Browsing by Author "Ferreira, Paulo J.T."
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- 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.
- Cytotoxicity Assessment of Novel Cellulose Nanomaterials on Intestinal CellsPublication . Vital, Nádia; Pinto, Fátima; Rolo, Dora; Pedrosa, Jorge; Ferreira, Paulo J.T.; Kranendonk, Michel; Silva, Maria João; Louro, HenriquetaCellulose nanomaterials (CNMs) emerged as an important group of nanomaterials with potential applications in multiple food-related products, as zero-calorie filler/thickener/stabilizer, or as a substitute for petroleum-based food packaging materials. Human oral exposure to CNMs is increasing, but little is known about the potential adverse biological impact of CNMs on human gastrointestinal tract. To contribute to the development of innovative CNMs for the food sector and to their early-stage safety assessment, in this work new CNMs were prepared, and their cytotoxic effects were investigated in human intestinal cell lines. Two cellulose micro/nanofibrillated (CNFs), were synthetized from industrial bleached Eucalyptus globulus kraft pulp using different pretreatments (enzymatic and TEMPO-mediated oxidation) followed by a high-pressure homogenization process. Potential cytotoxic effects were evaluated by the MTT assay using two human intestinal cell models (Caco-2 and HT29-MTX-E12). Since in humans the digestion process may modify the physicochemical properties of NMs, potentially influencing biological outcomes, the CNFs were subjected to a harmonized in vitro digestion method before cytotoxicity testing. No cytotoxic effect was observed after 24h exposure to the undigested CNFs in the concentration-range tested (3.1 µg/mL-200 µg/mL), irrespectively of the cell line used. Similar results were obtained for the digested CNFs for concentrations up to 14.3 µg/mL. The observation that the in vitro digestion mixture was cytotoxic by itself for concentrations above 7.6 % (v/v) (i.e., equivalent to CNMs exposure > 14.3 µg/mL) impaired cytotoxicity assessment at higher CNFs concentrations. Complementary cytotoxicity assays and future optimization of the in vitro digestion procedure to reduce its toxicity are underway, to refine the assessment of CNFs cytotoxicity, particularly after digestion. Furthermore, genotoxicity studies will increase the knowledge on the cellular effects of CNMs in the human intestine, contributing to the safety assessment of CNMs early in its development stage, towards sustainable innovation of nanomaterials, thereby protecting human health.
- Epigenomics as a novel approach to explore the toxic effects of nanomaterialsPublication . Ventura, Célia; Vieira, Luís; Valente, Ana; Fernandes, Camila; Silva, Catarina; Louro, Henriqueta; Ferreira, Paulo J.T.; Silva, Maria JoãoIn recent years, there has been a huge development of innovative engineered nanomaterials with potential use in industrial and biomedical applications. This increased widespread use raised concerns that nanomaterials may elicit human adverse health effects through occupational, environmental or consumer exposure. Many toxicity studies, mainly in vitro, have showed that some nanomaterials, such as carbon nanotubes or titanium dioxide nanoparticles (TiO 2 NP), may cause genotoxicity, inflammation, and associated adverse health effects. Nevertheless, few studies have focused on the nanomaterials effect s on the epigenome, namely, modifications of histone tails, microRNA expression or DNA methylation. Here we wil l present two “omics” studies based on next generation sequencing , one focusing on the effect of three nanocellulose s derived from Eucaliptus globulus kraft pulp on the microRNA expression of BEAS 2B, and an other one focusing on the effect of three types of TiO 2 NP on the DNA methylation of Caco 2 cells. Regarding the former 24h exposure to fibrillar micro/nanocellulose s did not induced significant (FDR ≤ 0.05) differentially expressed microRNAs, as compared to non exposed cells. By contrast, the crystalline nanocelul l ose induced the over and under expression of 22 and 30 microRNAs, respectively. These microRNAs can be f urther explored as potential biomarkers for human biomonitoring and co ntribute to elucidate the mechanisms of action of crystalline nanocellulose. As to the genome wide methylation study Reduced Representative Bisulfite Sequencing allowed the identification of significant ( p ≤ 0.05) differential methylation of 92, 70, and 88 gene sequences for the anatase, rutile and brookite phase TiO 2 NP exposures, respectively. Functional pathway analysis of these methylation changes showed that all TiO 2 NP may affect cell proliferation, differentiation, and survival, and suggested different molecular mechanisms of action for each type of TiO 2 NP. In conclusion, epigenomics revealed to be a powerful tool to understand the key molecular events underlying nanomaterials effects.
