Browsing by Author "Teixeira, Sara"
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- Exploring potential genotoxic effects of nanocelluloses versus multi-walled carbon nanotubes in co-cultures of human lung epithelial cells and monocyte-derived macrophagesPublication . Pinto, Fátima; Ventura, Célia; Teixeira, Sara; Lourenço, Ana Filipa; Fernandes, S.N.; da Rosa, R.R.; Godinho, M.H.; Ferreira, Paulo J.T.; Louro, Henriqueta; Silva, Maria JoãoCellulose nanomaterials (CNMs) are advanced materials exhibiting unique properties for innumerous industrial and biomedical applications. Human exposure to CNMs has been equally growing, which raises some concern, given the similarity of some CNMs size and morphology with that of multiwalled carbon nanotubes (MWCNTs) that induce lung toxicity. The genotoxic effects of three CNMs produced from Eucalyptus globulus bleached kraft pulp through different methods and, hence, harbouring different physicochemical properties (two micro/nano- fibrillated and one nanocrystalline), were investigated in human alveolar (A549) cells co-cultured with monocyte- derived (THP-1) macrophages and in conventional A549 cultures. Two MWCNT differing in diameter, length and flexibility, NM 401 and NM 402 (JRC Repository), were used as references. None of the CNMs was toxic to A549 cells. The results of the in vitro micronucleus assay showed that exposure of A549 cells (1.5 to 50 μg/cm2 48h) to each CNM or to NM 402), either in monoculture or in co-culture, did not produce significant alterations in the frequency of micronucleated binucleated cells (MNBNC), as compared to the control. In contrast, NM-401, the thickest, longest and more rigid nanofiber, was able to significantly increase the frequencies of MNBNC. The cytokinesis-block proliferation index of A549 cells was not affected by CNMs or MWNTs exposure. To conclude, although data from other endpoints is needed, the present in vitro data suggests that the studied CNMs are neither toxic nor genotoxic to lung cells, increasing the weight of evidence in favor of their biocompatibility.
- Exploring the toxicity of cellulose nanofibrils in a lung epithelial cell linePublication . Teixeira, Sara; Lourenço, Ana Filipa; Ventura, Célia; Louro, Henriqueta; Ferreira, Paulo; Silva, Maria JoãoNanotechnologies and nanomaterials (NMs) applications have been growing in recent years, bringing benefits to society but raising also some concerns about their safety to human health. Cellulose is a natural material that fits the global trend of sustainability: ecological, low cost, abundant and renewable nature. In particular, cellulose nanofibrils (CNF)1 are forest-derived products with advantageous mechanical, optical and rheological properties, assuming a high industrial potential, e.g., in paper, food, pharmaceutical and biomedical industries. With the innovative applications expanding, CNF synthesis and production has increasing, leading to concerns about occupational exposure, particularly by inhalation, or consumers exposure. The toxicity studies of other NMs, like MWCNT, have had a major impact on the understanding of the nanofibres health effects on humans. MWCNTs have been reported to cause adverse effects in vitro and in vivo, such as DNA damage and oxidative stress2. Because CNF show a high resemblance in terms of aspect ratio to MWCNT, our main focus is to identify if some of the CNF synthesized have a genotoxic or carcinogenic potential. This study aims to assess the safety of two types of CNF produced with different pre-treatments (TEMPO-mediated