An Evaluation of Neurotoxicity Following Fluoride Exposure from Gestational Through Adult Ages in Long-Evans Hooded Rats

Christopher A. McPherson, Guozhu Zhang, Richard Gilliam, Sukhdev S. Brar, Ralph Wilson, Amy Brix, Catherine Picut, and G. Jean Harry
Neurotoxicity Research (2018) DOI: https://doi.org/10.1007/s12640-018-9870-x PMID: 29404855

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Abstract

At elevated levels, fluoride (F-) exposure has been associated with adverse human health effects. In rodents, F- exposure has been reported to induce deficits in motor performance and learning and memory. In this study, we examined Long-Evans hooded male rats maintained on a standard diet (20.5 ppm F-) or a low F- diet (3.24 ppm F-) with drinking water exposure to 0, 10, or 20 ppm F- from gestational day 6 through adulthood. At postnatal day 25, brain F- levels were 0.048 or 0.081 μg/g and femur 235 or 379.8 μg/g for 10 and 20 ppm F-, respectively. Levels increase with age and in adults, levels for plasma were 0.036 or 0.025 μg/ml; for the brain 0.266 or 0.850 μg/g; and for the femur, 681.2 or 993.4 μg/g. At these exposure levels, we observed no exposure-related differences in motor, sensory, or learning and memory performance on running wheel, open-field activity, light/dark place preference, elevated plus maze, pre-pulse startle inhibition, passive avoidance, hot-plate latency, Morris water maze acquisition, probe test, reversal learning, and Y-maze. Serum triiodothyronine (T3), thyroxine (T4), and thyroid stimulating hormone (TSH) levels were not altered as a function of 10 or 20 ppm F- in the drinking water. No exposure-related pathology was observed in the heart, liver, kidney, testes, seminal vesicles, or epididymides. Mild inflammation in the prostate gland was observed at 20 ppm F-. No evidence of neuronal death or glial activation was observed in the hippocampus at 20 ppm F-.

Figures

Figure 1. Running Wheel activity in postnatal day (PND) 25 Long-Evans hooded male rats.

(a) Mean hourly wheel rotations during light and dark phases over 2 days. G1: standard chow/RO-H2O (n = 15); G2: low-F− chow/RO-H2O (n = 15); and G3: low-F− chow/10 ppm F− drinking water (n = 16). Drinking water exposure began on gestational day 6.
(b) Mean hourly wheel rotations over 5 days (mean ± SEM) for G2 (n = 10) and G4: low-F− chow/20 ppm F− drinking water (n = 10). Shaded areas represent dark phase of elevated activity

Figure 1 (5 MB)
Figure 1 supporting data: Running Wheel Shipment 1 2 (27 KB)
Figure 1 supporting data: Running Wheel Shipment 4 (29 KB)

Figure 2. Open-Field activity in postnatal day (PND) 40 Long-Evans hooded male rats.

(a) Ambulatory activity in open field in 5-min epochs (data represents mean ± SEM).
(b) Total distance (cm) traveled in the center of the arena and (c) total time spent in the margin of the arena over 45-min session (data represents mean ± SEM and individual animal response). G1: standard chow/ RO-H2O (n = 20); G2: low-F− chow/RO-H2O (n = 21); G3: low-F− chow/10 ppm F− drinking water (n = 21).
(d) Ambulatory activity,
(e) distance traveled in center, and (f) margin time for G2 (n = 17) and G4: low-F− chow/20 ppm F− drinking water (n = 16). Drinking water exposure began on gestational day 6

Figure 2 (7 MB)
Figure 2 supporting data: Motor Activity Ambulatory (36 KB)
Figure 2 supporting data: Motor Activity Summary Data (22 KB)

Figure 3. Light/dark place preference, hot-plate latency, passive avoidance, startle response, pre-pulse startle inhibition, Y-maze in Long-Evans hooded male rats.

