Nanomolar dose of bisphenol A rapidly modulates spinogenesis in adult hippocampal neurons

doi:10.1016/j.mce.2012.01.008

Molecular and Cellular Endocrinology

Volume 351, Issue 2, 4 April 2012, Pages 317-325

https://doi.org/10.1016/j.mce.2012.01.008 Get rights and content

Abstract

We demonstrated the rapid effects of 10 nM bisphenol A (BPA) on the spinogenesis of adult rat hippocampal slices. The density of spines was analyzed by imaging Lucifer Yellow-injected CA1 neurons in slices. Not only the total spine density but also the head diameter distribution of spine was quantitatively analyzed. Spinogenesis was significantly enhanced by BPA within 2 h. In particular, the density of middle-head spine (with head diameter of 0.4–0.5 μm) was significantly increased.

Hydroxytamoxifen, an antagonist of both estrogen-related receptor gamma (ERRγ) and estrogen receptors (ERα/ERβ), blocked the BPA-induced enhancement of the spine density. However, ICI 182,780, an antagonist of ERα/ERβ, did not suppress the BPA effects. Therefore, ERRγ is deduced to be a high affinity receptor of BPA, responsible for modulation of spinogenesis. The BPA-induced enhancement of spinogenesis was also suppressed by MAP kinase inhibitor, PD98059, and the blocker of NMDA receptors, MK-801. Washout of BPA for additional 2 h after 2 h BPA treatment abolished the BPA-induced enhancement of spinogenesis, suggesting that the BPA effect was reversible. ERRγ was localized at synapses as well as cell bodies of principal neurons. ERRγ at synapses may contribute to the observed rapid effect. The level of BPA in the hippocampal slices was determined by mass-spectrometric analysis.

Highlights

► The elevation of BPA level to 10 nM induced rapid increase in the density of middle-head spines. ► ERRγ was a high affinity receptor of BPA, mediating the modulation of spinogenesis. ► ICI, antagonist of ERα/ERβ, did not suppress the BPA-induced enhancement of spinogenesis.

Introduction

Low dose exposure to bisphenol A (BPA) may induce hormone-like effects on wildlife and humans. BPA is a widely used synthetic material included in polycarbonate resin used in water pipe sealant, dental prostheses, compact discs and baby bottles. Toxic effects of high dose BPA (mg/kg weight/day) have been investigated in relation to the development and functions of the reproduction systems (Fisher et al., 1999, Al-Hiyasat et al., 2002, Grote et al., 2004, Halldin et al., 2005). However, the low dose exposure to BPA (μg/kg/day or nanomolar doses) shows rather weak toxic effects on reproductive or endocrine functions in the peripheral tissues, probably due to the efficient detoxification of BPA by the liver. On the other hand, low dose exposure to BPA may significantly affect the brain function, because the detoxification of BPA in the brain is probably very weak, due to the extremely low expression of drug-metabolizing enzymes in the brain (Miksys and Tyndale, 2002, Kishimoto et al., 2004, Chinta et al., 2005).

The low dose exposure to BPA during fetal/neonatal stages has been extensively investigated. For example, fetal or neonatal exposure to BPA inhibits sexual differentiation of nonreproductive behaviors of adult animals, including maze learning behavior (Carr et al., 2003, Kubo et al., 2003, Fujimoto et al., 2006), at doses as low as 1/1000 of those required for the stimulation of uterine growth (Ashby, 2001). On the other hand, inadequate information is available for the low dose exposure to BPA in the adult stage, except some pioneer works in vivo (MacLusky et al., 2005, Leranth et al., 2008, Hajszan and Leranth, 2010).

The high affinity functional receptor for BPA has not been identified yet. Although ERα is one candidate of BPA receptor, the affinity of ERα for BPA is very low, in the order of 1/100–1/1000 of that for 17β-estradiol (E2) (Kuiper et al., 1997, Morohoshi et al., 2005). On the other hand, estrogen-related receptor gamma (ERRγ) is a high affinity binding protein for BPA (Takayanagi et al., 2006). However, ERRγ has not been recognized as a BPA receptor, because ERRγ shows constitutive transcriptional activity even without any ligand (Coward et al., 2001).

We here performed the investigation on the rapid modulation by nanomolar doses of BPA on the density and morphology of dendritic spines in the adult hippocampal slices, including investigations of BPA receptors. In order to observe the direct effects of BPA on hippocampal neurons, we used isolated ‘acute’ hippocampal slices which do not have projections of cholinergic or serotonergic neurons from outside of the hippocampus.

