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Evaluation of the effects of secondary microplastics on the respiratory system of BALBc mice and on human bronchial epithelial cell culture BEAS-2B

Abstract

Plastic is a polymer with a characteristic that makes its use highly convenient. Consequently, plastic debris is found all over the world and in all ecosystems. Plastics are environmentally persistent, however, once released into the environment, they are exposed to continuous processes such as chemical weathering, photo-oxidation, biological decomposition and mechanical forces, which affect their structural integrity and result in fragmentation to micrometer levels in size, microplastics (MPs) and are those that represent the greatest concern in relation to possible damage to exposed organisms. MPs were identified in foods intended for human consumption, in indoor and outdoor air samples and in human lung tissue samples. Studies on the effect of microplastics on the respiratory system are very scarce. Most of the current knowledge about the effects of MPs on the human respiratory system comes from studies based on occupational exposure that report an increased risk of worsening latent asthma, the risk of exacerbation and the risk of developing asthma. In vitro studies of the respiratory system show changes in the production of pro-inflammatory cytokines, increased oxidative stress and changes in the expression of proteins associated with the cell cycle and pro-apoptosis. To the best of our knowledge, the only published in vivo study demonstrated that the exposure of mice to MP particles led to an increase in inflammatory cell infiltrate, aggregation of bronchoalveolar macrophages, increased TNF-± level in bronchoalveolar lavage (BAL) and increased plasma IgG1 production in mice. In this study, as in the in vitro ones, the primary MPs (MPp) are studied, those that were not exposed to chemical and physical processes and to various interactions with other pollutants in the environment. In the present study, we will evaluate the effects of naturally degraded secondary MP particles (MPd) and MPp on the respiratory system of mice, male and female, and in human epithelial cell cultures. Methods: About 50 grams of the plastic was removed from the open environment where it remained for approximately 84 months, brushed to remove soil residues, and crushed using a planetary ball mill, obtaining particles with a size in the order of 1 µm. Fourier transform infrared spectroscopy was used to identify the polymer that makes up the MPd sample. The characterization of the elemental composition of the MPs samples will be done by neutron activation (INAA). BALB c mice, 30 females and 30 males, divided into 6 groups, will receive daily for 28 days by intranasal instillation of 20 µL saline (control) and, for the exposed groups, 20 µL of saline with 300 µg of MPd or MPp. On the 29th day, pulmonary mechanics parameters will be captured and blood collected to quantify IgG1 and IgE immunoglobulins, and interleukins, IL-13, IL-33, TNF-± and IFN-³ in plasma by cytometric bead array (CBA). After euthanasia, BAL will be collected for quantification of inflammatory markers Th1, Th2 and Th17 in the supernatant and for differential cytology in the precipitate. The lungs will be removed and the distal and proximal portions will be prepared for quantification of inflammatory cells and differential quantification of mucus (histology), quantification of macrophage markers and oxidative stress (immunohistochemistry). Human bronchial epithelial cells, BEAS-2B, will be cultivated in a culture medium containing MPs at a concentration determined from the MTT cell viability test. The CBA will be used to quantify the cytokines IL-1B, IL-6, IL-8, and TNF-alpha and GM-CSF in the culture supernatant. PCR-Real Time will be used to identify the CYP1A1 genes, the AhR gene and B-actin. (AU)

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