(a–b) Light/dark place preference at postnatal day (PND) 42. (a) Number of entries and (b) percent total time spend in lighted side. G1: standard chow/RO-H2O (n = 20); G2: low-F− chow/RO-H2O (n = 21); G3: low-F− chow/10 ppm F− drinking water (n = 21) and G2 (n = 16) and G4: low-F− chow/20 ppm F− drinking water (n = 17). (c) Hot-plate latency at PND60 (G1: n = 13; G2: n = 13; G3: n = 14; and G2: n = 16, G4: n = 14). (d) Passive avoidance latency over days (G1: n = 13; G2: n = 13; G3: n = 14; and G2: n = 16, G4: n = 14). (e) 120 dB startle amplitude (Vmax) over blocks of 5 trials and (f) percent inhibition of 120 dB response from pre-pulse stimulus intensities of 3, 6, 12, or 15 dB above background. (PND 61–63; G1: n = 18, G2: n = 17; G3: n = 21; G2: n = 12, G4: n = 14). (g) Y-maze number of entries and percent alternation at PND60 from G2 (n = 10) and G4 (n = 9). Drinking water exposure began on gestational day 6. Data represent means ± SEM and individual animal responses. *p < 0.05

Figure 3 (7 MB)
Figure 3 supporting data: Hot Plate Latency (17 KB)
Figure 3 supporting data: Light Dark Preference (13 KB)
Figure 3 supporting data: Passive Avoidance (21 KB)
Figure 3 supporting data: Prepulse Inhibition and Startle (238 KB)
Figure 3 supporting data: Y Maze (31 KB)

Figure 4. Morris water maze performance in adult Long-Evans hooded male rats.

(a) Cued learning across 2 days and spatial training across 7 days. Data expressed as mean latency ± SEM (over three trials per day) to escape onto platform in the goal quadrant (GQ). A significant decrease in latency was observed over training (G1 vs. G2: F(6,228) = 26.28; p < 0.0001; G2 vs. G3: F(6,240) = 20.81; p < 0.0001; G2 vs. G4: F(6,180) = 32.07; p < 0.0001). *significantly different (p < 0.01) as compared to G2. (b) Probe trial initial latencies to enter GQ or platform zone (PZ). (c–d) Probe trial distribution to quadrants across 90- and 30-s epochs for (c) # of visits and (d) duration of time in each quadrant. Data represents mean ± SEM and individual animal responses. G1: standard chow/ RO-H2O (n = 20); G2: low-F− chow/RO-H2O (n = 20); G3: low-F− chow/10 ppm F− drinking water (n = 22) and G2 (n = 15) and G4: low-F− chow/20 ppm F− drinking water (n = 17). Drinking water exposure began on gestational day 6. Distinctions of the individual quadrants: GQ: Northwest goal quadrant; NE, Northeast; SW, Southwest; SQ, Southeast start quadrant

Figure 4 (7 MB)
Figure 4 supporting data: Morris Water Maze Cued Learning (22 KB)
Figure 4 supporting data: Morris Water Maze Spatial (103 KB)
Figure 4 supporting data: Morris Water Maze Spatial Probe Test 30s Epoch DG24 (50 KB)
Figure 4 supporting data: Morris Water Maze Spatial Probe Test 30s Epoch DG123 (78 KB)

Figure 5. Morris water maze reversal learning of adult Long-Evans hooded male rats.

(a) Acquisition as measured by mean latency (three trials per day) ± SEM to escape onto platform located in new goal quadrant (GQ). A significant decrease in latency was observed over training (G1 vs. G2: F(3,114) =16.85; p<0.0001; G2 vs. G3: F(3,120) =19.27; p <0.0001; G2 vs. G4: F(3,90) =20.29; p <0.0001). *p < 0.01 as compared to G2. (b) Probe trial initial latencies to enter GQ or platform zone (PZ). Probe trial distribution to quadrants across 90- and 30-s epochs for (c) # visits and (d) duration of time in each quadrant. Data represents mean ± SEM. G1: standard chow/RO-H2O (n = 20); G2: low-F− chow/RO-H2O (n = 20); G3: low-F− chow/10 ppm F− drinking water (n = 22) and G2 (n = 15) and G4 low-F− chow/20 ppm F− drinking water (n = 17). Drinking water exposure began on gestational day 6. Distinction of the individual quadrants: GQ, Southeast goal quadrant; SW, Southwest; NE, Northeast; SQ, Northwest start quadrant

Figure 6. Representative images of GFAP+ astrocytes in the hippocampus of adult Long-Evans hooded male rats.

