A predictive toxicology approach to characterize potential respiratory effects of functionalized nanocellulose fibres in a co-culture system

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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

The use of the in vitro micronucleus assay on the genotoxicity assessment of cellulose nanofibrils in mammalian cell lines

Publication . Ventura, Célia; Teixeira, Sara; Marques, Catarina; Vilar, Madalena; Pinto, Fátima; Lourenço, A.F.; Sousa Mendes, A.; Ferreira, Paulo J.T.; Louro, Henriqueta; Silva, Maria João

With 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.

2020-09-12Conference object

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New “Omics” Approaches as Tools to Explore Mechanistic Nanotoxicology

Publication . Ventura, Célia; Torres, Vukosava; Vieira, Luís; Gomes, Bruno; Rodrigues, António Sebastião; Rueff, José; Penque, Deborah; Silva, Maria João

In the last years, “omics” approaches have been applied to study the toxicity of nanomaterials (NM) with the aim of obtaining insightful information on their biological effects. One of the most developed “omics” field, transcriptomics, expects to find unique profiles of differentially expressed genes after exposure to NM that, besides providing evidence of their mechanistic mode of action, may also be used as biomarkers for biomonitoring purposes. Moreover, several NM have been associated with epigenetic alterations, i.e., changes in the regulation of gene expression caused by differential DNA methylation, histone tail modification and microRNA expression. Epigenomics research focusing on DNA methylation is increasingly common and the role of microRNAs is being better understood, either promoting or suppressing biological pathways. Moreover, the proteome is a highly dynamic system that changes constantly in response to a stimulus. Therefore, proteomics can identify changes in protein abundance and/ or variability that lead to a better understanding of the underlying mechanisms of action of NM while discovering biomarkers. As to genomics, it is still not well developed in nanotoxicology. Nevertheless, the individual susceptibility to NM mediated by constitutive or acquired genomic variants represents an important component in understanding the variations in the biological response to NM exposure and, consequently, a key factor to evaluate possible adverse effects in exposed individuals. By elucidating the molecular changes that are involved NM toxicity, the new “omics” studies are expected to contribute to exclude or reduce the handling of hazardous NM in the workplace and support the implementation of regulation to protect human health.

2022Book part

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