Aplicação de métodos inovadores na avaliação do potencial alergênico de nanomateriais
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2019-05-30
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Universidade Federal de Goiás
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Introduction: The excessive use of fullerene (C60), titanium dioxide (TiO2) and single-wall carbon nanotubes (SWCNTs) in several areas of the industry has resulted in increased human and environmental exposures, evidencing the need for the assessment of toxicological profile of these nanomaterials (NMs). Studies have shown that the interaction of these materials in different systems, including the immune system, may cause several alterations at the cellular and tissue level, demonstrating the need to assess the allergenic potential of these NMs. Nonetheless, few data are available regarding the applicability of in vitro evaluation platforms for NMs toxicity assessment. Objective: The aim of this study was to investigate the skin sensitization potential of NMs (TiO2, SWCNTs and C60) through innovative animal-free methods with regulatory and/or scientific validation. Methods: after dispersion in aqueous solvent, the samples were characterized through the Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA) techniques, while the internalization assays were carried out using the dark field microscopy technique (Cytoviva). For the assessment of skin sensitization potential, validated methodologies for regulatory/scientific purposes were used. The key-event 1 of skin sensitization adverse outcome pathway (AOP) (reactivity with skin peptides) was evaluated using the micro-Direct Peptide Reactivity Assay (micro-DPRA) technique and the key event 3 (dendritic cells activation) using the U-SENSTM assay followed by cytokine quantification through the Cytometric Bead Array method by cytometer of flux. Results: NMs were well dispersed when sonicated allowing the evaluation of the allergenicity and modulation of cytokines tests. With the PDI values SWCNTs (0.148), C60 (0.105) e TiO2 (0.548). The internalization assays demonstrated that NMs were internalized by fibroblasts, achieving the intracellular compartment. The mDPRA demonstrated that the NMs interacts potentially with skin peptides, demonstrating a significant percentage of depletion, characterizing these materials as moderate positive sensitizers according to the first key-event of sensitization. Data from the U-SENSTM assay also demonstrated an increase in CD86 marker expression with Stimulation Index (SI) higher than 150 in a concentration-dependent manner, with EC150 for SWCNTs (189.48 μg/mL), C60 (91.72 μg/mL) and TiO2 (183.05 μg/mL). Additionally, cytokine quantitation assays demonstrated a significant production increase of IL-8 by U937 monocytic cell line exposed to NMs, which is a known biomarker of dendritic cell activation. Conclusion: The results demonstrated that micro-DPRA and U-SENSTM methods are applicable for skin sensitization potential assessment of nanomaterials. Furthermore, the evaluated materials presented a positive profile for skin sensitization regarding the first and the third key-event of the Adverse Outcome Pathway of allergenicity.
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BEZERRA, S. F. Aplicação de métodos inovadores na avaliação do potencial alergênico de nanomateriais. 2019. 65 f. Dissertação (Mestrado em Ciências Farmacêuticas) - Universidade Federal de Goiás, Goiânia, 2019.