Approved Abstracts

UNRAVELING MOLECULAR MECHANISMS OF FLUORIDE NEUROTOXICITY ON U87 GLIAL-LIKE CELLS: INSIGHTS FROM TRANSCRIPTOME AND PROTEMOMIC APPROACHES



Author(s): Universidade Federal do Pará; Universidade Federal do Pará; Universidade Federal do Pará; Universidade de São Paulo; Universidade de São Paulo; Universidade de São Paulo; Universidade Federal do Pará; Universidade Federal do Pará;
Presenter: Leonardo Oliveira Bittencourt

Despite being widely used for caries disease control, the over-exposure to fluoride (F) is frequently associated to dental and skeletal fluorosis. Recently, animal-based models have also suggested that F has potential to lead to damage on the central nervous system (CNS) and that glial cells are more susceptible than neurons to F toxic effects in vitro. However, when it comes to F effect on human CNS this subject still controversial on literature. Several studies have raised the debate about the real safety to human-being in a scenario where there is an increase of daily exposure to F-based products. The F over exposure happens from different sources as oral care products, food supplement and tap water. In this way, we aimed to investigate changes on gene and protein expression on a human glial cell in vitro model to understand the molecular mechanism of F toxicity on one of the main CNS cells. For that, U87 human glial-like cells were exposed to two F concentration (0.095µg/mL and 0.22µg/mL) for 10 days to mimic an over-exposure exposure. The low-dose (0.095µg/mL) and higher-dose (0.22µg/mL) concentration are based on human plasma levels from population living on fluorosis endemic area. Genes and protein expression modulation after F exposure were accessed by transcriptomic (based on one-color microarray) and proteomic analysis (based on mass spectrometry). Microarray data analysis were performed following a quality control and quantile normalization using limma package and differentially expressed genes were identified based on an absolute log2 fold change level >2 and the p-value adjusted by FDR p<0.05. The over-representation analysis for differently expressed genes of gene ontology (GO) terms or KEGG pathways were also done with limma package. Over-represented p- values were adjusted by Bonferroni method and only adjusted p-values <0.05 were considered. Proteomic data analysis was performed after protein identification. Categorization Cytoscape 3.6.1 (Java®) software was used for bioinformatic analyses with ClusterMarker plugin for protein-interaction network and ClueGO plugin for determination of biological processes groups. A total of 62 genes were regulated after 0.095µg/mL F while 209 were regulated after 0.22µg/mL F. Concerning protein modulation, 20 proteins were regulated after 0.095µg/mL and 10 proteins after 0.22µg/mL. Gene Ontology annotation have shown main terms related to cellular metabolism and cell death regulation pathways as MAPK, ERK1/ERK2 cascade in a non-dependent concentration manner. The proteomic has confirmed the changes on energy metabolism suggesting a potential molecular adaptation of U87 glial-like cells. Protein modulation also provided evidences of changes on proteins related to cytoskeleton components as actin filaments and tubulin. Our results not only reveal that F has potential to modulate gene and protein profile in human U87 glial-like cells overexposed to F, but also have identified a possible role of F on cytoskeleton disorganization. This study was financed in part by the Programa Nacional de Cooperação Acadêmica na Amazônia – PROCAD/Amazônia from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - This research was also supported by Pró-Reitoria de Pesquisa da UFPA (PROPESP, UFPA, Brazil) and Brazilian National Council for Scientific and Technological Development (CNPq).

Keywords: Fluoride; Neurotoxicology; Central Nervous System

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