- Evaluating the genotoxicity of cellulose nanofibrils in a co-culture of human lung epithelial cells and monocyte-derived macrophagesPublication . Ventura, Célia; Lourenço, Ana Filipa; Sousa-Uva, António; Ferreira, Paulo J.T.; Silva, Maria JoãoCellulose nanofibrils (CNF) are manufactured nanofibres that hold impressive expectations in forest, food, pharmaceutical, and biomedical industries. CNF production and applications are leading to an increased human exposure and thereby it is of utmost importance to assess its safety to health. In this study, we screened the cytotoxic, immunotoxic and genotoxic effects of a CNF produced by TEMPO-mediated oxidation of an industrial bleached Eucalyptus globulus kraft pulp on a co-culture of lung epithelial alveolar (A549) cells and monocyte-derived macrophages (THP-1 cells). The results indicated that low CNF concentrations can stimulate A549 cells proliferation, whereas higher concentrations are moderately toxic. Moreover, no proinflammatory cytokine IL-1β was detected in the co-culture medium suggesting no immunotoxicity. Although CNF treatment did not induce sizable levels of DNA damage in A549 cells, it leaded to micronuclei formation at 1.5 and 3 μg/cm2. These findings suggest that this type of CNF is genotoxic through aneugenic or clastogenic mechanisms. Noteworthy, cell overgrowth and genotoxicity, which are events relevant for cell malignant transformation, were observed at low CNF concentration levels, which are more realistic and relevant for human exposure, e.g., in occupational settings.
- Evaluation of cytotoxic and genotoxic effects of functionalized nanocellulose in a co-culture system approaching the lung environmentPublication . Pinto, Fátima; Ventura, Célia; Lourenço, Ana Filipa; Ferreira, Paulo J.T.; Louro, Henriqueta; Silva, Maria JoãoIntroduction: Nanocelluloses, obtained from different sources and by various methods and holding different functionalization are innovative environmentally friendly materials in both pure and composite forms that hold great promise for industrial or advanced biomedical applications. Based on previous knowledge on other nanofibers e.g., carbon nanotubes, revealing toxicity at cellular and organismal levels, there is a need to evaluate the toxic potential of new nanocelluloses, particularly cellulose nanofibrils (CNF), before entering the market. In this work, three different types of nanocelluloses produced from Eucalyptus globulus bleached kraft pulp but differing in important physicochemical properties such as the carboxyl content (CCOOH), degree of polymerization (DP), morphology, specific surface area and size of fibrils, were investigated regarding cyto- and genotoxic effects in human alveolar cells and in a co-culture of human alveolar cells (A549 cells) and THP-1 differentiated macrophages. Results: A preliminary evaluation of the cytotoxicity in A549 by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay revealed that all nanocellulose samples showed no toxicity over the examined concentration range during a 24h exposure period. The genotoxicity of the nanocellulose samples was assessed through the cytokinesis-blocked micronucleus assay in A549 co-cultured with THP-1 cells and exposed to 1.5, 3, 6, 12.5, and 25 μg/cm2 of each nanocellulose for 24h. Preliminary results showed no alterations in the frequency of micronucleated binuceated cells for all tested concentrations in one sample of CNF produced with a catalytic oxidation with TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) radical (T1300). Moreover, the cytokinesis-block proliferation index of exposed A549 cells did not show differences as compared with non-exposed cells, in line with the MTT results, suggesting that the tested nanocelluloses do not affect A549 cells proliferation. Conclusions: Preliminary results suggest that T1300 sample do not present cytotoxicity or genotoxicity in the examined concentration range and exposure time, as compared to controls. The other two samples are still being investigated.
