Chronic exposure to environmentally relevant concentrations of bisphenol S differentially affects cognitive behaviors in adult female zebrafish

Journal Pre-proof Chronic exposure to environmentally relevant concentrations of bisphenol S differentially affects cognitive behaviors in adult female zebrafish Mohammad Naderi, Arash Salahinejad, Anoosha Attaran, Douglas P. Chivers, Som Niyogi PII:

S0269-7491(19)34895-X

DOI:

https://doi.org/10.1016/j.envpol.2020.114060

Reference:

ENPO 114060

To appear in:

Environmental Pollution

Received Date: 29 August 2019 Revised Date:

2 December 2019

Accepted Date: 22 January 2020

Please cite this article as: Naderi, M., Salahinejad, A., Attaran, A., Chivers, D.P., Niyogi, S., Chronic exposure to environmentally relevant concentrations of bisphenol S differentially affects cognitive behaviors in adult female zebrafish, Environmental Pollution (2020), doi: https://doi.org/10.1016/ j.envpol.2020.114060. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

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Chronic exposure to environmentally relevant concentrations of bisphenol S differentially

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affects cognitive behaviors in adult female zebrafish

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Mohammad Naderi1*, Arash Salahinejad1, Anoosha Attaran1, Douglas P. Chivers1, Som Niyogi1,2

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Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK S7N 5E2, Canada 2 Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK S7N 5B3, Canada

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* Corresponding Author: Mohammad Naderi

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Email: [email protected]

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Tel: 1 306 850 7337

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Abstract

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Evidence is emerging that environmental exposure to bisphenol S (BPS), a substitute for

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bisphenol A (BPA), to humans and wildlife is on the rise. However, research on the

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neurobehavioral effects of this endocrine disruptive chemical is still in its infancy. In this study,

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we aimed to investigate the effects of long-term exposure to environmentally relevant

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concentrations of BPS on recognition memory and its mechanism(s) of action, especially

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focusing on the glutamatergic/ERK/CREB pathway in the brain. Adult female zebrafish were

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exposed to the vehicle, 17β-estradiol (E2, 1 µg/L), or BPS (1, 10 and 30 µg/L) for 120 days. Fish

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were then tested in the object recognition (OR), object placement (OP), and social recognition

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tasks (SR). Chronic exposure to E2 and 1 µg/L of BPS improved fish performance in OP task.

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This was associated with an up-regulation in the mRNA expression of several subtypes of

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metabotropic and ionotropic glutamate receptors, an increase in the phosphorylation levels of

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ERK1/2 and CREB, and an elevated transcript abundance of several immediate early genes

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involved in synaptic plasticity and memory formation. In contrast, the exposure to 10 and 30

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µg/L of BPS attenuated fish performance in all recognition memory tasks. The impairment of

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these memory functions was associated with a marked down-regulation in the expression and

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activity of genes and proteins involved in glutamatergic/ERK/CREB signaling cascade.

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Collectively, our study demonstrated that the long-term exposure to BPS elicits hermetic effects

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on the recognition memory in zebrafish. Furthermore, the effect of BPS on the recognition

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memory seems to be mediated by the glutamatergic/ERK/CREB signaling pathway.

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Keywords:

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Bisphenol S; Recognition Memory; Zebrafish; glutamatergic system; ERK; CREB

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1. Introduction

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In recent years, overwhelming evidence indicates that bisphenol A (BPA), a raw material of

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polycarbonate and epoxy resins, can cause a series of detrimental health effects on humans and

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wildlife (Rochester, 2013; Vandenberg et al., 2009). As a result, BPA production, import, and

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use have been banned or restricted in several countries including the US and Canada (Chen et al.,

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2016). Consequently, this has spurred a demand for BPA replacements.

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Bisphenol S (BPS) is one of the main BPA alternatives, which is increasingly used in the

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production of multitudinous products including polycarbonate plastics, personal care products,

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epoxy resins, baby bottles, and thermal papers (Wu et al., 2018). BPS is a structural analog of

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BPA, but with higher resistance to sunlight and high temperature and less leachable from related

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products compared to BPA (Viñas et al., 2010). However, the widespread use of BPS

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progressively contributes to its dispersion throughout the environment. Accumulating evidence

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indicates the occurrence of BPS in several media, including sewage sludge, sediments, indoor

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dust, thermal receipt papers, food, and beverages as well as human tissues (Wu et al., 2018). For

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instance, 81% of urine samples collected from the United States and several Asian countries

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contained detectable amount of BPS (mean concentration of 0.654 µg/L) (Liao et al., 2012). Like

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many other contaminants, the aquatic environment acts as the final sink for the BPS released in

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the environment. The concentrations of BPS detected in surface water of some lakes and rivers in

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China, Japan, and Korea, have been found to range from no detectable to 65.60 µg/L (Huang et

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al., 2018; Wu et al., 2018).

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In addition to the widespread distribution of BPS in the environment, the structural similarity of

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BPS to BPA raises new concerns about the endocrine disruptive effects of BPS on humans and

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wildlife. The estrogenic, anti-estrogenic, androgenic, and anti-androgenic effects of BPS has

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been documented in both in vitro and in vivo studies (Chen et al., 2016; Rochester and Bolden,

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2015). In line with this, burgeoning evidence shows that BPS can cause multiple adverse effect

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to human health and animals, such as reproductive abnormalities, cytotoxicity, genotoxicity,

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immunotoxicity, and teratogenicity (Ho et al., 2017; Wan et al., 2018; Zhang et al., 2016). Fish

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are among the most sensitive organisms to BPS toxicity. Several lines of studies, including ours

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have documented that the exposure to BPS leads to a suite of reproductive, metabolic, visual, and

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developmental abnormalities in fish (Ji et al., 2013; Liu et al., 2018; Moreman et al., 2017; Mu et 3

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al., 2018; Naderi et al., 2014; Wei et al., 2018; Zhang et al., 2017). Nonetheless, research on the

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adverse effects of BPS is in its infancy and more studies are required to elucidate the possible

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mechanism(s) by which this unregulated contaminant can cause toxicity.

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Apart from their roles in the regulation of a broad spectrum of reproductive functions, sex steroid

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hormones also have profound effects on a myriad of neurobehavioral functions (Ervin et al.,

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2013; McEwen, 2002). 17β-Estradiol (E2), the predominant form of estrogens, is the best-

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characterized hormone that acts as a modulator of brain tissue. E2, through interaction with

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estrogen receptors (ERs; ERα and ERβ), regulates a large array of cellular mechanisms involved

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in synaptic connectivity, dendritic arborization, and synaptic plasticity in cognitive regions of the

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brain in both males and females (Frankfurt and Luine, 2015; McEwen, 2002). In recent years,

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growing evidence indicates that the E2 effects on hippocampal related behaviors are mainly

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mediated via the activation of the extracellularly regulated kinase/mitogen-activated protein

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kinase (ERK/MAPK) (Boulware et al., 2013).

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The ERK/MAPK pathway is a central cellular signaling pathway that connects the membrane

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receptors to the numerous extracellular signals, cascading down to transcription factors, which

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controls the transcription of genes involved in learning and memory (Medina and Viola, 2018).

