Evaluating the genotoxicity of cellulose nanofibrils in a co-culture of human lung epithelial cells and monocyte-derived macrophages

Abstract

Cellulose 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/cm. 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.

TEMPO-Nanocellulose/Ca2+ Hydrogels: Ibuprofen Drug Diffusion and In Vitro Cytocompatibility

“…In particular, in our study, percentage cell viability measured for cells cultured with C1 eluates was slightly higher than cells cultured in culture medium without TOUS-CNFs samples, for all the considered time points. This result is in accordance with the percentage cell viability described by other authors [62] that cultured lung epithelial alveolar cells (A549) in the presence of low concentrations of TOUS-CNFs. In particular, the authors demonstrate that cells directly exposed to low concentrations of TOUS-CNFs are characterized by increased proliferation, thus leading to a percentage of cell viability higher than the control (i.e., cells cultured in culture medium without TOUS-CNFs).…”

Human hazard potential of nanocellulose: quantitative insights from the literature

“…Menas et al (2017) however did find that CNC induced a significant inflammatory response in A549 cells, greater than the two CNF materials also investigated in the study. Also using A549 cells as test systems, Ventura et al (2018) similarly did not observe any inflammatory response tied to TEMPOmediated CNF. In a study interested in safety surrounding friction grinding and spray drying of CNF, no inflammatory effects on human peripheral blood mononuclear cells (PBMCs) and murine macrophages (RAW 264.7) were observed (Vartiainen et al 2011).…”

“…Ruby cotton was the only CNF source that did not induce any significant DNA breaks in either cell types. Similarly, Ventura et al (2018) assessed genotoxicity using the Comet and Micronucleus assays in a co-culture of A549 cells (adenocarcinomic human alveolar basal epithelial) and THP-1 macrophages. A genotoxic effect was observed from the Comet assay results at the highest tested concentration (range 1.5-25 mg/cm 2 ), although DNA damage was low, as was the case for de Lima et al (2012).…”

Genotoxicity assessment of cellulose nanofibrils using a standard battery of in vitro and in vivo assays

“…Several genotoxicity assessments have previously been performed by a single test, resulting in varying test materials and results. In vitro micronucleus assay data illustrated that CNF produced by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation of an industrial bleached Eucalyptus globulus were genotoxic at low, but not high, concentrations [ 15 ]. A bacterial reverse mutation test (Ames test) using Salmonella typhimurium showed no genotoxic effect of fractionated fibrillated cellulose [ 16 ].…”

“…Furthermore, considering the nanofibrous structure of CNFs, it is important to assess mutagenicity and genotoxicity potencies. While several genotoxicity assessments using single tests have been reported [ [15] , [16] , [17] , [18] ], using a standard battery of genotoxicity tests covering a wide range of mechanisms is essential to further clarify CNF genotoxicity. A standard battery of genotoxicity tests are defined by the following two options for testing pharmaceuticals based on the guidance of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use [ 19 ].…”