- 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.
- Genotoxicity of Three Micro/Nanocelluloses with Different Physicochemical Characteristics in MG-63 and V79 CellsPublication . Ventura, Célia; Marques, Catarina; Cadete, João; Vilar, Madalena; Pedrosa, Jorge F.S.; Pinto, Fátima; Fernandes, Susete Nogueira; da Rosa, Rafaela Raupp; Godinho, Maria Helena; Ferreira, Paulo J.T.; Louro, Henriqueta; Silva, Maria JoãoBackground: Nanocellulose is an innovative engineered nanomaterial with an enormous potential for use in a wide array of industrial and biomedical applications and with fast growing economic value. The expanding production of nanocellulose is leading to an increased human exposure, raising concerns about their potential health effects. This study was aimed at assessing the potential toxic and genotoxic effects of different nanocelluloses in two mammalian cell lines; Methods: Two micro/nanocelluloses, produced with a TEMPO oxidation pre-treatment (CNFs) and an enzymatic pre-treatment (CMFs), and cellulose nanocrystals (CNCs) were tested in osteoblastic-like human cells (MG-63) and Chinese hamster lung fibroblasts (V79) using the MTT and clonogenic assays to analyse cytotoxicity, and the micronucleus assay to test genotoxicity; Results: cytotoxicity was observed by the clonogenic assay in V79 cells, particularly for CNCs, but not by the MTT assay; CNF induced micronuclei in both cell lines and nucleoplasmic bridges in MG-63 cells; CMF and CNC induced micronuclei and nucleoplasmic bridges in MG-63 cells, but not in V79 cells; Conclusions: All nanocelluloses revealed cytotoxicity and genotoxicity, although at different concentrations, that may be related to their physicochemical differences and availability for cell uptake, and to differences in the DNA damage response of the cell model.
- 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.
- On the toxicity of cellulose nanocrystals and nanofibrils in animal and cellular modelsPublication . Ventura, Célia; Pinto, Fátima; Lourenço, Ana Filipa; Ferreira, Paulo J.T.; Louro, Henriqueta; Silva, Maria JoãoThe need for reaching environmental sustainability encourages research on new cellulosebased materials for a broad range of applications across many sectors of industry. Cellulosic nanomaterials obtained from different sources and with different functionalization are being developed with the purpose of its use in many applications, in pure and composite forms, from consumer products to pharmaceutics and healthcare products. Based on previous knowledge about the possible adverse health effects of other nanomaterials with high aspect ratio and biopersistency in body fluids, e.g., carbon nanotubes, it is expected that the nanometric size of nanocellulose will increase its toxicity as compared to that of bulk cellulose. Several toxicological studies have been performed, in vitro or in vivo, with the aim of predicting the health effects caused by exposure to nanocellulose. Ultimately, their goal is to reduce the risk to humans associated with unintentional environmental or occupational exposure, and the design of safe nanocellulose materials to be used, e.g., as carriers for drug delivery or other biomedical applications, as in wound dressing materials. This review intends to identify the toxicological effects that are elicited by nanocelluloses produced through a topdown approach from vegetal biomass, namely, cellulose nanocrystals and nanofibrils, and relate them with the physicochemical characteristics of nanocellulose. For this purpose, the article provides: (i) a brief review of the types and applications of cellulose nanomaterials; (ii) a comprehensive review of the literature reporting their biological impact, alongside to their specific physicochemical characteristics, in order to draw conclusions about their effects on human health.
- 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.