Prenatal Bisphenol A (BPA) Exposure Alters the Transcriptome of the Neonate Rat Amygdala in a Sex-specific Manner: a CLARITY-BPA Consortium Study
Sheryl E. Arambula, Dereje Jima, and Heather B. Patisaul.
NeuroToxicology (2018) DOI: https://doi.org/10.1016/j.neuro.2017.10.005 PMID: 29097150
Bisphenol A (BPA) is a widely recognized endocrine disruptor prevalent in many household items. Because experimental and epidemiological data suggest links between prenatal BPA exposure and altered affective behaviors in children, even at levels below the current US FDA No Observed Adverse Effect Level (NOAEL) of 5mg/kg body weight (bw)/day, there is concern that early life exposure may alter neurodevelopment. The current study was conducted as part of the CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) program and examined the full amygdalar transcriptome on postnatal day (PND) 1, with the hypothesis that prenatal BPA exposure would alter the expression of genes and pathways fundamental to sex-specific affective behaviors. NCTR Sprague-Dawley dams were gavaged from gestational day 6 until parturition with BPA (2.5, 25, 250, 2500, or 25000μg/kg bw/day), a reference estrogen (0.05 or 0.5μg ethinyl estradiol (EE2)/kg bw/day), or vehicle. PND 1 amygdalae were microdissected and gene expression was assessed with qRT-PCR (all exposure groups) and RNAseq (vehicle, 25 and 250μg BPA, and 0.5μg EE2 groups only). Our results demonstrate that that prenatal BPA exposure can disrupt the transcriptome of the neonate amygdala, at doses below the FDA NOAEL, in a sex-specific manner and indicate that the female amygdala may be more sensitive to BPA exposure during fetal development. We also provide additional evidence that developmental BPA exposure can interfere with estrogen, oxytocin, and vasopressin signaling pathways in the developing brain and alter signaling pathways critical for synaptic organization and transmission.
Figure 1. Effects of gestational BPA or EE2 on neonatal amygdalar expression of selected genes.
Esr1 was unaffected by BPA or EE2 (A). Esr2 was increased by 2.5 μg BPA in males and 250 μg BPA in females (B). Oxtr was increased by 250 and 250 μg BPA in males and 2.5, 25, 250, and 25,000 μg BPA and 0.5 μg EE2 in females (C). Male Avpr1a was decreased by 2.5 μg BPA and increased by 25 and 250 μg BPA (D). Ar was increased by 25 μg BPA in males and 250 μg BPA and 0.5 μg EE2 in females (E). Female Gadd45b was increased by 0.5 μg EE2 (F). Camk4 was upregulated in males by 25 μg BPA and in females by 2.5, 25, 250, and 25000 μg BPA and 0.05 and 0.5 μg EE2 (G). Grm5 was increased by 25 μg BPA in males and 250, 2500, 25000 μg BPA and 0.05 μg EE2 in females (H). Graphs depict mean ± SEM (*p ≤ 0.05, **p ≤ 0.01, and §p ≤ 0.08).
- Figure 1 (636 KB)
Figure 2. Top canonical pathways enriched by differentially expressed genes.
The x-axis represents negative log p values based on the probability that molecules in the uploaded dataset were included in the predefined IPA canonical pathways by true association as opposed to inclusion of molecules based on chance alone. For each male (A) and female (B) exposure group, only the top 10 pathways with the largest negative log p values are shown. The dashed line indicates the threshold of significance for a p-adjusted value of 0.05.
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Figure 3. Prenatal exposure to BPA and EE2 result in common and unique differently expressed genes.
Differentially expressed genes (padj ≤ 0.05) were identified in males (A) and females (B) prenatally exposed to 25 and 250 μg BPA and 0.5 μg EE2. Venn diagrams were created using Venny (http://bioinfogp.cnb.csic.es/tools/venny/).
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Figure 4. Sex differences in amygdala expression of selected genes.
Relative differences in gene expression between male and female control (unexposed) groups with male gene expression set as baseline. No a priori selected genes were sexually dimorphic in the neonate amygdala. (A-F). Expression of Camk4 was significantly higher in males than females (G). No sex difference in Grm5 was detected (H). Graphs depict mean ± SEM (**p ≤ 0.01 and §p ≤ 0.08).
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Table 1. Rationale for genes of interest selected a priori.
- Table 1 (32 KB)
Table 2. qRT-PCR outcomes and descriptive statistics for genes found to be significantly altered by BPA or EE2 exposure.
Sample sizes for each group are in parentheses.
- Table 2 (69 KB)
Table 3. Differentially expressed genes identified by RNAseq within selected canonical pathways identified by IPA analysis in exposed females.
- Table 3 (53 KB)
- Supplementary Figure 1 (171 KB)
- Supplementary Figure 2 (393 KB)
- Supplementary Figure Legend (24 KB)
- Supplementary Table 1 (32 KB)
- Supplementary Table 2 (135 KB)
- Supplementary Table 3 (79 KB)