Section snippets

Chemicals

BPA, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), cycloheximide, nicardipine, N-methyl-d-aspartate (NMDA), PD98059, ICI 182,780 (ICI), MK-801 and Lucifer Yellow were purchased from Sigma (USA). 4-hydroxy-tamoxifen (OH-Tam) was from Calbiochem (Germany). Other chemicals used were of highest purity commercially available. Polyclonal anti-ERRγ antibody (against C-terminal of ligand binding site) was prepared by Dr. Shimohigashi at Kyushu Univ. (Tokunaga et al., 2006).

Animals

Adult male Wistar rats (12

Rapid effect of BPA on spinogenesis

We analyzed the effect of BPA on the modulation of the density and head diameter of spines in the CA1 region. To do this, single spine imaging was performed for Lucifer Yellow-injected neurons in hippocampal slices from adult male rats. We analyzed secondary branches of the apical dendrites located 100–200 μm distant from the pyramidal cell body around the middle of the stratum radiatum of CA1 region.

Discussion

Current investigations of the rapid effects of BPA on spinogenesis in isolated hippocampal slices lead to the finding of ERRγ-mediated BPA action at synapses of pyramidal neurons.

Acknowledgments

We thank Prof. J.A. Rose (Ritsumeikan University) and Dr. Anna Barron (Univ. of Tokyo) for critical reading of the manuscript. Drs. S. Homma and M. Okuyama (Aska Pharma Medical) are acknowledged for technical assistance of mass-spectrometric analysis.

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BPA was found to have a very low affinity with ERs (about 1/100–1/1000 for 17β-E2), but have a high affinity with ERRγ (Takayanagi et al., 2006). Further pre-treatment of Tam, an antagonist of both ERs and ERRγ, completely suppressed the promotion of BPA to glutamate-elevated [Ca2+]i. Tanabe et al. (2012) recently found that BPA-induced enhancement of the spine density in CA1 neurons of adult hippocampal slices was blocked by Tam rather than ICI at 1 μM. Therefore, ERRγ is deduced to mediate BPA modulating glutamate-elevated [Ca2+]i in the hippocampal neurons.
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Our previous and present studies in hippocampal neurons in vitro showed that BPA alone enhanced the densities of dendritic filopodium, spine, and synapse but inhibited 17β-E2- or DHT-induced increases in the densities of dendritic spine and synapse (Xu et al., 2010, 2014). Using hippocampal slices of adult rat, Tanabe et al. demonstrated the rapidly enhancement of 10 nM BPA in spinogenesis within 2 h, in particular, the density of middlehead spine (Tanabe et al., 2012). BPA was also rapidly enhance LTP and LTD in DG or CA1 of hippocampus slice in vitro of adolescence or adult male rats (Chen et al., 2017; Ogiue-Ikeda et al., 2008; Tanabe et al., 2005) but inhibited the 17β-E2-promotion in LTP when it was co-treatment with 17β-E2 (Chen et al., 2017).
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The brain develops over an extended period of time during which it is extremely sensitive to environmental influences and to endogenous hormones that modulate brain plasticity and behavior. Endocrine disrupting chemicals (EDCs) interfere with physiological endocrine actions by a variety of mechanisms including epigenetic modification. In common with known neurotoxicants EDCs induce neurobehavioral alterations in the developing organisms that may not become apparent until later in life. The scientific criteria for proper risk assessment of EDCs are still the subject of intense debate among scientists and regulators worldwide: major issues concern the definition of endocrine function and relative adverse effects, the existence of thresholds, the significance of non-monotonic dose–response relationships.
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The adverse effects induced by a “low dose” of BPA include neuronal and behavioral alterations: documented effects of prenatal exposure to BPA are abnormal development of the neocortex in terms of differentiation and neuronal migration [19,38], aberrant positions and connections between thalamus and cortex [39], inhibition of the proliferation of neural progenitor cells [21], loss of sexual dimorphism in terms of brain structure and behavior [24], increased anxiety and cognitive deficits [45,54]. Part of these results are justified by the strong affinity of BPA to the dopamine receptor, the estrogen-like receptor-β type [8] and the estrogen-like receptor-γ type present in hippocampal neurons [51]. In all these hypotheses the normal synaptic communication was altered.
Over 50% of the causes of fetal malformations in humans are still unknown. Recent evidence suggests the relationship between environmental exposure to endocrine disruptors and fetal malformations. Our study aims to establish the role of Bisphenol A (BPA), if any, in altering human reproduction. We enrolled 151 pregnant women who were divided into two groups: case group (CS, n = 101), women with established diagnosis of developmental defect, and control group (CL, n = 50), pregnant women with normally developed fetus. Total, free and conjugated BPA were measured in their blood using GC–MS with isotopic dilution. The results show a correlation between environmental exposure to BPA and the genesis of fetal malformations. Conjugated BPA, which was higher in the CL, casts light on the hypothesis that a reduced ability to metabolize the chemical in the mother can concur to the occurrence of malformation. In a more detailed manner, in case of chromosomal malformations, the average value of free BPA appears to be nearly three times greater than that of the controls. Similarly, in case of central and peripheral nervous system non-chromosomal malformations, the value of free BPA is nearly two times greater than that of the controls.
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Our previous study indicated that perinatal exposure to low-dose BPA, one of the most common environmental endocrine disrupters, alters behavioral development in offspring mice. Given that synaptic structure of the hippocampus is closely related to behaviors, in the present study, we examined the effects of perinatal exposure to BPA (0.04, 0.4, and 4.0 mg kg⁻¹ day⁻¹) on the synaptic density and the synaptic structural modification of pyramidal cells in hippocampus region CA1 and the expressions of synaptic proteins such as synapsin I and PSD-95 and glutamate NMDA and AMPA receptors in male offspring mice on postnatal day (PND) 14, 21, and 56. The results of electron microscope measurement showed that BPA significantly reduced the numeric synaptic density and altered the structural modification of synaptic interface of pyramidal cells with the enlarged synaptic cleft, the shortened active zone, and the thinned postsynaptic density (PSD) on PND 14, 21, and 56 and the increased curvature of synaptic interface on PND 14 and 21. Further analyses of Western blot indicated that BPA markedly reduced the levels of synapsin I and PSD-95 on PND 14, 21, and 56 and down-regulated NMDA receptor subunit NR1 and AMPA receptor subunit GluR1 during development and young adulthood. These results suggest that perinatal exposure to low level of BPA inhibits synaptogenesis and affects synaptic structural modification after birth. The reduced expressions of synaptic proteins synapsin I and PSD-95 and glutamate NMDA and AMPA receptors may be involved in the negative changes in the synaptic plasticity.
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Prevalent use of bisphenol-A (BPA) in the manufacture of resins, plastics and paper products has led to frequent exposure of most people to this endocrine disruptor. Some rodent studies have suggested that BPA can exert detrimental effects on brain development. However as rodent models cannot be relied on to predict consequences of human exposure to BPA during development, it is important to investigate the effects of BPA on non-human primate brain development. Previous research suggests that BPA preferentially targets dopamine neurons in ventral mesencephalon and glutamatergic neurons in hippocampus, so the present work examined the susceptibility of these systems to low dose BPA exposure at the fetal and juvenile stages of development in non-human primates. Exposure of pregnant rhesus monkeys to relatively low levels of BPA during the final 2 months of gestation, induced abnormalities in fetal ventral mesencephalon and hippocampus. Specifically, light microscopy revealed a decrease in tyrosine hydroxylase-expressing (dopamine) neurons in the midbrain of BPA-exposed fetuses and electron microscopy identified a reduction in spine synapses in the CA1 region of hippocampus. In contrast, administration of BPA to juvenile vervet monkeys (14–18 months of age) was without effect on these indices, or on dopamine and serotonin concentrations in striatum and prefrontal cortex, or on performance of a cognitive task that tests working memory capacity. These data indicate that BPA exerts an age-dependent detrimental impact on primate brain development, at blood levels within the range measured in humans having only environmental contact with BPA.