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Several studies suggested that E2, via a functional increase in abundance and activity of

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glutamate receptors, activates ERK/MAPK signaling (Boulware et al., 2013; Boulware et al.,

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2005; Wang et al., 2007). Once activated, ERK transduces into the nucleus to activate several

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transcription factors such as cAMP response element-binding protein (CREB). Substantial

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evidence points to phosphorylation of CREB as a necessary step in the estrogen-dependent

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synaptic plasticity and long-term memory formation (Boulware et al., 2013). Indeed, activation

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of CREB unleashes the transcription of numerous genes which are involved in dendritic growth

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and synaptic plasticity (Finkbeiner et al., 1997; Wang et al., 2007). Thus, this pathway can be a

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prime target for environmental xenoestrogens. For example, recent studies have shown that BPA

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impairs different forms of learning and memory in rodents through disruption of

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glutamatergic/ERK/CREB pathway (Chen et al., 2017; Wang et al., 2016; Xu et al., 2014).

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Moreover, BPS has also been reported to interfere with membrane-initiated E2-induced ERK

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signaling, leading to improper cell proliferation and cell death in rat pituitary cells (Viñas and

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Watson, 2013). Although there is ample evidence that BPS adversely affects different aspects of

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the neuroendocrine system, to date very few studies have attempted to evaluate the

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neurobehavioral effects of this contaminant. Recent studies show that short-term embryonic

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exposure to low concentrations of BPS increased hypothalamic neurogenesis and altered

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locomotor behavior in zebrafish larvae (Gu et al., 2019; Kinch et al., 2015).

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The brain of adult teleost fish exhibits an intense activity and expression of ERs and

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steroidogenic enzymes indicating that fish brain is a true steroidogenic organ (Diotel et al.,

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2011). The present study was aimed to investigate the effects of low concentrations of BPS on

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cognition in adult female zebrafish. Extra-gonadal steroids such as E2 plays a pivotal role in

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sculpting the fish brain and behavior, particularly in females (Ramsey et al., 2011). In a

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preliminary study (data not shown), we found that E2 and its receptor agonists differentially

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affect learning and memory in males and females, with a more prominent effect on female fish.

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Indeed low concentrations of E2 rapidly impacted the recognition memory in adult female

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zebrafish. Therefore, female zebrafish may also be highly susceptible to BPS neurotoxicity. The

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zebrafish is now widely accepted as a robust animal model to study different aspects of learning

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and memory in vertebrate (Meshalkina et al., 2017). Among the learning paradigms, zebrafish

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have shown a remarkable learning ability in recognition tasks. Intact recognition memory is of

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great importance to aquatic organisms, as it enables successful navigation of physical and social

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environments which is fundamental for appropriate antipredator behaviors and mate choice

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(Brown et al., 2011). In order to dissect mechanisms underlying the neurobehavioral effects of

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BPS, we have mainly focused on the glutamate receptors/ERK/CREB signaling pathway. This

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signaling cascade controls the transcription of synaptic activity-inducible immediate early genes

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(IEGs), which are an integral part of synaptic plasticity and memory formation (Sun and Lin,

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2016). Different components of this signaling pathway have been implicated in learning and

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memory in zebrafish (Naderi et al., 2018b; Nam et al., 2004; Ng et al., 2012).

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2. Materials and Methods

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2.1. Zebrafish maintenance and exposure

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Adult female wild-type (AB) zebrafish (Danio rerio, 9 months of age) were acquired from our

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breeding colony in R.J.F Smith Center for Aquatic Ecology of the University of Saskatchewan.

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A total of 140 fish (0.38 ± 0.02 g and 3.53 ± 0.06 cm) were used in this study. Fish were kept in

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groups of 7 fish per tank and 4 tanks were randomly assigned to each treatment group (28 fish 5

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per treatment). Fish were maintained under 14/10 h light/dark period with a constant temperature

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(26 ± 1˚C) in aerated aquaria. Moreover, animals were fed with a commercial flake food

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(Nutrafin Max flakes, Germany) complemented with bloodworm (Hikari BIO-PURE®,

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California, USA) twice a day. Zebrafish were acclimated to the aforementioned conditions for 2

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weeks prior to the experiment. No fish mortalities were observed during the experiment.

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BPS (purity > 99%) and E2 were obtained from Alfa Aesar (UK) and Sigma-Aldrich (USA)

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respectively. Stock solutions of BPS and E2 were prepared (every 2-3 days) in dimethyl

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sulfoxide (DMSO, VWR, CA) and stored in the dark at 4˚C. Fish were exposed to 1, 10, and 30

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µg BPS/L (nominal concentrations) for 120 days. The selected concentration range was

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environmentally relevant and has been found to induce behavioral/physiological effects in

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zebrafish (Ji et al., 2013; Wei et al., 2018; Yamazaki et al., 2015; Zhang et al., 2017). A group of

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fish was also exposed to 1 µg/L of E2 (as positive control) while the control group (solvent

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control) only received DMSO. The solvent concentration was kept at 0.01% DMSO (v/v)

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throughout the experiment. A semi-static approach (with 100% water renewal per day) was

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adopted to retain stable concentrations of the test compounds in the exposure tanks.

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2.2. Chemical analysis of BPS

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To determine actual concentrations of BPS, water samples (50 ml, n=2) from each treatment

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group were collected before and 24 h after the renewal of exposure solution on days 59 and 119.

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Water samples were filtered using 0.22 µm filters (Fisher Scientific, USA) and the BPS

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concentrations were quantified by High Performance Liquid Chromatography (HPLC) system

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(Agilent Technologies 1200 series HPLC system; USA) using a device fitted with a Discovery

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C18 reversed-phase column (Eclipse XDB-C18 column, 4.6 mm × 250 mm, particle size 5 µm)

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at 30 ℃. The composition of the mobile phase used in the present study consisted of a mixture of

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acetonitrile and water (20:80, V/V) at a flow rate of 0.6 mL/min. Samples were analyzed with a

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UV/VIS detector at a wavelength of 258 nm. The detection limit for BPS was ~ 0.25 µg/L.

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2.3. Learning paradigms

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In order to evaluate long-term recognition memory in zebrafish, we employed 3 learning

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paradigms, including object recognition (OR), object placement (OP), and social recognition

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(SR) tests. These learning paradigms were originally developed for rodent studies (Tuscher et al.,

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2015), and have recently been extended to zebrafish (Gaspary et al., 2018; Oliveira et al., 2015).

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These tasks are based on the natural tendency of zebrafish to explore novel or moved

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objects/stimuli more than familiar/unmoved stimuli. To this end, fish are exposed to two

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identical objects. Those fish who remember the identity or location of the training objects will

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spend more time exploring the novel/moved object, which indicates memory for the previously

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encountered objects (i.e. familiar/unmoved objects). However, if exploration of the novel/moved

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and familiar/unmoved objects is the same, this can be interpreted as a memory deficit. All fish

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(n=28) from each treatment group were subjected to each learning task with a minimum interval

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of 4 days (OR>OP>SR).

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2.4. Object recognition paradigm

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The task has been evolved to assess fish ability to discriminate between old and new objects. The

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OR task was evaluated based on the modified version of protocols previously described (Faillace

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et al., 2017; Gaspary et al., 2018). The experimental apparatus was a 10 L – white plastic tank

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(35 cm × 35 cm × 10 cm; L× W× H, respectively) which was filled with 6 cm of water (Figure

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1A). The OR consisted of four phases, including (1) a habituation phase in which individual fish

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was acclimated to the empty experimental apparatus for 5 min twice a day over 5 consecutive

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days, (2) a training phase on the 6th day in which subjects explored two identical objects for 15

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min (two blue LEGO® plastic blocks, 4 cm × 2 cm × 2 cm; L× W× H, respectively), (3) a 24 h

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retention interval, and (4) a 15 min test phase where fish were allowed to freely explore the tank

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while one of the objects (i.e. the familiar object) was replaced by a totally new item (a yellow

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LEGO® plastic block). All objects presented similar textures and sizes but had distinctive colors.