Representative images of GFAP+ astrocytes in the hippocampus of adult Long-Evans hooded male rats exposed to low-F− chow/RO-H2O or low-F− chow/20 ppm F− drinking water beginning on gestational day 6. (a) Suprapyramidal blade of the dentate gyrus. (b) CA1 pyramidal cell layer. Cells displayed normal process-bearing morphology with no evidence of hypertrophy. 3,3-diaminobenzidine staining (brown). Hematoxylin counterstain (blue) showed no disruption of the normal morphology of the hippocampal regions and no evidence of neuronal death. (n = 6). Scale bar = 100 μm

Figure 6 (9 MB)

Figure 7. Representative images of Iba-1+ microglia in the hippocampus of adult Long-Evans hooded rats.

Representative images of Iba-1+ microglia in the hippocampus of adult Long-Evans hooded rats exposed to low-F− chow/RO-H2O or low-F− chow/20 ppm F− drinking water beginning on gestational day 6. (a) Suprapyramidal blade of the dentate gyrus. (b) CA1 pyramidal cell layer. Cells displayed normal process-bearing morphology with no evidence of reactivity or activation. 3,3-diaminobenzidine staining (brown). Hematoxylin counterstain (blue) (n = 6). Scale bar = 100 μm

Figure 7 (8 MB)

Figure 8. Representative images of prostate from adult male rats.

Representative images of prostate from adult male rats receiving to (G2) low-F− chow/RO-H2O or (G4) low-F− chow/RO-H2O 20 ppm F− drinking water beginning on gestational day 6. Chronic inflammation is evident as dense infiltrating immune cells in the (a) ventral and (b) dorsal lobes with infiltration of mononuclear cells, expansion of interstitium, and concretions within the glandular lumen. Hematoxylin and eosin staining. A higher incidence of prostate inflammation was observed in G4 receiving low-F− chow/20 ppm F− (7 out of 13) as compared to G1 receiving standard chow/RO-H2O (0 out of 11), G2 receiving low-F− chow/RO-H2O (2 out of 6), or G3 receiving low-F− chow/ 10 ppm F− (0 out of 8) drinking water

Figure 8 (2 MB)

Tables

Table 1. Elevated plus maze performance of Long-Evans hooded rats following drinking water exposure to fluoride.

Table 1 (180 KB)
Table 1 supporting data: Elevated plus maze (75 KB)

Table 2. Fluoride levels in Long-Evans hooded rats following drinking water exposure to fluoride.

Table 2 (39 KB)
Table 2 supporting data: Tissue NaF levels (37 KB)

Table 3. T3, T4, and TSH levels in Long-Evans Hooded rats following drinking water exposure to fluoride.

Table 3 (27 KB)
Table 3 supporting data: Serum T3 T4 levels (16 KB)
Table 3 supporting data: Serum TSH levels (16 KB)

Table 4. Summary of selected publications of fluoride levels in rats following fluoride exposure.

Table 4 (104 KB)

Supplemental Materials

Supplementary Data

Figure S1 (495 KB)
Figure S2 (1 MB)
Figure S2 supporting data: Morris Water Maze Spatial Distance DG 2 4 (59 KB)
Table S1 (421 KB)
Table S1 supporting data: Moto Activity Body Weight (1 KB)
Table S1 supporting data: Passive Avoidance Body Weights (1 KB)
Table S1 supporting data: PPI Body Weight (1 KB)
Table S2 (341 KB)