Prolonged exposure to high fluoride levels during adolescence to adulthood elicits molecular, morphological, and functional impairments in the hippocampus

Abstract

Fluoride is added to water due to its anticariogenic activity. However, due to its natural presence in soils and reservoirs at high levels, it could be a potential environmental toxicant. This study investigated whether prolonged exposure to fluoride from adolescence to adulthood-at concentrations commonly found in artificially fluoridated water and in fluorosis endemic areas-is associated with memory and learning impairments in mice, and assessed the molecular and morphological aspects involved. For this endeavor, 21-days-old mice received 10 or 50 mg/L of fluoride in drinking water for 60 days and the results indicated that the increased plasma fluoride bioavailability was associated with the triggering of short- and long-term memory impairments after high F concentration levels. These changes were associated with modulation of the hippocampal proteomic profile, especially of proteins related to synaptic communication, and a neurodegenerative pattern in the CA3 and DG. From a translational perspective, our data provide evidence of potential molecular targets of fluoride neurotoxicity in the hippocampus at levels much higher than that in artificially fluoridated water and reinforce the safety of exposure to low concentrations of fluoride. In conclusion, prolonged exposure to the optimum fluoride level of artificially fluoridated water was not associated with cognitive impairments, while a higher concentration associated with fluorosis triggered memory and learning deficits, associated with a neuronal density reduction in the hippocampus.

Figure 1

Methodological scheme summarizing the experimental and analytical steps of the study. In (A) the protocol of fluoride exposure during 60 days in male mice and groups division. In (B) behavioral assessments performed after exposure period to analyze cognitive functions. After euthanasia, blood and hippocampus were collect to assess the plasma fluoride levels (C) and global proteomic profile of the hippocampus (D). A set of animals were perfused for immunohistochemical analyses by investigating anti-NeuN immunostaining in the hippocampal regions for cell density determination.

Figure 2

The effects of 60 days of fluoride exposure (10 or 50 mg/L) from adolescence to adulthood on the plasma fluoride level of mice (A) and body weight (g) during the experimental period (B). The data are presented as mean ± standard error of the mean (SEM). Statistical analysis: in A, one-way analysis of variance (ANOVA) with Tukey’s post hoc test; in (B), two-way ANOVA. Different letters indicate a significant difference (p < 0.05).

Figure 3

Circos plot of the protein–protein networks in the hippocampus of mice exposed to 10 or 50 mg/L of fluoride. The networks are associated with biological processes including cellular component organization (yellow), nervous system development (light blue), response to stimulus (pink), metabolic process (greyish blue), nervous system process (green) and synaptic signalling (beige), based on Gene Ontology annotations. Each protein is described with its respective UniProt accession ID, and each coloured rectangle indicates a different comparison, with a log2ratio ranging from − 1 to 1.

Figure 4

The effects of fluoride exposure from adolescence to adulthood on the mature neuronal density in mouse hippocampus. NeuN immunohistochemistry in the CA1 (A–D), CA3 (E–H), dentate gyrus (I–L) and hilus (M–P) of mouse hippocampus. The data are presented as the mean ± standard error of the mean (SEM) of the neuronal density. Different letters indicate a significant difference (p < 0.05, one-way analysis of variance with Tukey’s post hoc test). The scale bar is 50 µm.

Figure 5

The effects of fluoride exposure from adolescence to adulthood on step-down inhibitory avoidance test performance of mice. (A) and (B) show the step-down latency (seconds) in the short-term (1.5 h) and long-term (24 h) memory assessments, respectively. Different letters indicate a significant difference (p < 0.05, one-way analysis of variance with Tukey’s post hoc test).

Figure 6

The effects of fluoride exposure from adolescence to adulthood on Morris’s water maze performance of mice. (A) and (B) show the escape latency time (ELT, seconds) in the first and fourth training sessions, respectively. (C) shows the arrival latency time (ALT, seconds) in the target quadrant (Q4). (D) shows the time spent (seconds) in the target quadrant (Q4). Different letters indicate a significant difference (p < 0.05, one-way analysis of variance with Tukey’s post hoc test).