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The exploration time was calculated as the duration of time that zebrafish (whole-body) entered

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the “exploration area” which was defined as 8 × 8 cm area around each object. In this study, we

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employed blue and yellow colors to avoid any innate color preference by zebrafish as described

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previously (Faillace et al., 2017).

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2.5. Object placement paradigm

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This task assessed the spatial memory of zebrafish using a protocol described previously

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(Faillace et al., 2017; Gaspary et al., 2018). The fish were tested in an experimental apparatus

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with the same dimensions as the maze used for the OR task, except that the maze walls were

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made of black plastic sheets containing the spatial cues (Figure 1B). The visual cues composed 7

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of square and diamond-shaped white paper cuts with the same color and size, which enabled

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zebrafish to estimate the location of the object within the apparatus. Similar to the assessment of

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OR task, OP paradigm also consisted of 5 days of habituation, a 15 min training, a 24 h interval,

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and a 15 min test phase. During the training phase, two objects with same shape and colour were

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placed in two consistent locations against one side of the maze. However, during the test, one of

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the objects was moved to a new location (to the opposite side of its previous location). The

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amount of time spent by fish in the exploration area was recorded.

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2.6. Social recognition paradigm

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The task is designed to assess memory for past interactions with different individuals. We used a

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learning paradigm equivalent to that recently introduced by (Madeira and Oliveira, 2017). The

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experimental apparatus consisted of a white plastic tank (60 cm × 20 cm × 20 cm; L× W× H,

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respectively), divided into three equally sized compartments separated by a removable

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transparent partition. Each of the lateral compartments of the tank contained a clear Plexiglas

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parallelepiped with small perforations at the bottom (Figure 1C). This test was also comprised of

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habituation, training, and a test phase separated from each other by 24 h. During the habituation

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phase, each focal fish was placed in the central compartment of the tank for 15 min. In the

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training phase, one stimulus fish was placed in each clear Plexiglas parallelepiped in the middle

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of lateral compartments. After 5 min of habituation, the partitions were removed and the focal

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fish was allowed to explore the two lateral compartments, each containing a nonfamiliar

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conspecific, for 20 min. The stimulus fish were from the same origin, age, and size as the

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experimental fish. Stimulus fish were never in physical, chemical, or visual contact with

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experimental fish. Only female fish were used as stimulus fish in order to avoid possible bias

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towards opposite sex. The test phase was conducted after 24 h. During the test phase, however,

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one of the two stimulus fish was replaced with a novel fish. The amount of time that fish spent in

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each compartment was recorded.

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2.7. Behavioral data analysis

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Fish behavior during the test phase of each learning task was video recorded using an overhead

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HD digital camera (Logitech C922x Pro Stream, USA). MATLAB software (Academic version

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R2014a) with image processing and computer vision toolbox was employed to extract the

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parameters of interest from video footages. The exploration ratio was calculated as = N/(N+F), 8

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where N is the amount time that zebrafish explore the novel or moved stimuli/objects and F is

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the amount of time that fish investigate the familiar or unmoved stimuli/objects. Exploration

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ratio during training typically fluctuates around ∼0.5 (chance). A deviation from 0.5 indicates a

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discrimination while a score greater than 0.5 indicates discrimination and preference for the

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novel object/place (Tuscher et al., 2015). In order to minimize the possibility of using intra- or

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extra maze cues by fish or bias towards a certain lateral compartment, the direction of maze and

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location of stimulus fish was changed. Moreover, the position of the familiar and new

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object/stimulus fish. Locomotion (total distance traveled by fish) was also measured. At the

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conclusion of behavioral tests (SR test), fish were immediately euthanized using Aquacalm

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(Syndel Laboratories, Canada) and the whole-brain was dissected out (within 1-2 minutes see

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Naderi et al., 2018b for detail), and stored at -80 °C until further analysis.

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2.8. Biochemical Assays

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Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are the most extensively studied and

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best-known ERK family members (Medina and Viola, 2018). These proteins are activated by the

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phosphorylation of tyrosine and threonine residues in the regulatory sites. Therefore, the

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activation of ERK1/2 was determined by an ELISA kit (Abcam, UK) comparing the level of

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phosphorylated-ERK1/2 (p-ERK1/2) with the total ERK1/2 in the zebrafish whole-brain (pools

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of 2 brains, n=4). We also measured the total CREB activation in the zebrafish brain. To this

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end, nuclear protein extracts were isolated from brain homogenates (pools of 2 brains, n=4) using

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a commercial Nuclear Extraction Kit (Cayman Chemical, USA). The activated CREB in nuclear

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extracts was further quantified by a CREB (Phospho-Ser133) Transcription Factor Assay Kit

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according to the manufacturer's instructions (Cayman Chemical, USA).

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2.9. Quantitative real-time polymerase chain reaction

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We monitored the transcript profile of zebrafish ERs, including the ERα (esr1) and ERβ (esr2a

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and esr2b). To gain further insights into the mechanisms by which BPS may affect cognitive

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functions in zebrafish, the transcript abundance of genes encoding the metabotropic receptors

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(mGluRs) and ionotropic glutamate receptors (iGluRs) was quantified in the brain tissue. To this

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end, we measured the expression level of different mGluR1 subtypes (grm1a, grm1b, grm5a, and

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grm5b). With respect to iGluRs, the transcript levels of gene encoding different subtypes of N-

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methyl-d-aspartate (NMDA) (grin1a, grin1b, grin2da, and grin3a) and α-amino-3-hydroxy-59

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methyl-4 isoxazolepropionic acid (AMPA) receptors (glur1a, glur1b, glur2a, glur2b, glur3a,

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glur3b, glur4a, and glur4b) was quantified. These receptors are involved in synaptic plasticity

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and long-term memory formation in adult zebrafish (Studzinski et al., 2015). In addition to these

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receptors, the expression levels of ERK1 (also known as MAPK3, erk1), ERK2 (also known as

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MAPK1, erk2), CREB1a (creb1a), brain-derived neurotrophic factor (BDNF; bdnf), neuronal

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PAS domain protein 4a (NPAS4; npas4), c-Fos (c-fos), and wingless-type MMTV integration

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site family, member 3 (wnt3) genes were determined in the whole-brain tissues. These genes are

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well-established markers of synaptic plasticity, neurogenesis, and memory formation in a diverse

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range of organisms, including zebrafish (da Silva Peixoto et al., 2017; Naderi et al., 2018a).

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Total RNA extraction from the zebrafish whole-brain (pools of 2 brains, n = 4) was carried out,

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immediately after thawing the brain tissue, using the using RNeasy Mini Kit (Qiagen, Germany).

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Adequate RNA quality and concentration were confirmed using NanoDrop (Thermo Scientific,

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USA). cDNA was synthesized using the Quantiscript reverse transcription kit (Promega

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Corporation, USA) according to the manufacturer’s instructions. The expression levels of

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candidate genes were measured in triplicates on an iCycler Thermal Cycler (Bio-Rad, USA)

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using SYBR Green PCR Master Mix (SensiFAST, SYBR No-ROX Kit, Bioline, USA) as

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described previously.(Naderi et al., 2018b) Specific primers for each gene were designed using

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IDT PrimerQuest software (Integrated DNA Technologies Inc., USA) based on available

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sequences in NCBI (Table S1). The gene encoding β-actin (β-actin) was selected as the reference

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gene since its expression remains unaffected by estrogen treatment (Bosma et al., 2001).

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Therefore, the relative expression of genes of interests was normalized to the expression of β-

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actin using the 2-∆∆CT method as described by Livak and Schmittgen (2001).

