Low-dose exposure to bisphenol A and replacement bisphenol S induces precocious hypothalamic neurogenesis in embryonic zebrafish

Abstract

Bisphenol A (BPA), a ubiquitous endocrine disruptor that is present in many household products, has been linked to obesity, cancer, and, most relevant here, childhood neurological disorders such as anxiety and hyperactivity. However, how BPA exposure translates into these neurodevelopmental disorders remains poorly understood. Here, we used zebrafish to link BPA mechanistically to disease etiology. Strikingly, treatment of embryonic zebrafish with very low-dose BPA (0.0068 μM, 1,000-fold lower than the accepted human daily exposure) and bisphenol S (BPS), a common analog used in BPA-free products, resulted in 180% and 240% increases, respectively, in neuronal birth (neurogenesis) within the hypothalamus, a highly conserved brain region involved in hyperactivity. Furthermore, restricted BPA/BPS exposure specifically during the neurogenic window caused later hyperactive behaviors in zebrafish larvae. Unexpectedly, we show that BPA-mediated precocious neurogenesis and the concomitant behavioral phenotype were not dependent on predicted estrogen receptors but relied on androgen receptor-mediated up-regulation of aromatase. Although human epidemiological results are still emerging, an association between high maternal urinary BPA during gestation and hyperactivity and other behavioral disturbances in the child has been suggested. Our studies here provide mechanistic support that the neurogenic period indeed may be a window of vulnerability and uncovers previously unexplored avenues of research into how endocrine disruptors might perturb early brain development. Furthermore, our results show that BPA-free products are not necessarily safer and support the removal of all bisphenols from consumer merchandise.

Keywords: androgen receptor; aromatase; endocrine disruption; hyperactivity.

Fig. 1.

BPA exposure induces hyperactive behaviors in larval zebrafish during the window of hypothalamic neurogenesis. (A) Treatment paradigm for BPA exposure. (B) Representative locomotor activity scribes with fish (green) and movement (red) shown. Locomotion in 5-dpf controls and groups exposed to 0.1 μM BPA from 10–16 hpf is compared with locomotion in groups exposed to 0.1 μM BPA from 16–24 hpf and 24–36 hpf. (C) Quantified locomotor activity for 5-dpf zebrafish treated with 0.1 μM BPA from 10–16 hpf, 16–24 hpf, and 24–36 hpf. (D) Locomotor activity in 5-dpf zebrafish coexposed from 0–5 dpf to 0.0068 μM BPA + 1 μM ICI. (E) Locomotor activity in 5-dpf AroB morphants exposed from 0–5 dpf to 0.0068 μM BPA. (F) Locomotor activity in 5-dpf zebrafish coexposed from 24–48 hpf to 1 μM BPA + FAD. (D and E) BPA exposure in AroB morphants and BPA+ICI treatment were run in the same experiment. Results have been separated for simplicity, and controls are shown twice. Data in C–F are shown as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 [one-way ANOVA, Tukey’s Honestly Significant Difference test (Tukey's HSD)]; n = 5–10 fish per group.

Fig. 2.

Precocious neurogenesis in the hypothalamus following BPA exposure is independent of ER signaling. (A) Cartoon of the experimental design and sample neurogenic curves for BPA-exposed (blue) and control (black) groups. (B) Quantification of neurons born at each time point in BPA (0.0068 μM) relative to vehicle treatment (set to 100%). Neuronal birth was identified by EdU and α-HuC colabeling in BPA-treated and control coronal sections through the hypothalamus. Data are shown as mean ± SEM; *P < 0.05 (Student’s t test); n = 3–11 fish per group. (C) Representation of ICI interaction with ERs. *ICI 182,780 is an antagonist to genomic ERs and membrane-bound ER (mER) but is an agonist toward GPR30. (D) Neuronal birth in 5-dpf zebrafish exposed to 0.0068 μM estradiol or 0.0068 μM BPA or coexposed to 0.0068 μM BPA + 1 μM ICI and pulsed with EdU at 24 hpf. (E) Neuronal birth in 5-dpf zebrafish exposed to 0.0068 μM 17β-Estradiol or 0.0068 μM BPA and pulsed with EdU at late neurogenesis (36 hpf). Data in D and E are shown as mean ± SEM; *P < 0.05, **P < 0.01, ****P < 0.0001 (ANOVA, Tukey’s HSD); n = 3–11.

Fig. 3.

BPA exposure induces precocious neuronal birth in the hypothalamus. Representative immunohistochemistry images of rostral hypothalamus in larval zebrafish exposed to 0.0068 μM BPA at 0–5 dpf. EdU (red), α-HuC (green), and merged hypothalamic sections are shown. The red box in the DAPI (blue)-stained image marks the hypothalamus. Mth, mouth. (Scale bar: 50 μm.)

Fig. 4.

BPA exposure does not cause precocious neurogenesis in other brain regions. (A) Quantification of neurons born at 24 hpf in the thalamus of larvae exposed to 0.0068 μM BPA at 0–5 dpf. Data are shown as mean ± SEM; P > 0.05 (Student’s t test); n = 3 or 4 fish. (B) Representative immunohistochemistry images of the thalamus of a larval zebrafish exposed to 0.0068 μM BPA at 0–5 dpf and pulsed with EdU at 24 hpf. Mth, mouth; ot, optic tract. (Scale bar: 50 μm.)

Fig. 5.

BPA-induced precocious neurogenesis is mediated via ARs and aromatase. (A) Quantification of neuronal birth in 5-dpf zebrafish coexposed from 0–5 dpf to 1 μM FAD + 0.0068 μM BPA and pulsed with EdU at 24 hpf. (B) Neuronal birth in 5-dpf zebrafish exposed from 0–5 dpf to 0.1 μM GSK4716 alone or coexposed to BPA + 50 nM amiodarone (AMIO) or to BPA + 6.17 μM flutamide (FLU). The red arrow indicates exposure at 8–48 hpf. The hash mark (#) indicates the AroB morphant (AroB-MO) exposed to BPA. (C) Neuronal birth in 5-dpf zebrafish exposed from 0–5 dpf to 0.0068 μM BPA or 1 μM DHT or coexposed to 1 μM DHT from 0–5 dpf and to 6.17 μM FLU (red arrow) for 8–48 hpf or to 1 μM ICI from 0–5 dpf. (D) Log-transformed relative AroB (cyp19a1b) expression at 48 hpf in zebrafish exposed to 0.0068 μM BPA or 1 μM DHT or coexposed to BPA or 1 μM DHT + 6.17 μM FLU or 1 μM ICI at 8–48 hpf. Data in A–D are shown as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (ANOVA, Tukey’s HSD); n = 3–13. (E) Diagram illustrating targets of various pharmacological agents and AroB MO.

Fig. 6.

BPS exposure increases neurogenesis and hyperactive behavior via a mechanism similar to that of BPA. (A) Chemical structures of BPA and BPS. (B) Neuronal birth in 5-dpf zebrafish exposed from 0–5 dpf to 0.0068 μM BPS. Data are shown as mean ± SEM; *P < 0.05 (Student’s t test); n = 6. (C) Locomotor activity in fish coexposed from 0–5 dpf to 0.0068 μM BPS + 1 μM ICI. (D) Locomotor activity in 5-dpf controls and AroB morphants exposed from 0–5 dpf to 0.0068 μM BPS. Data in C and D are shown as mean ± SEM; *P < 0.05 (ANOVA, Tukey’s HSD); n = 3–9. (C and D) BPS exposure in AroB morphants and BPS+ICI treatment were run in same experiment. Results have been separated for simplicity, and controls are shown twice.