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¹: These authors contributed equally.

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Nanomolar dose of bisphenol A rapidly modulates spinogenesis in adult hippocampal neurons

Abstract

Highlights

Introduction

Section snippets

Chemicals

Animals

Rapid effect of BPA on spinogenesis

Discussion

Acknowledgments

Toxicol. Lett.

Neuroscience

Genomics

Brain Res.

Toxicology

Front Neuroendocrinol.

Brain Res. Bull.

Methods Enzymol.

Neurosci. Res.

Neuroscience

Brain Res. Mol. Brain Res.

Toxicol. In Vitro

Brain Res. Rev.

Toxicol. Lett.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Toxicol. Appl. Pharmacol.

Effects of bisphenol A on adult male mouse fertility

Eur. J. Oral Sci.

Increasing the sensitivity of the rodent uterotrophic assay to estrogens, with particular reference to bisphenol A

Environ. Health Perspect.

Impact of early-life bisphenol A exposure on behavior and executive function in children

Pediatrics

Effect of neonatal rat bisphenol A exposure on performance in the Morris water maze

J. Toxicol. Environ. Health A

Constitutive expression and localization of cytochrome P-450 1A1 in rat and human brain: presence of a splice variant form in human brain

J. Neurochem.

The structure of postsynaptic densities isolated from dog cerebral cortex. I. Overall morphology and protein composition

J. Cell Biol.