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2.10. Statistical analysis

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Data were analyzed statistically using SPSS software (version 23.0, IBM SPSS Inc., USA) and

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are expressed as mean ± S.E.M. unless stated otherwise. Normality of data and homogeneity of

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the variances were tested using the Kolmogorov-Smirnov one-sample test and Levene’s test,

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respectively. For statistical analysis of exploration ratios, data were arcsin-transformed (figures

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represent original ratio data). One-sample t-tests were conducted to determine if fish memory

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performance in each group differs from chance performance (0.5). This analysis determines

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whether learning occurred within each treatment group. To determine between-group differences 10

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in recognition memory, one-way ANOVA and Tukey's post hoc tests was performed. In the case

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of heteroscedasticity, Welch’s test with the Games-Howell post hoc test was conducted. The

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alpha level was set at 0.05.

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3. Results

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3.1. BPS Exposure Concentrations

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Chemical analysis of exposure solutions revealed a small deviation between (less than 25%) the

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actual and nominal concentrations of BPS (see Table S2). BPS concentration was undetectable in

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the control group. For simplification, all BPS treatments are referred by the nominal

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concentrations.

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3.2. Learning performance of zebrafish

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3.2.1. Object recognition

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One-sample t-tests were applied to evaluate each treatment group’s change in OR memory

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relative to the control group. Fish in the control group (t(27)=2.31, p<0.028) and the groups

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exposed to 1 µg/L of E2 (t(27)=4.67, p<0.001), and 1 µg/L of BPS (t(27)=13.05, p<0.001) spent

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significantly more time exploring the novel object during the test phase (Figure 1E). However,

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fish treated with the 10 (t(27)=1.83, p=0.078) and 30 µg/L of BPS (t(27)=1.98, p=0.057) spent

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similar amounts of time with the familiar and novel objects, demonstrating an impaired OR

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memory. Moreover, one℃way ANOVA followed by Tukey's test revealed significant differences

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in exploration ratios among treatments (F(4, 135)=19.98, p<0.001; Figure 1E). While fish in the

312

groups treated with 10 and 30 µg/L of BPS spent significantly less time with the novel object

313

(p<0.001), there was no difference in the exploration ratio among the other treatment groups

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compared to the control group (p>0.05). Moreover, locomotor activity did not differ among

315

treatments compared to the control (p>0.05, Figure S1A).

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3.2.2. Object placement

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Our results showed that the control fish (t(27)=4.45, p<0.001) treated with the vehicle or fish

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treated with E2 (t(27)=9.35, p<0.001), 1 (t(27)=10.06, p<0.001) and 10 µg/L of BPS (t(27)=7.25,

319

p<0.001) spent significantly more time than chance (0.5) exploring the moved object. However,

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fish exposed to 30 µg/L of BPS (t(27)=-5.48, p<0.001) spent less time than chance with the

11

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moved object (Figure 1F). One-way ANOVA also yielded significant group differences in the

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exploration ratio among different treatment groups (F(4,

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treated with E2 and 1 µg/L of BPS exhibited an improved OP memory (both p=0.028), the

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exposure to 30 µg/L of BPS significantly attenuated OP memory performance compared to the

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control group (p<0.001; Figure 1F). There was no significant difference in the exploration ratio

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between the control group and the group treated with 10 of BPS (p=0.46). Locomotion also did

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not significantly differ among treatment groups (p>0.05; Figure S1B).

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3.2.3. Social recognition

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Fish in groups treated with vehicle (t(27)=6.82, p<0.001), E2 (t(27)=6.83, p<0.001), and the lowest

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BPS concentration (t(27)=6.89, p<0.001) spent a longer time investigating the novel conspecific,

331

indicating an intact SR memory. Fish treated with 10 µg/L of BPS did not perform better than

332

random chance (t(27)=-1.40, p=0.174). However, fish in the group exposed to 30 µg/L spent

333

significantly less time than chance exploring the unfamiliar conspecific (t(27)=-5.61, p<0.001).

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The exploration ratios differed significantly among treatment groups (F(4, 135)=31.21, p<0.001;

335

Figure 1G). Fish in the groups exposed to 10 and 30 µg/L of BPS spent significantly less time

336

investigating the novel stimulus fish compared to the control group (p<0.001). However, the

337

exploration ratio in the groups exposed to 1 µg/L of E2 and BPS was not significantly different

338

from that of the control group (p>0.05). Fish locomotion remained unchanged among different

339

treatment groups (p>0.05; Figure S1C).

340

3. 4. Alterations in phosphorylation levels of ERK1/2 and CREB

341

Our results indicated that chronic exposure to E2 and BPS influenced ERK phosphorylation in a

342

dose-specific manner (F(4, 15)=41.14, p<0.001; Figure 2A). While the exposure to 1 µg/L of E2

343

and BPS increased the ratio of p℃ERK1/2/total ERK1/2 (p<0.001 and p=0.035, respectively),

344

the highest concentration of BPS significantly decreased this ratio (p=0.001). The exposure to

345

BPS and E2 also impacted activity level of CREB (F(4, 15)=39.66, p<0.001; Figure 2B). There

346

was a significant increment in the phosphorylation level of CREB in fish treated with E2

347

(p=0.005). The phosphorylation level of CREB in the group treated with 1 µg/L of BPS was not

348

statistically distinguishable from that of the control group (p=0.051). In contrast, the exposure to

349

30 µg/L of BPS decreased the phosphorylation level of CREB (p=0.017).

12

135)=37.06,

p<0.001). While groups

350

3. 5. Effect of BPA exposure on whole-brain gene expression

351

The exposure to E2 up-regulated the expression of E2 receptor subtypes including esr1 and

352

esr2a, and esr2b (esr1: F

353

6.92)=8.31,

354

and esr2b (both p≤0.026). The transcriptional activity of these genes in groups treated with10

355

and 30 µg/L of BPS remained unchanged when compared to the control group (p<0.05). The

356

expression of glutamate receptors was also affected by exposure to E2 and BPS (Table 1).

357

With respect to mGluR1, the expression level of grm1a (F

358

15)=34.43,

359

significantly changed. E2 exposure increased the expression of grm1a, grm1b, and grm5b

360

(p≤0.026). The exposure to 1 µg/L of BPS only up-regulated the expression of grm1b and grm5b

361

subtypes (both p≤0.001). In contrast, a significant decrease in the mRNA expression of grm5b

362

was found in the group exposed to 10 µg/L of BPS (p=0.003). The mRNA expression of grm5a

363

and grm5b was also down-regulated in fish exposed to 30 µg/L of BPS (p=0.046 and p<0.001,

364

respectively). An alteration in the mRNA expression of iGluRs was also found across different

365

treatment groups (glur1a: F

366

6.47)=8.42,

367

levels of AMPA receptors (glur1a, glur1b, and glur2b) was found in fish exposed to E2 (all

368

p≤0.001). The exposure to the lowest concentration of BPS also up-regulated the expression

369

levels of glur1a and glur1b in the zebrafish brain (p≤0.007). However, a marked down-

370

regulation in the expression of glur3a was observed in fish exposed to the highest concentration

371

of BPS (p=0.043). The exposure to BPS and E2 also affected the transcriptional activity of

372

grin1a (F

373

µg/L of E2 resulted in an up-regulation in the expression level of grin1a and grin1b (both

374

p≤0.005). An up-regulation in the expression of grin1b was recorded in the group exposed to 1

375

µg/L of BPS (p=0.011). In contrast, there was a significant decrease in the mRNA abundance of

376

grin1a in fish treated with the highest BPS concentration (p=0.046). However, no statistically

377

significant change was recorded in the expression levels of grin1b in the 30 µg/L of BPS

378

exposed group relative to the control group (p>0.05).