oxidation and enzymatic hydrolysis) of an industrial bleached Eucalyptus globulus kraft pulp, through the characterization of its cytotoxicity and genotoxicity in human cells. The CNF cytotoxicity was assessed using lung epithelial alveolar (A549) cells by two methodologies, the MTT and the clonogenic assay, whereas the genotoxicity was assessed by the cytokinesis-block micronucleus assay. Dose-range finding experiments were performed using the MTT (24h, 48h and 72h exposure) and the clonogenic (8 days exposure) assays, which revealed that both CNF were not cytotoxic at concentrations between 3,125 and 100 μg/ml. On the contrary, both CNFs were able to increase cell viability at the highest concentrations tested (50 and 100 μg/ml). This effect had been previously observed in the same cell line exposed to CNF produced by TEMPO-mediated oxidation, but at the lowest concentration level3. The potential of the CNF to induce chromosomal alterations, either chromosome breaks or loss is being analysed through the micronucleus assay and the results will be presented. Overall, this study is expected to uncover potential adverse outcomes of CNF to human health, in order to promote the design of safer CNF and CNF-based products that will allow a more sustainable and responsible industrial development. References: 1) Gamelas, J., Pedrosa, J., Lourenço, A., Mutjé, P., González, I., Chinga-Carrasco, G., Singh, G. and Ferreira, P. (2015). On the morphology of cellulose nanofibrils obtained by TEMPO-mediated oxidation and mechanical treatment. Micron, 72, 28-33. 2) Louro, H., Pinhão, M., Santos, J., Tavares, A., Vital, N. and Silva, M. (2016). Evaluation of the cytotoxic and genotoxic effects of benchmark multi-walled carbon nanotubes in relation to their physicochemical properties. Toxicology Letters, 262, 123-134. 3) Ventura, C., Lourenço, A., Sousa-Uva, A., Ferreira, P. and Silva, M. (2018). Evaluating the genotoxicity of cellulose nanofibrils in a co-culture of human lung epithelial cells and monocyte-derived macrophages. Toxicology Letters, 291, 173-183.
- A predictive toxicology approach to characterize potential respiratory effects of functionalized nanocellulose fibres in a co-culture systemPublication . Ventura, Célia; Teixeira, Sara; Louro, Henriqueta; Lourenço, Ana Filipa; Ferreira, Paulo J.T.; Silva, Maria JoãoCellulose nanofibrils (CNF) have an enormous potential for industrial and biomedical applications, assuming a great economic value. Because other nanofibres, e.g. carbon nanotubes (CNT), have revealed toxicity 1,2, there is a need to comprehensively evaluate the toxic potential of CNF along the value chain, before they enter the market. This project is aimed at a safety evaluation of several CNF comparatively to CNT, in a co-culture of human-derived alveolar epithelial cells and macrophages3. A predictive toxicology approach is used, i.e., the toxicity will be characterized alongside the specific fibre-associated mode of action, including immunotoxicity, genomic and epigenetic effects. The data obtained for two CNF synthesized from Eucalyptus gobulus but using different pre-treatments will be presented. Future work includes the use of omics-based tools adapted to the toxicity assessment of CNF and other NMs that will give some insights on cellular and molecular mechanisms underlying CNF toxicity. The overall results will be used to ensure the safety of these CNF or to allow the modification of toxic CNF in order to reduce the adverse outcomes, thereby complying with the safer-by-design approach.
