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Analysis of the In Vitro Toxicity of Nanocelluloses in Human Lung Cells as Compared to Multi-Walled Carbon Nanotubes

Publication . 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 João

Cellulose 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.

2022-04-22Journal article

Open access

Genotoxicity of Three Micro/Nanocelluloses with Different Physicochemical Characteristics in MG-63 and V79 Cells

Publication . 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 João

Background: 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.

2022-04-21Journal article

Open access

Cyto-genotoxic effects of distinct micro/nanocelluloses in human osteoblastic and mice fibroblastic cells

Publication . Ventura, Célia; Marques, Catarina; Cadete, João; Vilar, Madalena; Pedrosa, J.F.S.; Pinto, P.; Rosa, R.R.; Fernandes, S.N.; Godinho, M.H.; Ferreira, P.J.T.; Louro, Henriqueta; Silva, Maria João

The unique characteristics of micro/nanocelluloses (MNCs) have been boosting their use in a wide variety of industrial and biomedical applications. With the expected decrease of their production cost, it is likely to observe an even faster growing adoption of these materials in the coming years. With the increasing use of MNCs comes an increased likelihood of human exposure to these materials, raising concerns about potential human health effects. The number of studies on the potential adverse effects of MNCs is still scarce and sometimes inconclusive. The aim of the present study is to shed some light on the impact of human exposure to cellulose-based nanomaterials. For that, three MNCs were produced from bleached Eucalyptus kraft pulp using different pretreatments. A microfibrillated (CMF) and a nanofibrillated cellulose (CNF) were respectively obtained by enzymatic and TEMPO-mediated oxidation pretreatments followed by high-pressure homogenization. In addition, cellulose nanocrystals (CNC) were obtained by acid hydrolysis with diluted sulfuric acid. The potential cytotoxicity of the MNCs was evaluated in vitro in two mammalian cell lines (human MG-63 osteoblasts and mice V79 lung fibroblasts) by the MTT and the clonogenic assays, and the genotoxicity was assessed by the micronucleus assay. Cytotoxicity was observed by the clonogenic assay in V79 cells, particularly for CNC, 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. From the results, it was concluded that under the tested conditions all MNCs present cytotoxicity and genotoxicity, although at different concentration levels, which may be related to their physicochemical differences, to the availability for cell uptake, and to differences in the DNA damage response of the cell model.

2022-06-28Conference object

Restricted access

Exploring potential genotoxic effects of nanocelluloses versus multi-walled carbon nanotubes in co-cultures of human lung epithelial cells and monocyte-derived macrophages

Publication . 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 João

Cellulose 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.

2021-09-22Conference object

Restricted access