(4, 15)=7.47,

p=0.001; esr2a: F

(4, 6.2)=21.66,

p<0.001 and esr2b: F

(4,

p=0.009). The exposure to 1 µg/L of BPS also increased the transcript levels of esr2a

p<0.001), grm5a (F

(4, 6.2)=11.53,

(4, 15)=25.40,

(4, 15)=13.24,

p=0.005), and grm5b (F

p<0.001), grm1b (F

(4, 7.35)=63.05,

(4,

p<0.001)

p<0.001, glur1b: F (4, 15)=23.92, p<0.001, glur2b: F

(4,

p<0.001, glur3a: F (4, 6.51)=9.78, p=0.007). A significant up-regulation in the transcript

(4, 6.94)=28.70,

p=0.007) and grin1b (F

13

(4, 15)=13.17,

p=0.003). Chronic exposure to 1

379

As shown in Table 1, the expression of erk1 mRNA (F

380

reduced in the group exposed to the highest concentration of BPS (p=0.014). In contrast, a

381

relative increase in the mRNA level of erk2 (F (4, 15)=17.01, p<0.001) was observed in the groups

382

exposed to 1 µg/L of E2 and BPS (both p=0.001). There was a significant change in the mRNA

383

expression of creb1a (F

384

expression of creb1a. There was not statistically meaningful change in the transcript abundance

385

of creb1a in other treatment groups (p>0.05).

386

Our results also revealed an alteration in the mRNA expression of IEGs associated with the

387

synaptic plasticity and memory formation (bdnf: F

388

p≤0.009; c-fos: F

389

transcriptional up-regulation of BDNF gene was found in the fish exposed to 1 µg/L of E2 and

390

BPS (p=0.028 and 0.034, respectively), while this gene was down-regulated in the group treated

391

with 30 µg/L of BPS (p=0.046). A modest albeit non-significant decrease in the mRNA

392

expression of bdnf was also observed in the group treated with 10 µg/L of BPS (p=0.087).

393

Similarly, fish exposed to 1 µg/L of E2 and BPS exhibited an increase in the transcript

394

abundance of wnt3 (both p≤0.031). There was also a significant increase in the mRNA

395

expression of c-fos in fish exposed to 1 µg/L of BPS (p=0.005). Additionally, a significant up-

396

regulation in the mRNA expression of npas4 was found following exposure to 1 µg/L of E2 and

397

BPS (both p≤0.027). While there was an apparent down-regulation in the transcript abundance of

398

npas4 in fish exposed to 30 µg/L of BPS, it did not significantly differ from the control group

399

(p=0.09).

400

4. Discussion:

401

In recent years, BPS has emerged as a potential BPA replacement alternative. However, wide-

402

spread consumer and commercial use of BPS coupled with the lack of regulatory guidelines on

403

its use inevitably leads to the release of this substitute compound into the environment and

404

thereby presenting a new threat to humans and animals. The results of the present study, for the

405

first time, provide evidence that the chronic exposure to ecologically relevant concentrations of

406

BPS dose-specifically affects recognition memory in adult female zebrafish. Recognition

407

memory plays a pivotal role in animals’ perception to approach or avoid predators, reproductive

408

advantages, and foraging strategies (Brown et al., 2011). Therefore, impaired recognition

(4, 15)=17.99,

(4, 6.30)=19.53,

(4, 6.50)=22.44,

p=0.001) significantly

p<0.001). The exposure to E2 also up-regulated the

(4, 6.99)=26.88,

p=0.001; npas4: F

14

p<0.001; wnt3: F

(4, 6.17)=21.63,

(4, 6.08)=9.52,

p=0.001). A marked

409

memory can bring about a wide array of adverse consequences for animals in their natural

410

environment. Furthermore, our results also illustrated that neurobehavioral effects of BPS are

411

likely to be mediated through changes in the expression of genes and proteins involved in

412

recognition memory, such as glutamate receptors, ERK, CREB, and several IEGs.

413

The results of the present study showed that the vehicle treated group (control) spent more time

414

exploring the novel/moved stimuli in OR, OP, SR tasks after a 24h delay indicating a long-term

415

recognition memory in zebrafish as demonstrated by others (Faillace et al., 2017; Madeira and

416

Oliveira, 2017; Pinheiro-da-Silva et al., 2017). Moreover, our data demonstrate that chronic

417

exposure to a low concentration of E2 enhanced long-term recognition memory in adult female

418

zebrafish tested in OP memory task. This was evident by a marked increase in the exploration

419

ratio of the displaced object during the test phase. These findings are in agreement with previous

420

studies in mammals indicating that E2 treatment generally improves OP memory (Boulware et

421

al., 2013; Walf et al., 2008). Different from OP memory assessed, however, E2 did not affect

422

fish performance in OR and SR tasks. Estrogen enhancement of object and social recognition has

423

been demonstrated in several studies (see Ervin et al., 2013). Therefore, this discrepancy may be

424

due to time, task, or dose-specific effects of E2 on learning and memory in zebrafish, as

425

repeatedly demonstrated in mammals. Moreover, the route of E2 delivery may also explain these

426

discordant results. For instance, dorsal hippocampal administration of E2 did not facilitate SR

427

memory in ovariectomized female mice, while systemic administration of E2 did (Phan et al.,

428

2013). Nevertheless, it seems that chronic waterborne exposure to 1 µg/L of E2 was not effective

429

in facilitating OR and SR memory in adult female zebrafish. Collectively, our results suggest that

430

E2 may not influence all cognitive functions to the same degree.

431

Interestingly, the present study demonstrated that, similar to E2, the chronic exposure to BPS

432

also alters the recognition memory in adult female zebrafish. The lowest concentration of BPS

433

used in this study augmented zebrafish performance in OP memory task in the same magnitude

434

as E2. However, the exposure to 1 µg/L of BPS did not alter OR and SR memories in zebrafish,

435

as observed with exposure to E2 as well. This suggests that the molecular machinery underlying

436

OR and SR memory is to some extent different from that of spatial OP task. The effects of BPS

437

at lower concentrations appeared to be opposite to that in higher concentrations. Indeed, the

438

long-term exposure to the higher concentrations of BPS (10 and 30 µg/L) attenuated OP, OR and

15

439

SR memories in adult zebrafish. These data suggest that BPS has a biphasic effect on recognition

440

memory in female zebrafish. Currently, there are only a handful of studies showing the

441

neurobehavioral effects of BPS on fish. Recently, it has been shown that embryonic exposure to

442

BPS alters locomotor behavior in zebrafish larvae (Gu et al., 2019; Kinch et al., 2015).

443

Behavioral abnormalities associated with BPS have also been demonstrated in few studies in

444

mammals, including the increased anxiety, altered exploration activity, decreased interest in

445

social interactions, and compromised maternal care in mice and rats (Catanese and Vandenberg,

446

2016; da Silva et al., 2019; Kim et al., 2015). In addition to BPS studies, our findings are in

447

striking agreement with previous studies reporting various neurobehavioral abnormalities in fish

448

and rodents exposed to BPA. For example, BPA induced-learning deficits and impaired social

449

behaviors have been reported in zebrafish (Saili et al., 2012; Weber et al., 2015). Rodent studies,

450

however, reported antithetical actions of BPA on cognitive functions, ranging from

451

enhancements to deficits. For example, several studies have documented that BPA enhanced

452

passive avoidance memory, contextual fear memory, and spatial memory in rats and mice

453

(Matsuda et al., 2013; Xu et al., 2015; Xu et al., 2011; Zhang et al., 2014). In contrast, BPA

454

induced-memory disruption, including OR and OP memories has also been documented in other

455

studies (Eilam-Stock et al., 2012; Jardim et al., 2017; Wang et al., 2016). Taking these results

456

into account and considering the biphasic effects of BPS on the recognition memory in zebrafish

457

observed in the present study, we postulate that BPS induces hormetic effect on zebrafish

458

memory and cognitive behavior.