- Safety assessment of nanofibrillated cellulose to human health using cellular modelsPublication . Ventura, Célia; Lourenço, Ana Filipa; Vilar, Madalena; Teixeira, Sara; Ferreira, Paulo J.T.; Silva, Maria JoãoNanomaterial - A natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm - 100 nm. In specific cases and where warranted by concerns for the environment, health, safety or competitiveness the number size distribution threshold of 50% may be replaced by a threshold between 1 and 50%. (2011/696/EU)
- Study on cellulose nanofibrils biological effects in different cellular modelsPublication . Pinto, Fátima; Ventura, Célia; Teixeira, Sara; Vilar, Madalena; Lourenço, Ana Filipa; Ferreira, Paulo J.T.; Louro, Henriqueta; Silva, Maria JoãoCellulose nanofibrils (CNFs), is an innovative environmental friendly material that has been incorporated into several types of materials, in both pure and composite forms and holds great promise in different fields of application. This leads to a rising level of human exposure and draws considerable concerns regarding its potential toxicity in humans, which are specially motivated by the physicochemical resemblance of cellulose nanofibrils with multi-walled carbon nanotubes and asbestos that have shown deleterious health effects. Currently, several in vitro toxicological studies have been performed with the aim of predicting health effects caused by exposure to CNFs. A literature review was performed to gather and analyse recent data regarding the potential cytotoxic, genotoxic, immunotoxic and epigenetic effects, in different in vitro cell models, triggered by the exposure to CNFs produced from vegetal biomass and consequently differing in critical physicochemical characteristics. The main findings of this review work demonstrate that different functionalization affects CNFs hydrophobicity, surface charge and chemistry, which may either facilitate or difficult the uptake and interaction of its functional groups with the cell membrane, affecting the biological responses. Generally, CNFs are not phagocytized and do not cause inflammatory response, but revealed in vitro genotoxicity. In fact, our data have shown that CNFs may induce chromosome damage in some cell models, e.g., a co-culture of epithelial lung alveolar cells and macrophages. However, more studies are required to assess CNFs toxicity and to understand the relationship between their physicochemical properties, their behaviour in biological media and mechanism of action, which are of outmost importance to predict their nanosafety.
- The use of the in vitro micronucleus assay on the genotoxicity assessment of cellulose nanofibrils in mammalian cell linesPublication . Ventura, Célia; Teixeira, Sara; Marques, Catarina; Vilar, Madalena; Pinto, Fátima; Lourenço, A.F.; Sousa Mendes, A.; Ferreira, Paulo J.T.; Louro, Henriqueta; Silva, Maria JoãoWith the expansion of innovative cellulose nanofibrils (CNF) applications, either for industrial (e.g., paper industry) or biomedical purposes (e.g., tissue regeneration and dentistry), human exposure has been increasing, raising concerns about their potential health outcomes . Being persistent high aspect-ratio nano-objects, CNFs effects may resemble those of some multi-walled carbon nanotubes, e.g., the MWCNT-7, which has been reported to induce chromosome instability, cytoskeleton alterations and dysfunction of several cancer-related cell signaling pathways . The in vitro cytokinesis-block micronucleus (CBMN) assay is a sensitive and reliable assay for analysis of chromosome damage in mammalian cells, with the potential of providing mechanistic information and predicting carcinogenic effects. In this work we evaluated the use of the CBMN assay in different mammalian cell systems to characterize the genotoxicity of a CNF produced from an industrial bleached Eucalyptus globulus kraft pulp through TEMPO-mediated oxidation followed by a high-pressure homogenization process . The CNF sample was fully characterized in order to assess the most relevant physicochemical properties. It presented a wide distribution of diameters, with the mode in the 20–25 nm range and a length of several micrometers. Preliminary doserange finding through assessment of the cytotoxic effects (MTT and clonogenic assays) in cell lines representative of the respiratory tract (A549, A549 co-cultured with macrophage-like THP-1 cells and V79 cells) and bone (MG63 osteoblasts) showed that the CNF was not cytotoxic, regardless of the exposure period. The results of the CBMN assay (following OECD guideline 487) showed that exposure to low CNF concentrations induced a significant increase of micronuclei in co-cultured alveolar (A549) cells and in osteoblasts, while no significant effect was observed in A549 cells in monoculture. These results support the suitability of the in vitro micronucleus assay to assess the genotoxicity of nanofibers like CNF, highlighting the relevance of the cell system selected. This and other factors related to the experimental design are discussed, pointing to the need of nano-specific guidelines for this assay application in nanogenotoxicology. References Ventura et al. 2020. Cellulose, DOI 10.1007/s10570-020-03176-9 Ventura et al. 2020. Nanotoxicology, 14:479 Ventura et al.2020. Toxicol Lett, 328: 7 Fenech, M. 2007. Nature Protocols 2: 1084. Lourenço et al. 2017. Cellulose 24: 349.