Effects of TBBPA Exposure on Neurodevelopment and Behavior in Mice

Abstract

Tetrabromobisphenol A (TBBPA) is a brominated flame retardant widely used in consumer products. TBBPA is often detected in soil, water, organisms, and even in human blood and breast milk. Hence, it is accessible to developing fetuses and nursing offspring after maternal exposure. The reported evidence for the endocrine disruption of TBBPA in the brain has raised concerns regarding its effects on neurodevelopmental and behavioral functions. This study investigated the effects of TBBPA exposure on neurodevelopment. A cell-based developmental neurotoxicity assay was performed to determine whether TBBPA is a developmental neurotoxicant. The assay revealed TBBPA to be a developmental neurotoxicant. C57BL/6N maternal mice were administered TBBPA at 0, 0.24, and 2.4 mg/kg during pregnancy and lactation, and their offspring underwent behavioral testing. The behavioral experiments revealed sex-specific effects. In females, only a deterioration of the motor ability was observed. In contrast, deteriorations in motor function, memory, and social interaction were noted in males. Furthermore, we validated changes in the expression of genes associated with behavioral abnormalities, confirming that perinatal exposure to TBBPA, at the administered doses, can affect neurodevelopment and behavior in offspring. These findings highlight the need for more in-depth and multifaceted research on the toxicity of TBBPA.

Keywords: Tetrabromobisphenol A; behavioral disorders; developmental neurotoxicity; endocrine disrupting chemicals; neurodevelopment.

Figure 1

Determination of the developmental neurotoxicity of TBBPA through a Sox1-GFP-based cell screening assay. (A) Results of cell viability test, n = 6 for each concentration. The coefficient of determination, R², was 0.9813. (B) GFP intensity from neuronal differentiation test, n = 8 for each concentration. The R² value was 0.9997. No statistical tests were performed.

Figure 2

Effects of TBBPA on locomotor activity and anxiety-like behavior. (A,B) Rotarod test results. n = 15 males/11 females for vehicle, 19 males/16 females for TBBPA 0.24 mg/kg, 7 males/13 females for TBBPA 2.4 mg/kg. (C–J) Open-field test results: (C,D) distance moved; (E,F) velocity; (G,H) time spent in the center; (I,J) number of entries into the center. Open-field test groups used the same n as that in the rotarod test. The data are presented as the means ± SEM. Statistical significance was determined by a one-way ANOVA with a Dunnett’s multiple comparisons test. Data were analyzed per individual animal and not on a litter basis. * p < 0.05 vehicle vs. TBBPA 0.24 mg/kg, **** p < 0.0001 vehicle vs. TBBPA 0.24 mg/kg, # p < 0.05 vehicle vs. TBBPA 2.4 mg/kg, ## p < 0.01 vehicle vs. TBBPA 2.4 mg/kg, #### p < 0.0001 vehicle vs. TBBPA 2.4 mg/kg.

Figure 3

Effects of TBBPA on depression-related behaviors. (A) Representative image of the nest building test. (B,C) Nest building test scores. n = 15 males/11 females for vehicle, 19 males/16 females for TBBPA 0.24 mg/kg, 7 males/13 females for TBBPA 2.4 mg/kg. (D,E) Immobile time in the forced swimming test (FS). (F,G) Immobile time in the tail suspension test (TS). FS and TS groups used the same n as in the nest building test. The data are plotted as the means ± SEM. The statistical analysis was determined by a one-way ANOVA with a Dunnett’s multiple comparisons test. Data were analyzed per individual animal and not on a litter basis.

Figure 4

Effects of TBBPA on learning and memory in offspring mice. (A–G) Morris water maze test (MWM) results: n = 15 males/11 females for vehicle, 19 males/16 females for TBBPA 0.24 mg/kg, 7 males/13 females for TBBPA 2.4 mg/kg. (A,B) Escape latency of mice for nine consecutive training periods. (C) Representative image of mouse movement tracking in the test session. (D,E) Frequency of crossing the platform. (F,G) Time spent in the platform area. (H) Representative image of mouse movement tracking of novel object recognition (NOR). (I,J) Recognition index for the mouse offspring. NOR groups used the same n as that in MWM. The data were evaluated as the means ± SEM. Statistical significance of MWM results was determined by a one-way ANOVA with a Dunnett’s multiple comparisons test. NOR results were analyzed using an unpaired two-tailed Student’s test. Data were analyzed per individual animal and not on a litter basis. # p < 0.05 vs. vehicle (A); * p < 0.05, ** p < 0.01 vehicle vs. TBBPA 0.24 mg/kg (D,F); ** p < 0.01, *** p < 0.001 increased recognition index for familiar object vs. novel object; ns, not significant (I,J).

Figure 5

Effects of TBBPA on social interaction behaviors. (A–E) Three-chamber social test (3C) results. (A) Representative heatmap images of sociality and social novelty session. (B,C) Preference index of sociality session, n = 13 males/8 females for vehicle, 16 males/13 females for TBBPA 0.24 mg/kg, 7 males/13 females for TBBPA 2.4 mg/kg. (D,E) Preference index of social novelty session, n = 13 males/7 females for vehicle, 14 males/13 females for TBBPA 0.24 mg/kg, 2 males/13 females for TBBPA 2.4 mg/kg. (F–K) Social interaction test (SI) results: n = 15 males/11 females for vehicle, 19 males/16 females for TBBPA 0.24 mg/kg, 7 males/13 females for TBBPA 2.4 mg/kg. (F,G) The number of general sniffing events. (H,I) The number of anogenital sniffing events. (J,K) The number of following behavior. The data were evaluated as the means ± SEM. The statistical significance was calculated using a one-way ANOVA with a Dunnett’s multiple comparisons test. Data were analyzed per individual animal and not on a litter basis. * p < 0.05 vehicle vs. TBBPA 2.4 mg/kg.

Figure 6

Changes in the expression of behavior-related genes in the brain. (A,B) Relative gene expression compared with Gapdh. n = 15 males/11 females for vehicle, 19 males/16 females for TBBPA 0.24 mg/kg, 7 males/13 females for TBBPA 2.4 mg/kg. The statistical significance was determined by one-way ANOVA with a Dunnett’s multiple comparisons test. Data were analyzed per individual animal and not on a litter basis. * p < 0.05, *** p < 0.001 vs. vehicle.