459

Synaptic plasticity of neuronal circuits in the brain, mainly in the hippocampus, is the basis of

460

learning and memory (Bailey et al., 2015). Estradiol and its receptors have been strongly

461

implicated in influencing this process through changes in synaptogenesis, neuronal network

462

connectivity, and synaptic transmission (Frick, 2015). It is generally postulated that abnormal

463

expression and activation of ERs is central to BPS’s mode of action (Rochester and Bolden,

464

2015). Consequently, exposure to BPS may elicit widespread effects on the structure and

465

function of the brain. The results of the present study showed that the chronic exposure to E2 and

466

BPS alters the expression of ERs in the zebrafish brain. While E2 up-regulated all zebrafish ERs,

467

only the lowest concentration of BPS increased the mRNA abundance of esr2a and esr2b in the

468

zebrafish brain. This finding is consistent with previous studies in mammals and zebrafish

469

documenting that BPS is more active on the ERβ than ERα (Le Fol et al., 2017; Molina-Molina 16

470

et al., 2013). Moreover, a wealth of data indicates that the cognitive effects of E2 on the CNS are

471

mainly mediated through ERβ (Liu et al., 2008). Thus, the induced expression of esr2a and esr2b

472

might be responsible for the facilitative effects of BPS on OP memory in adult female zebrafish.

473

The higher BPS concentrations, however, slightly decreased the expression of esr2a and esr2b.

474

Based on these observations, we suggest that long-term exposure to higher concentrations of

475

BPS might have led to desensitization of ERs or desensitization of signal transduction

476

mechanisms coupled to these receptors. Another possibility could be attributed to the non-

477

estrogenic effects of BPS in higher concentrations as suggested previously (Le Fol et al., 2017).

478

Indeed, BPS may produce various effects on the CNS depending on the concentration. Further

479

studies are required to elucidate these dose-dependent effects of BPS on the transcriptional

480

activity of ERs in zebrafish.

481

Glutamate receptors are essential mediators of the behavioral plasticity in zebrafish, including

482

recognition memory (Gaspary et al., 2018). The expression and activity of glutamate receptors

483

are required for the increase in dendritic growth, spine formation, and synaptic efficacy (Riedel

484

et al., 2003). Therefore, alterations in the transcript levels of glutamate receptors may lead to

485

functional and/or structural changes in neuronal circuits underlying memory. Convergent

486

evidence also suggests that ERs interact with glutamate receptors to regulate multiple aspects of

487

morphological and functional plasticity in the brain (Boulware et al., 2013; Boulware et al.,

488

2005; Liu et al., 2008). Our results showed that the exposure to E2 and/or the lowest

489

concentration of BPS up-regulated the several subtypes of mGluR1s and iGluRs in the zebrafish

490

brain. The increment in the expression and phosphorylation levels of glutamate receptors along

491

with the improved memory performance has also been reported in rodents exposed to BPA (Xu

492

et al., 2011; Zhang et al., 2014). Therefore, it seems that the lowest BPS concentration used in

493

this study might have up-regulated the transcriptional activity of glutamate receptors through an

494

estrogenic mode of action leading to memory enhancement. An opposite trend, however, was

495

observed in the expression level of different variants of mGluR1s (grm5a and grm5b) and

496

iGluRs (glur3a and grin1a) in fish treated with the higher concentrations of BPS. Consistent

497

with this, a concomitant decrease in the transcription of ERβ and different NMDA receptor

498

subtypes were reported in rats developmentally exposed to BPA (Xu et al., 2010). In our study,

499

while a marked down-regulation in the expression of several glutamate receptor subtypes was

500

recorded in fish treated with the high concentrations of BPS, the mRNA levels of ERs remained 17

501

unchanged. One possible explanation for this finding draws on transient effects of BPS on the

502

transcript abundance of ERs or desensitization of these receptors as discussed earlier. Indeed, the

503

higher concentrations of BPS may have provoked only a short-term change in the expression

504

level of ERs. Alternatively, the BPS induced learning impairment might have occurred due to the

505

non-estrogenic effects of this compound as suggested elsewhere (Ullah et al., 2019; Zhang et al.,

506

2016). Alterations in the transcriptional activity of different glutamate receptors in the group

507

treated with the highest BPS concentration, relative to the groups treated with E2 or lowest

508

concentration of BPS, lend further support to this notion.

509

The ERK1/2 are components of the ERK/MAPK signaling cascade, which their activity in the

510

brain is primarily associated with long-term memory formation (Adams and Sweatt, 2002;

511

Medina and Viola, 2018). It also catalyzes the phosphorylation process of CREB, a primary

512

regulator of expression of genes associated with synapse re-modeling, synaptic plasticity and

513

memory (Kandel, 2012). The present study indicated an up-regulation in the transcript levels of

514

ekr2 in fish treated with E2 and 1 µg/L of BPS. Our results further revealed that chronic

515

exposure to E2 and 1 µg/L of BPS resulted in an increase in the relative phosphorylation levels

516

of ERK1/2 (p℃ERK1/2/ERK1/2), indicating the stimulated activity of this protein kinase. This is

517

in line with a previous study demonstrating that BPS at femtomolar to picomolar concentrations

518

had the same effect as E2 to initiate phosphoactivation of ERK in rats pituitary cells (Viñas and

519

Watson, 2013). However, the highest concentration of BPS (30 µg/L) used in our study

520

noticeably reduced the mRNA abundance of erk1 and the phosphorylation of ERK1/2. Down-

521

regulation of ERK/MAPK signaling can be an initial step in survival of ERK-dependent

522

pathways (Hunter, 1995). Therefore, the reduced phosphorylation of ERK might be a protective

523

mechanism against harmful estrogenic stimulation caused by the high concentration of BPS as

524

suggested previously (Viñas and Watson, 2013). A similar trend was also noticed for CREB. E2

525

up-regulated the transcript levels of creb1a and phosphorylation levels of CREB. The transcript

526

levels and activity of CREB in fish treated with the lowest concentration of BPS remained

527

unchanged. In contrast, exposure to 30 µg/L of BPS suppressed the activity of this transcription

528

factor. Overall, our results indicate that low concentrations of E2 and BPS induce the expression

529

and activity of ERK and CREB, whereas the exposure to the highest concentration of BPS

530

reduces the ERK and CREB expression and activity in the zebrafish brain. This is consistent with

531

previous studies that suggested that the neurotoxicity of BPA is mainly correlated with the 18

532

disruption of ERK/CREB pathway (Chen et al., 2017; Xu et al., 2014). Jang et al. (2012)

533

demonstrated that developmental exposure of female mice to BPA reduced the hippocampal

534

levels of phosphorylated ERK and CREB (Jang et al., 2012). BPA was also found to impair both

535

OR and OP memory in adult male rats through alterations in cytosolic and nuclear levels of

536

CREB (Eilam-Stock et al., 2012). An antagonistic effect of BPA on the ERK/CREB pathway

537

and subsequent OR memory impairment was also reported in male rats maternally exposed to

538

this xenoestrogen (Wang et al., 2016). Based on all of these observations, it is reasonable to

539

suggest that alterations in ERK and CREB activity are one of the main mechanisms underlying

540

neurobehavioral effects of BPS in zebrafish.

