Browsing by Author "Marques, Catarina A.C."
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- Avaliação da genotoxicidade de nanomateriais através do ensaio do micronúcleo: Uma comparação interlaboratorial para a sua validação.Publication . Marques, Catarina A.C.; Silva, Maria João; Martins, Francisco Rente de PinaNanomaterials (NMs) have unique physico-chemical characteristics that make them ideal for various applications in numerous areas. With the increase in the use of NMs in some industries and consumer products, among others, human and environmental exposure to these substances has increased more and more, and further studies are needed to understand the potential risks associated with such exposure. Currently, the in vitro micronucleus assay (MN) in cytokinesis-blocked cells is one of the most widely used tests for genotoxic substances assessment and is considered the gold standard when compared to other similar tests. Even though it is an appropriate test, the comparability of the MN assay results is difficult due to inconsistent results reported. Thus, there is a need to develop harmonised protocols and select the most sensitive cell lines, given the particularities of these substances, in order to validate techniques like this one This project, which is part of a larger international study involving interlaboratorial comparisons, aims to optimize the in vitro micronucleus assay to obtain a more sensitive and reliable assay for the evaluation of nanomaterial genotoxicity in mammalian cell lines. The toxicity of nanomaterials was evaluated, namely, cerium dioxide (CeO2), barium sulfate (BaSO4), silica (SiO2), cellulose nanofibrillated (CNF) and cellulose nanocrystalline (CNC), all of them with varied applications in industry and consumer products. Chinese hamster lung fibroblasts (V79 cells) were exposed to different concentrations of each NM during 1 cell cycle (17 hours) to assess cytotoxicity through the MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and for 4 days for the clonogenic assay. Genotoxicity was assessed by the MN assay after exposure to the same concentration-range of nanomaterials for 17 hours. The results showed a significant decrease in cell viability after exposure to BaSO4 at the highest concentrations (64, 128 and 256 μg/mL) tested. The exposure for 4 days to BaSO4, CNF or CNC led to a decrease in cell proliferation, showing positive cytotoxic effects for these NMs. As for genotoxic effects, assessed by the MN assay, the results revealed a significant increase in the frequency of micronucleated cells after exposure to CNF, as well as to the lowest concentration of BaSO4. CeO2 and SiO2 nanomaterials were not genotoxic. The knowledge generated through this project will contribute to data set on the use of the in vitro micronucleus assay for the genotoxicity assessment of nanomaterials. The results obtained for the several NMs assessed suggest that the protocol used for the MN assay allowed to distinguish differential effects regarding their genotoxicity. Furthermore, the results generated will add to the safety assessment of the NMs under study, thereby contributing to their safe application in industry, consumer products and nanotheranostics. A future interlaboratory comparison will allow to assess the assay reproducibility and adequacy, considering the physical-chemical characteristics of the NMs under test, and the use of standardized protocols will improve results comparability.