Approved Abstracts

Long-term exposure to fluoride during adolescence to adulthood modulates hippocampal proteome and causes long-term memory impairments associated with a neurodegenerative pattern in mice



Author(s): Bittencourt LO; Dionizio A; Ferreira MKM; Aragão WAB; Queiroz, LY; Cartágenes SC; Fernandes LMP; Puty B; Buzalaf MAR; Maia CSF; Lima RR;
Presenter: Leonardo Oliveira Bittencourt

The fluoride (F) is present in human daily life through various forms, as water supply and dentifrices, due to its effectiveness in controlling caries disease. Moreover, it is also used in pharmaceutical factory and in the aluminum production, and found in naturally fluoridated water reservoirs. Due to the possibility of characterizing it as an environmental pollutant, caused by natural or anthropogenic actions, there is a big effort to investigate the F toxic effects beyond the skeletal and dental fluorosis, and for that reason, an intense debate has grown regarding the safety of water fluoridation and its possible association with F-induced neurotoxicity, especially regarding cognitive functions. Therefore, this study aimed to investigate the effects of long-term and systemic exposure to F on the hippocampus, a fundamental neural structure for cognitive processes, of mice through molecular and morphological parameters associated with cognitive functions of memory and learning. For this, thirty-six male Swiss mice were exposed to two different F concentrations through the drinking-water: 10mg/L of F, equivalent to the concentrations of artificially fluoridated water supply and 50mg/L of F, equivalent to the concentration of F in water of endemic regions of fluorosis, both adapted to rodent’s metabolism, for a period of 60 days during the maturation of central nervous system of mice (beginning at the 21st postnatal day). Control group received only the vehicle for the same period. After the experimental period, the hippocampal functions were assessed by Morris Water Maze and Step-down Inhibitory Avoidance tests, to evaluate the short- and long-term memories. Then, the F content was determined in blood plasma and hippocampus, and the hippocampal global proteomic profile was based on a label-free proteomic analysis in a nanoACQUITY UPLC system coupled to a mass spectrometer (UPLC/MS). It was performed bioinformatic approaches as Gene Ontology Term Enrichment by Cytoscape software and Over-representation analysis using R programing language. Also, it was evaluated the neuronal density by anti-NeuN labeling in CA1, CA3, Dentate Gyrus (DG) and Hilus regions of hippocampus through immunohistochemistry. The results were analyzed by 1-way ANOVA with Tukey's post-hoc test, adopting p<0.05; the results obtained from the UPLC/MS were analyzed by the ProteinLynx GlobalSERVER software, using the Monte-Carlo algorithm. The results evidenced that both concentrations elevated the F bioavailability in plasma, but only 50 mg/L of F group had higher F levels in hippocampal parenchyma. The proteomic approach revealed that 10mg/L of F modulated 444 proteins and 50mg/L of F, 388 proteins, both compared to control group. The proteins are mainly related to energy metabolism, synaptic transmission and hippocampal development. Moreover, it was observed a pattern of neurodegeneration only in 50 mg/L of F group in DG and CA3 regions. No short-term memory impairment was observed in both exposed groups, but the highest F concentration triggered long-term memory deficits. In conclusion, it was observed that the long-term exposure to 10 mg/L of F does not affect hippocampal integrity and cognitive functions, possibly due to the neuroplasticity mechanisms and synaptic signaling rearrangement observed in the proteomic analyses, while 50 mg/L of F elicits long-term memory impairments associated with neurodegeneration and negative modulation of proteins of synaptic transmission. These data, in a translation perspective, reinforce the safety of water fluoridation with proper regulation and highlight the risks of long-term exposure to F at high concentrations. This study was financed in part by the Brazilian National Council for Scientific and Technological Development (CNPq).

Keywords: Fluoride; Cognition ; Neurotoxicity

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