paulo; Gonçalves, Lídia; Bettencourt, Ana; Silva, Catarina C.; Fernandes, Susete N.; Godinho, Maria Helena; Vieira, Luís; Jordan, Peter; Ferreira, Paulo J.T.; Louro, Henriqueta; Silva, Maria JoãoMicro/nanofibrillated (CMF/CNF) and nanocrystalline (CNC) celluloses are innovative materials with enormous potential for industrial and biomedical applications. Their expanding production/application urges the investigation of their safety for human health. This study aimed at investigating the potential respiratory outcomes of two CMF/CNF and one CNC produced from bleached Eucalyptus globulus kraft pulp using human alveolar epithelial (A549) cells grown in monoculture or co-cultured with THP-1 monocyte-derived macrophages, by assessing their cellular uptake, cytotoxic, immunotoxic, genotoxic, and epigenetic effects. The nanocelluloses were characterized for their physicochemical properties: CMF displays a low percentage of nanofibrils while CNF comprises 100% fibrils with a diameter (D) circa 11 nm; CNC consists of nanorods with D of 4-5 nm and aspect ratio around 42. TEM analysis evidenced that CMF and CNF were internalised into A549 cells whereas CNC were not. Neither cytotoxicity (colorimetric and clonogenic assays) nor ROS induction was observed for any of the nanocelluloses. CMF caused chromosomal alterations (in vitro micronucleus assay) in A549 cells while negative results were obtained in co-culture and for the other micro/nanocelluloses in mono- or co-culture. Results in progress of DNA damage and gene mutation analyses will complement mutagenesis assessment. Additionally, potential inflammatory and epigenetic effects are being evaluated. These results contribute to the weight of evidence of nanocelluloses biological effects and knowledge of the underlying molecular mechanisms. Such information will drive the synthesis of the safest nanocelluloses,thus minimising potential negative impacts of their use on human and environmental health.
- Investigation of the genotoxicity of digested titanium dioxide nanomaterials in human intestinal cellsPublication . Vieira, Adriana; Vital, Nádia; Rolo, Dora; Roque, Rossana; Gonçalves, Lídia M.; Bettencourt, Ana; Silva, Maria João; Louro, HenriquetaThe widespread use of titanium dioxide nanomaterials (TiO2 NMs) in food and consumer products such as toothpaste or food contact materials, suggests the relevance of human oral exposure to these nanomaterials (NMs) and raises the possibility of adverse effects in the gastrointestinal tract (GIT). We previously showed that the in vitro digestion of TiO2 NMs may increase their toxicity in intestinal cells. In this work, we analyzed the genotoxicity and the intracellular reactive oxygen species induction by physiologically relevant concentrations of three different TiO2 NMs (NM-102, NM-103 and NM-105) in Caco-2 and HT29-MTX-E12 intestinal cells, while considering the potential influence of the digestion process in the NMs' physiochemical characteristics. The results evidenced a DNA-damaging effect dependent on the NM, more relevant for the rutile/anatase NM-105, possibly due to its lower hydrodynamic size in the cells medium. In addition, the results of the micronucleus assay suggest effects on chromosomal integrity, an indicator of cancer risk, in the HT29-MTX-E12 cells, for all the tested TiO2 NMs, especially after the in vitro digestion. This work supports the evidence for concerns on the use of TiO2 NMs as a food additive, recently reported by EFSA, and for their use in applications in consumer products that may drive human exposure through ingestion.