541

As a transcription factor, CREB has been implicated in the transcriptional regulation of a variety

542

of genes involved in neuronal survival, memory consolidation, and synaptic plasticity. BDNF is

543

best known transcriptional target of CREB, which acts as a potent modulator of synaptic

544

transmission and plasticity (Sakamoto et al., 2011). The expression of this neurotrophin is crucial

545

for the consolidation of long-term memories, including the recognition memory (Bekinschtein et

546

al., 2008). In the present study, we observed that the expression level of bdnf gene was increased

547

in female fish exposed to E2 and 1 µg/L of BPS. However, the transcript levels of this gene were

548

down-regulated in fish treated with higher BPS concentrations. A great deal of research shows

549

that E2 and BDNF work in concert to promote dendritic spine density and enhance cognition in

550

animals (Luine and Frankfurt, 2013). Therefore, it is likely that the lowest dose of BPS used in

551

this study improved OP memory in adult female zebrafish through an elevation in BDNF levels.

552

In contrast, impaired OP, OR, and SR memory performance of zebrafish in groups treated with

553

higher concentrations of BPS (10 and 30 µg/L) can be attributed to the suppressed expression of

554

this neurotrophic factor in the brain.

555

c-Fos and Wnt3 are other downstream targets of CREB signaling (Lakhina et al., 2015). c℃Fos

556

is a transcription factor which is widely used as a generic marker of neuronal activation and

557

associated synaptic plasticity and memory (Minatohara et al., 2016). A positive correlation

558

between c℃Fos expression and improvement of recognition memory has been reported in

559

mammals (Callaghan and Kelly, 2012). Analysis of c℃Fos expression in the present study

560

revealed a marked up-regulation of this gene in groups treated with 1 µg/L of BPS as well as a

561

slight but non-significant increase in the mRNA levels of this gene in E2 treated fish. Given that

19

562

up-regulation of c-Fos expression reflect increases in neuronal activity, these data suggest that

563

E2 and the lowest concentration of BPS may improve fish performance by increasing the central

564

processing of spatial information in OP task. This finding is also supported by previous in vitro

565

and in vivo studies showing the E2-induced increased expression of c℃Fos (Hennessy et al.,

566

2005; Maggiolini et al., 2004). BPA-induced increase in the c℃Fos expression through activation

567

of the ERK/MAPK pathway has also been reported in several studies (Sheng et al., 2013;

568

Steinmetz et al., 1998). Dong et al. (2011) reported that BPA evoked the expression of c-Fos

569

through increase in the expression of the G protein-coupled estrogen receptor (GPER) and

570

subsequent activation of ERK1/2 pathway. (Dong et al., 2011). GPER mediates non-genomic

571

signaling by estrogens. Therefore, the lowest BPS concentration used in this study up-regulated

572

the expression of c-Fos probably via the non-genomic signaling leading to OP memory

573

improvement in zebrafish. This hypothesis needs to be elaborated by further studies. An apparent

574

but non-significant decrease in the expression of c-Fos gene was also detected in fish treated

575

with the highest BPS concentration. The rapid induction and transient nature of c-Fos, as well as

576

its neuroprotective roles, might have restored down-regulation of c-Fos to normal levels, at least

577

to an extent.

578

Wnt signaling is critically involved in a host of neurological processes, including neurogenesis,

579

synapse formation, and synaptic connectivity in the adult brain (Oliva et al., 2013). Wnt3 is

580

functionally necessary for acquisition and consolidation of different forms of memory including

581

the recognition memory (Fortress et al., 2013). In this study, we recorded an increase in the

582

mRNA levels of Wnt3 in fish exposed to E2 and 1 µg/L of BPS. This likely points to a

583

stimulated neurogenesis process in fish treated with E2 and 1 µg/L of BPS. This is consistent

584

with previous studies that showed E2 and BPA can promote Wnt3 expression and protein levels

585

in rat’s hippocampus and zebrafish embryos (Zhang et al., 2008). Changes in the transcript

586

abundance of Wnt3 in fish treated with higher concentrations of BPS was not statistically

587

significant. It has recently been shown that BPA inhibits hippocampal neurogenesis in rats

588

through inhibition of Wnt3 gene expression and protein synthesis (Tiwari et al., 2016).

589

Therefore, a slight decrease or even inhibition of neurogenesis in the present study might explain

590

the reduced cognitive performance of fish treated with higher concentrations of BPS. NPAS4 is a

591

neuron-specific transcription factor which regulates the development of excitatory and inhibitory

592

synapses to control homeostatic excitatory/inhibitory balance in neurons. NPAS4 is required for 20

593

normal social interaction and long-term memory formation (Coutellier et al., 2012; Sun and Lin,

594

2016). Reduced NPAS4 transcript abundance has been suggested to contribute to impaired

595

neurogenesis, spatial recognition memory, and social behavior in several organisms including

596

zebrafish (Coutellier et al., 2012; Naderi et al., 2018b). In our study, we observed a marked

597

increase in the mRNA expression of NPAS4 in fish treated with E2 and 1 µg/L of BPS, while a

598

slight decrease in the transcript abundance of this gene was observed in fish treated with higher

599

BPS concentrations. This might reflect a modification in the homeostasis of neuronal excitation

600

and inhibition in the zebrafish brain, which either facilitates or impairs recognition memory in

601

adult zebrafish. It is also of note that down-regulation in the expression of these neuronal

602

markers may indirectly indicate that exposure to high concentrations of BPS can lead to

603

cytotoxicity and neuronal death in the zebrafish brain. One of the unfortunate limitations of this

604

study was the using of the whole brain tissue to analyse genetic and biochemical parameters

605

associated with memory and behavior. In fact, there was no available study about the role of

606

different parts of the brain involved in the regulation of OR, OP, and SR memories.

607

5. Conclusions

608

Although the results of the present study showed that E2 and the lowest BPS concentration

609

improved OP memory by modulating glutamatergic/ERK/CREB signaling cascade, OR and SR

610

memories were apparently independent from such changes. In other words, one may raise the

611

question that why the up-regulation in the expression and activity of genes and proteins involved

612

in this signaling pathway did not impact OR and SR memories, while they were in line with the

613

improved OP memory performance. This might be due to the fact that different cognitive

614

functions are governed by different neural circuits and molecular mechanisms. Another

615

explanation for these findings may lie with the transient effects of E2 and the lowest BPS

616

concentration on OR and SR memories. Indeed, the effects of E2 and the lowest BPS

617

concentration might have been occurred earlier in the study (before behavioral tests). Obviously,

618

perturbations of glutamatergic/ERK/CREB signaling cascade in the groups treated with higher

619

BPS concentrations can be a major mechanism by which this chemical induces adverse effects

620

on OP, OR, and SR memories in adult female zebrafish. Collectively, these data are the first to

621

suggest chronic exposure to environmentally relevant concentrations of BPS affects learning and

622

memory in adult female zebrafish through alterations in glutamatergic/ERK/CREB signaling

21

623

pathway. Recognition memory is a fundamental facet of fish ability to decide whether to

624

approach or avoid the environment, social interactions, potential mates, feeding, and predators.

625

Therefore, the impaired recognition memory can be life-threatening for aquatic organisms.

626

Supporting Information

627

Primer sequences used in this study (Table S1), nominal and measured concentrations of BPS

628

(Table S2), and fish locomotion (Figure S1).

629

Acknowledgments

630

This research was funded by Natural Sciences and Engineering Research Council of Canada

631

(NSERC) Discovery Grants to DC and SN. We kindly thank Mr. Mohammad Shabani and BIG

632

Laboratory of Computer Science Department of the University of Saskatchewan for construction

633

and implementation of MATLAB codes to analyze video data.

634 635 636 637 638 639 640 641 642 643 644 645 646

22

647 648 649 650 651 652

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655 656

Bailey, C.H., Kandel, E.R., Harris, K.M., 2015. Structural components of synaptic plasticity and memory consolidation. Cold Spring Harbor perspectives in biology 7, a021758.

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Bekinschtein, P., Cammarota, M., Katche, C., Slipczuk, L., Rossato, J.I., Goldin, A., Izquierdo, I., Medina, J.H., 2008. BDNF is essential to promote persistence of long-term memory storage. Proceedings of the National Academy of Sciences 105, 2711-2716.

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Bosma, P.T., Blázquez, M., Fraser, E.J., Schulz, R.W., Docherty, K., Trudeau, V.L., 2001. Sex steroid regulation of glutamate decarboxylase mRNA expression in goldfish brain is sexually dimorphic. Journal of neurochemistry 76, 945-956.

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810 811

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879 880 881 882 883 884 885 886 887

Table 1. The mean fold change in the expression level of genes involved in glutamatergic/ERK/CREB pathway as well as the expression levels of several immediate early genes involved in the synaptic plasticity and memory formation. Gene

Control

E2 1µg/L

BPS 1µg/L

BPS 10µg/L

esr1

1.00±0.35

2.66±0.43*↑

1.01±0.19

0.56±0.20

0.82±0.11

esr2a

1.00±0.18

5.05±0.81*↑

3.11±0.45*↑

0.58±0.18

0.31±0.03

esr2b

1.00±0.14

3.35±0.57*↑

2.62±0.32*↑

0.78±0.23

0.44±0.08

grm1a

1.00±0.05

2.58±0.45*↑

1.88±0.28

0.49±0.15

0.53±0.07

grm1b

1.00±0.14

3.57±0.41*↑

2.71±0.34*↑

0.44±0.09

0.19±0.03

grm5a

1.00±0.14

3.59±0.73

2.23±0.57

0.52±0.13

0.28±0.02*↓

grm5b

1.00±0.07

3.10±0.33*↑

2.32±0.11*↑

0.30±0.07*↓

0.34±0.09*↓

glur1a

1.00±0.20

4.00±0.47*↑

3.18±0.42*↑

0.84±0.26

0.39±0.04

glur1b

1.00±0.14

3.91±0.45*↑

2.63±0.32*↑

0.78±0.23

1.00±0.08

glur2a

1.00±0.32

0.79±0.08

0.75±0.13

0.99±0.40

0.78±0.05

glur2b

1.00±0.05

6.73±1.13*↑

3.71±1.08

2.80±0.54

1.16±0.11

glur3a

1.00±0.14

2.60±0.93

1.86±0.33

0.93±0.28

0.28±0.05*↓

glur3b

1.00±0.17

1.88±0.93

1.65±0.27

0.97±0.08

0.87±0.80

29

BPS 30µg/L

888 889 890 891

glur4a

1.00±0.21

2.12±0.98

1.46±0.40

1.17±0.22

1.15±0.10

glur4b

1.00±0.24

1.64±0.42

1.75±0.31

0.77±0.16

1.14±0.05

grin1a

1.00±0.14

3.30±0.28*↑

3.31±0.50

0.44±0.13

0.30±0.07*↓

grin1b

1.00±0.17

3.54±0.78*↑

3.29±0.44*↑

0.70±0.17

0.33±0.07

grin2da

1.00±0.09

1.80±0.93

1.43±0.34

0.94±0.19

0.89±0.09

grin3a

1.00±0.16

1.70±0.73

1.33±0.28

1.28±0.25

1.28±0.19

erk1

1.00±0.11

2.78±1.01

2.14±0.32

0.96±0.12

0.20±0.03*↓

erk2

1.00±0.19

2.84±0.37*↑

2.87±0.35*↑

1.28±0.18

0.61±0.11

creb1a

1.00±0.21

3.90±0.36*↑

2.24±0.43

1.45±0.26

0.52±0.25

bdnf

1.00±0.14

2.45±0.26*↑

2.76±0.33*↑

0.40±0.04

0.27±0.05*↓

wnt3

1.00±0.21

3.20±0.75*↑

3.41±0.67*↑

0.68±0.15

0.33±0.02

c-fos

1.00±0.13

2.53±0.49

3.40±0.65*↑

2.33±0.31

0.32±0.04

npas4

1.00±0.18

5.43±0.77*↑

5.89±0.79*↑

0.53±0.25

0.24±0.04

To illustrate the difference in gene expression levels among the treatments, the basal expression level of the control group was set to 1. Asterisks denote significant differences from the control (p<0.05). Data are mean ± SEM (n=4). Arrow up “↑” and arrow down “↓” indicate either up-regulation or down-regulation in the gene expression, respectively.

892 893 894 895 896 897 898 899 900

30

901 902 903 904 905 906 907

Figure Captions:

908

Figure 1. Schematic drawing of behavioral paradigms, consists of OR (A), OP (B), and SR (C)

909

tasks used in this study. T1 and T2 stand for training and test phase, respectively. In SR

910

paradigm, the black fish refers to the novel fish stimulus. Exploration ratio of fish treated with

911

vehicle, E2 and different concentrations of BPS in OR (E), OP, (F), and SR (G) tasks. The

912

dashed line at 0.5 indicates chance performance. The pound (#) denotes significant difference

913

from chance (p<0.05). Asterisks above bars indicate a significant difference in exploration ratio

914

in comparison to the control group (p < 0.05; n=28). Gray fish indicates novel stimulus fish.

915

Figure 2. The ratio of phospho-ERK1/2 to total ERK (p-ERK1/2/ERK) in the zebrafish brain

916

treated with vehicle, E2, and different concentrations of BPS (A; one-way ANOVA; n=4).

917

Relative phosphorylation levels of CREB in the zebrafish brain among different treatment groups

918

(B; n=4).

31

E

A

T1

T2 #

#

#

F

Figure 1

T1

T2 #

B

#

#

#

#

G

C

#

T1

T2

#

#

A

Figure 2

B CREB (Phospho-Ser133)

2.0

* *

1.5

1.0

*

0.5

0.0

Control

E2 (1)

BPS (1)

µg/l

BPS (10)

BPS (30)

Relative phosphorylation level

Relative phosphorylation level

p-ERK1/2/total ERK1/2 4

* 3

2

*

1

0

Control

E2 (1)

BPS (1)

µg/l

BPS (10)

BPS (30)

• E2 and the lowest BPS concentration improved OP memory. • Exposure to 10 and 30 µg/L of BPS impaired OR, OP, and SR memories. • The BPS effects on memory are mediated through the glutamatergic/ERK/CREB pathway.

Authorship Statement Manuscript Title: Chronic exposure to environmentally relevant concentrations of bisphenol S differentially affects cognitive behaviors in adult female zebrafish

All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this material or similar material has not been and will not be submitted to or published in any other publication before its appearance in the journal of Environmental Pollution. Authorship contributions Mohammad Naderi: Conception and design of study, acquisition of data, analysis, and/or interpretation of data, Drafting the manuscript Arash Salahinejad: Technical assistant, acquisition of data, Experiment Implementation Anoosha Attaran: Technical assistant, data analysis Doug Chivers: leading the project, financial support of the project, revising the manuscript Som Niyogi: leading the project, financial support of the project, editing the manuscript

Thank you for your consideration of this manuscript. Sincerely, Mohammad Naderi Ph.D. Department of Biology 112 Science Place University of Saskatchewan Saskatoon, Saskatchewan Canada, S7N 5E2

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: